Carbon Nanotubes as Drug Delivery Vehicles

Solid State Phenomena Vol. 222 (2015) pp 145-158 © (2015) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/SSP.222.145

Carbon Nanotubes as Drug Delivery Vehicles Shweta Aroraa, Vanish Kumarb,Shriniwas Yadavc, Sukhbir Singhd, Deepika Bhatnagare, Inderpreet Kaurf Biomolecular Electronics and Nanotechnology Division (BEND), Central Scientific Instruments Organisation (CSIR-CSIO), Sector-30C, Chandigarh - 160030, India. a

[email protected], [email protected], [email protected], [email protected], [email protected], [email protected] (corresponding author)

d

Keywords: Nanomaterials, Carbon Nanotubes, Drug Delivery, Cancer, Toxicity, Biomedical.

Abstract: Various biomedical applications of nanomaterials have been proposed in the last few years leading to the emergence of a new field in diagnostics and therapeutics. Most of these applications involve the administration of nanoparticles into patients. Carbon Nanotubes are enjoying increasing popularity as building blocks for novel drug delivery systems as well as for bioimaging and biosensing. The recent strategies to functionalize carbon nanotubes have resulted in the generation of biocompatible and water-soluble carbon nanotubes that are well suited for high treatment efficacy and minimum side effects for future cancer therapies with low drug doses. The toxicological profile of such carbon nanotube systems developed as nanomedicines will have to be determined prior to any clinical studies undertaken. Contents of Paper 1. Introduction 2. Cancer: The need for targeting 3. Carbon nanotubes 4. CNTs as Drug Delivery vehicles 4.1. Efficient drug delivery 4.2. Antitumor Immunotherapy 4.3. Local Antitumor HyperthermiaTherapy 4.4. Gene delivery systems 5. Cellular Internalization of CNTs 5.1. Endocytosis 5.2. Passive diffusion 6. CNT toxicity 6.1. Effect of Length and shape 6.2. Solubility issues 6.3. Effect of Impurities 7. Overcoming the toxicity of CNTs by functionalization 8. Bio-distribution mapping of CNTs in Animals 9. Measurement of anticancer activity of CNT-drug Conjugate 10. Conclusions Acknowledgements References

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1. Introduction Evolution in nanotechnology is taking biomedical industry to the next level. Diagnosis, implants, drug delivery are the three major areas in which nanotechnology is actively participating. The convergence of medical industry and nanotechnology represents various revolutionary researches to deal with the cure and diagnosis of complex diseases such as cancer etc. Cancer diagnosis and treatment are of great interest due to the widespread occurrence of the disease, high death rate, and recurrence after treatment. Cancer is characterized by uncontrolled growth of abnormal cells [1]. According to Reports, cancer is wide-spread among all races. Lung cancer, breast cancer and prostate cancer were the three leading causes of death. Current cancer treatment strategies employ a combination of surgery, radiation, and chemotherapy. The localized therapies, such as surgery and radiotherapy, succeed only when malignant cells are confined to the treated area. The use of chemotherapy is therefore essential for the systemic treatment of metastases accompanying the local and regional growth of tumors. But the prolonged use of chemotherapy results in lethal damage to proliferating non-cancerous cells. Despite several decades of intensive research, there is still a need for innovative approaches to design anticancer drugs, targeting of the drug leading to reduced toxicity and improved therapeutic indices [2]. Nanotechnology can help in development of such nanomedicines. Nanomedicine is an area with particular promise that may inspire the construction of nanostructured carriers for the targeted delivery of small-molecule ‘passengers’ to a desired area. Nanosized delivery vehicles may also offer better or more efficient use of an active molecule (i.e. drugs, antigens, proteins, enzymes and nucleic acids) by controlling release rates, by protecting it from unwanted metabolic processes or through targeted delivery processes that can reduce side effects. In this chapter we will discuss on the promising drug delivery vehicles proposed by nanomaterials to overcome the limitation of present medication techniques, since carbon materials ensures biocompatibility and biodegradability, hence major focus of the review will be on carbon nanotubes based drug delivery for targeted cancer treatment. The chapter would also bring insight to the toxicity issues of the material along with the uptake mechanisms of the drugs into cells. 2. Cancer: The need for targeting Cancer is one of the 5 leading causes of death in all age groups amongst both males and females. Cancer occurs due to genetic mutations that happen as a result of the aging process and lifestyle in general, rather than inherited mutations. These cancerous cells may invade the nearby tissues and spread to other parts of body through the blood and lymph systems which is called metastasis [3]. Metastasis is the primary cause of death in case of cancer. The anticancer drugs which have been used in chemotherapy are systemic anti-proliferative agents that preferentially kill the dividing cells. One of the main limitations of almost all cancer treatments so far is the lack of selectivity for tumor tissues. In fact cytotoxic agents (or other therapies) do not exert their action solely on cancer cells, but also healthy organs, resulting in severe side effects for patients, therefore limiting their compliance and the maximum administrable dose. Most chemotherapeutic agents enter normal tissues in the body with indiscriminate cytotoxicity and do not preferentially accumulate at tumor sites. At times the dose reaching the tumor may be as little as 5% to 10% of the doses accumulating in normal organs [4]. In this way, the anticancer effect is decreased and toxic effect to normal cells is increased. Another major problem related to anticancer therapy is Multi drug resistance (MDR) [5].Multidrug resistance, the principal mechanism by which many cancers develop resistance to chemotherapy drugs, during recurrence, tumors usually consist of mixed populations of malignant cells, some of which are drug-sensitive while others are drug-resistant. Chemotherapy kills drugsensitive cells, but leaves behind a higher proportion of drug-resistant cells. As the tumor begins to grow again, chemotherapy may fail because the remaining tumor cells are now resistant [6].

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Hence to reduce drug resistance and develop high impact of drug on cancerous cells with low side effects, the targeting with sustained release at the site of action of the chemotherapeutic drugs is highly needed. A targeted drug delivery system is usually designed to improve the pharmacological and therapeutic properties of drugs and to overcome problems such as limited solubility, poor distribution, lack of selectivity and tissue damage. Cell membranes act as barriers and allow only certain structures to pass which have the right hydrophilicity to hydrophobicity ratio. Carbon nanotubes because of their needle like structure and capacity to be functionalized with various groups have recently gained popularity as potential drug carriers of therapeutic agents. Scientists have already reported Intracellular internalization of biological cargos such as DNA, proteins, and drug molecules through needle-like shapesof carbon nanotubes [7]. Nanotubes offer some special advantages over spherical nanoparticles [8]:  Nanotubes have longer inner volume (relative to diameter of nanotube), which can be filled with desired chemical or biochemical species, ranging in size from small molecules to proteins.  Nanotubes have distinct inner and outer surface which can be differentially modified for chemical or biochemical functionalization. This makes the possibility of loading a moiety on inside of nanotube, and at the same time imparting chemical features to the outer surface that render it biocompatible.  Nanotubes have needle like structure which ensures greater cell permeability. 3. Carbon Nanotubes Carbon nanotubes (CNTs) have two main variants, as single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). SWNTs are the tubes of graphite which have a single cylindrical wall whereas MWNTs are larger and consist of many single walled tubes stacked inside the other (Fig. 1).

Fig. 1: Molecular structure of SWNTs (left), and MWNTs (right) To evaluate CNTs for drug delivery potential and biocompatibility, the first parameter of interest usually is particle size. Both length and diameter of nanotubes appear to be critical for function and avoidance of adverse effects [9]. The knowledge of in vivo fate, effect and clearance of CNTs from the body is critical for optimization of CNTs for therapeutic purposes. Studies in animals indicate that size, aggregation state and targeting group functionalization are major contributors to biodistribution and clearance of nanotubes. CNTs vary significantly in length and diameter depending on the method of synthesis. Diameters range from 0.7 nm to 3.0 nm for SWNTs and 10 nm to 200 nm for MWNTs. The length is typically several millimeters though this varies significantly. As a consequence of their small dimensions, CNTs have very high aspect ratios (length to diameter). The available surface area is dependent on length, diameter and degree of agglomeration.

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Size of nanotubes is one of the main deciding factors for its distribution and elimination from the body. Long tubes (i.e. 10-20 µm long) pose hindrance for the macrophages which will have problem in properly phagocytosing and removing them from cells and tissues. The cleansing mechanism will hence be slower and therefore resulting in accumulation in the body and leading to building of a dose and making it available to make contact with the cells [10]. In addition of favorable size, CNTs have high aspect ratio, capsule like structure, easy penetration, easily modifiable chemistry at surface and excreted out of the body through urine. 4. CNTs as Drug Delivery Vehicle The ability of functionalized carbon nanotube (f- CNT) to penetrate into the cells, and carry one or more therapeutic agents with recognition capacity, optical signal of imaging and/or specific targeting is of fundamental advantage, for example, in the treatment of cancer and different types of infectious diseases. Wu et al. [11] have attached an antifungal drug AmB to CNTs using the 1,3 cycloaddition reactions. They have shown that AmB covalently linked to CNTs can be taken up by mammalian cells without presenting any specific toxic effect. In addition, AmB bound to CNT preserves its high antifungal activity against a broad range of pathogens, including Candida albicans, Cryptococcus neoformans and Candida parapsilosi, showing that CNTs can be effective and efficient nano-carriers for drug delivery across cell membranes. Liu et al. [12] have attached a cancer chemotherapy drug doxorubicin (DOX) molecule on to prefunctionalized CNTs for in vivo cancer therapy. They have demonstrated that DOX-loaded prefunctionalized CNTs induce significant U87 cancer cell death and cell apoptosis, similar to free DOX. However, the main advantage of using functionalized CNTs as a drug carrier compared to free drug is their potential to target delivery for selective destruction of certain types of cells, reducing the toxicity to non-targeted cells. Methotrexate is another well-known anticancer drug, which suffers, as many others, from low bioavailability and toxic side effects. In a research conducted, this drug has been bound to MWNTs through cleavable linkers. The use of peptide which is recognized by intracellular proteases leads to a significant decrease in MCF-7 breast cancer cell viability, if compared to free methotrexate [13]. Insertion of Cisplatin (-Diammineplatinum (II) dichloride – CDDP) within MWNTs via capillary forces is studied and has proposed that MWNTs can be used as anticancer drug containers [14]. Physical loading of paclitaxel (PTX) is achieved onto CNTs through immersion of poly-ethyleneglycol (PEG)-graft-single walled CNTs (PEG-g-SWNTs) or PEG-g-multi-walled CNTs (PEG-gMWNTs) in a saturated solution of PTX in methanol. The PTX loaded PEG-g-CNTs are reported to be more promising for cancer therapeutics than free PTX. Paclitaxel to branched PEG chains on SWNTs, thereby overcoming drug resistance. Nanotube drug delivery promises for high treatment efficacy and minimum side effects for future cancer therapy with low drug doses. [15]. A multi-walled carbon nanotube (MWNTs)-based drug delivery system is developed by combining CNTs with antitumor agent 10-hydroxy Camptothecin (HCPT) by using hydrophilic spacer diaminotriethylene glycol between nanotube and drug moieties. They have demonstrated that obtained MWNTs-HCPT conjugates are superior in antitumor activity both in-vivo and in-vitro to clinical HCPT formulation [16] Li and coworkers have shown that SWNTs can be functionalized with p-glycoprotein antibodies and are loaded with the anticancer agent doxorubicin. Compared with free doxorubicin, this formulation demonstrates higher cytotoxicity by 2.4-fold against K 562R leukemia cells [17]. The release of drug from the nanotubes is of fundamental interest and has been studied by various groups in buffers of varying pH. It is observed that as the environmental pH becomes acidic, more doxorubicin is released from the CNT-doxorubicin conjugate. At a pH of 5.5, the approximate

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release of doxorubicin from the CNTs is approximately 40% in 1 day. Because the microenvironment of the extracellular tumor tissue and intracellular lysosomes and endosomes is acidotic, release of doxorubicin in these environments occurs with a higher magnitude [18]. Our group has also worked on similar domain and attached highly potent anticancer drug docetaxel with MWNTs and tested the in-vitro toxicity on human breast cancer cell lines. In vitro drug release studies were carried out to study the drug release at three pH buffers, i.e., 5.0, 6.8, 7.4 and deionised water as depicted in Fig. 2. The selection of the buffers was done in order to mimic the environment inside the cancer cells. In drug release studies for Conjugate, buffers with pH 5.0 (35%) and 6.8 (32.4%) have shown more drug release in 16 h as compared to the buffer of pH 7.4 (25%) and deionized water (12.5%). Despite appropriate sink conditions, no further release of drug is observed. The drug conjugate showed drug release evidently at pH 5.0 and 6.8, which is the pH of cancerous cells as compared to pH 7.4, which is present inside normal cells. The studies provide an in vitro evidence for the plausible hydrolysis of the ester linkage in the low acidic pH of the cancer cells, particularly in lysosomes and endosomes, resulting in accumulation of drug inside the cells. Compared to conjugate the free drug was released in a very short duration of time depicting the drug release. So it was proved that the nanotube conjugated drug will show better release profile inside cancerous cells rather than healthy cells. The figure given below shows the release profile of drug [19].

Fig. 2: Drug release study of drug conjugate and plain drug on different pH [19]. In nutshell, CNTs can be explored for anticancer activity in the following ways and research is already showing a great promise in this area. 4.1. Efficient Drug Delivery: CNTs can be used as drug carriers to treat tumors [20]. The efficacy of anticancer drugs used alone is restrained not only by their systemic toxicity and narrow therapeutic window but also by drug resistance and limited cellular penetration. Because CNTs can easily cross the cytoplasmic membrane and nuclear membrane, anticancer drug transported by this vehicle will be liberated in situ with intact concentration and consequently, its action in the tumor cell will be higher than drug administered alone by traditional therapy. Thus, the development of efficient delivery systems with the ability to enhance cellular uptake of existing potent drugs is needed. The high aspect ratio of CNTs offers great advantages over the existing delivery vectors, because the high surface area provides multiple attachment sites for drugs. Many anticancer drugs have been conjugated with functionalized CNTs and successfully tested in vitro and in vivo such as epirubicin, doxorubicin, cisplatin, methotrexate, quercetin, and paclitaxel [21]. 4.2. Antitumor Immunotherapy: Some studies have demonstrated that CNTs can be used as carriers in antitumor immunotherapy. Antitumor immunotherapy is a therapy which consists of stimulating the patient’s immune system to attack the malignant tumor cells. This stimulation can

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be achieved by conjugation CNTs with either a cancer vaccine or a therapeutic antibody. Some authors have validated the use of CNTs as vaccine delivery tools. Yang’s group observed that the conjugate of MWCNTs and tumor lysate protein (tumor cell vaccine) can considerably and specifically enhance the efficacy of antitumor immunotherapy in a mouse model bearing the H22 liver tumor [22]. 4.3. Local Antitumor Hyperthermia Therapy: The hyperthermia therapy using CNTs has been recently suggested as an efficient strategy for the cancer treatments. SWCNTs exhibit strong absorbance in the near-infrared region (NIR; 700–1100 nm). These nano-materials are considered as potent candidates for hyperthermia therapy since they generate significant amounts of heat upon excitation with NIR light [23].The photo-thermal effect can induce the local thermal ablation of tumor cells by excessive heating of SWCNTs shackled in tumor cells such as pancreatic cancer. Some progress in the technique has been achieved in recent years, and it has shown feasibility in clinical application. 4.4. Gene Delivery Systems: In a gene therapy treatment technique, the genetic materials (DNA or RNA) are delivered as pharmaceutical agents to help target cells to recover missing or defective genes and generate their own therapeutic proteins with minimal toxicity to cure a disease. This technology is a promising treatment option especially in cancer and is expected to be an alternative method to traditional chemotherapy. Functionalized-CNTs (f-CNTs) seem to be very suitable for gene therapy because they are easily able to cross cell membranes and deliver genes into cells. In many reports, DNA and RNA have been covalently and non-covalently attached to f-CNTs. It has been found that not only DNA molecules can be linked to the tips and walls of f-CNTs, but also can be encapsulated inside the structure. Release of DNA molecules from CNTs-based delivery systems using thermal properties of CNTs has been investigated and it has been found that DNAs can be released from f-CNTs transporters to the nucleus after the laser pulses [24-26]. 5. Cellular Internalization of CNTs In targeting the delivery of therapeutic molecules to cells, CNTs are first attached to the carrier by either covalent or non-covalent bonding. The conjugates are then directed to the targeted cells via passive targeting methods (i.e. a methodology primarily by minimizing interactions with non-target organs, tissues and cells, and it is without specific molecular recognition agent) or active targeting methods (i.e., the method by which the therapeutic agent is delivered to tumors by attaching with a ligand that binds to specific receptors that are over expressed on target cells). After reaching the targeted site (organs, tissues or cells), there are two possible mechanisms of CNT internalization. 5.1. Endocytosis Pathway: Endocytosis is a process in which cells engulf the material with a small area in the cell membrane to form a vesicle. The vesicle inside the cell then fuses with lysosomes forming endolysosomes. Then the lysosomal enzymes help digestion of the material. Since endocytosis is energy dependent, a low energy environment inhibits the endocytosis process. Fig. 3 represents the internalization of drug carrier conjugate through endocytotic pathway, following release of drug due to low pH inside the cancerous cell [27].

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Fig. 3: Represents internalization of drug-carrier conjugate through endocytosis, with low pH triggering the release of drug from the endosome followed by entry of drug inside the nucleus [28] 5.2. Passive Diffusion and Insertion: The CNTs enter cells via insertion and diffusion mechanism, which is a two-step process in which the tubes are first accommodated onto the lipid cell membrane and then adjust to adopt the transmembrane configuration. In this model, the internalization of nanotubes into the cells is spontaneous and mediated by the lipid membrane. The hydrophilic interactions or static charge interactions between the tubes and the lipid membrane drives the translocation of the nanotubes. CNTs can also enter cells under the application of external magnetic fields. Well known endocytosis inhibitors like sodium azide (NaN3), 2,4-dinitrophenol (DNP) or incubation at 4 °C inhibits the mechanism of endocytosis, leading to passive diffusion of CNTs [29]. It has been proved that MWCNTs and single-walled carbon nanotubes (SWCNTs) differ in their cell penetration mechanism. Confocal microscopy imaging has shown that SWCNTs have the ability to be internalized into cells, whereas MWCNTs are not capable to enter to the cells. The size of the CNTs also influences their cellular uptake and fate, because long SWCNTs (L-SWCNTs) have been shown to be localized in the cytoplasm, while short SWCNTs (S-SWCNTs) are transported into the nucleus [30]. 6. CNT Toxicity For successfully applying CNTs as biomedicine, it is essential to determine their pharmacological and toxicological profiles. Although the biological toxicity of CNTs has been evaluated by several groups, further investigations are still necessary for achieving comprehensive conclusion. Toxicity and immunogenicity of CNTs in vivo and in vitro studies has been attributed to various factors such as number of walls, length and aspect ratio, surface area, degree of aggregation, extent of oxidation, hydrophobicity, surface topology, method of administration, dispersibility, type and degree of functionalization, and method of manufacture (which can leave catalyst residues and produce impurities). Toxicity of CNTs is also dependent on their concentration, dose, duration and method that cells or organisms are exposed to them and even the utilized dispersant to solubilize the nanotubes. 6.1. Effect of Length and Shape: The length and shape of CNTs influence how well they cross the membrane of macrophages and determine the resulting immunologic response [31]. For example, shorter CNTs (less than 0.8 µm in length) were found to have better immunogenic responses than longer ones (greater than 0.8 µm in length). A complementary study has also showed similar results after injection of long and short length CNTs into mice [32].

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Despite a large number of studies performed in the past several years to explore the potential toxic effects of CNTs, the results are often contradictory. Some in vitro studies have reported toxicity of CNTs in a variety of relevant cell types, including macrophages, lung epithelial cell lines, and normal and malignant mesothelial cell lines [33], while other studies have found CNTs to be nontoxic [34]. 6.2. Solubility Issues: Another reason for the persistence of toxicity is its insolubility. It may be possible that it is partly due to the hydrophobic nature of the CNTs and also their propensity to aggregate and interact with the cell membranes. There are chances that these conditions may be mitigated by surface modifications of CNTs. According to one study, if the CNTs are modified to increase their solubility and decrease their surface hydrophobicity, eventually it results in decrease of the CNTs cytotoxicity [35]. Therefore, in order to attain a successful application in medicine, it becomes a prerequisite to purify and chemically modify the CNTs in order to increase their solubility and decrease the level of toxicity. Through the mechanism of chemical functionalization, water soluble CNTs can be modified. This helps to bind them to selective therapeutics or biologically relevant molecules [36]. 6.3. Effect of Impurities: Another important factor is the influence of impurities included in CNTs, especially iron that is used as a catalyst during CNT fabrication. In fact, the carcinogenic power of asbestos is increased by iron, which accelerates the generation of oxygen radical species. It has been generally agreed that f-CNTs represent a major improvement over unmodified, non-functionalized (pristine) CNTs. It has been found that pristine CNTs show toxicity on cell lines and causes necrosis and apoptosis deaths even after purification [37]. Fig. 4 gives a brief description of all the factors which determine the toxicity of CNTs and ways by which CNTs can be used to manage them efficiently.

Fig. 4: Parameters determining the pharmacological and toxicity profile of CNTs and the CNT characteristics [49].

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7. Overcoming the Toxicity of CNTs by Functionalization Compared to pristine CNTs, well-functionalized CNTs by biocompatible coatings such as PEGylation exhibit remarkably reduced in vivo toxicity after being intravenously injected into animals [38]. The size of the functional group also seems to be important. For example, it was indicated that SWCNTs functionalized with relatively large molecules (molecular weight 460 kDa) can increase toxicity. CNTs lend themselves to a range of chemical modifications. Both covalent and non-covalent functionalizations are possible at intact CNT sidewalls, at defect sites on sidewalls or at the tip of the nanotubes. The most common modification is the formation of carboxyl residues [39, 40]. The non-covalent functionalization of CNTs can be carried out by coating CNTs with amphiphilic surfactant molecules or polymers (poly-ethylene-glycol). 8. Bio-distribution Mapping of CNTs in Animals The pharmacokinetics and bio-distribution of CNTs is an important aspect to explore the potential of CNTs as a drug delivery vehicle. The blood circulation time and accumulation of CNTs in animal affect their biomedical applications and toxicity. On a positive side, CNTs can serve as a drug vehicle, circulating long in the blood leading to more accumulation in tumor, making them efficient for the cancer diagnostics and treatment. But on the other hand, the pharmacokinetic and bio-distribution map needs to be addressed thoroughly. The majority of the intravenously injected CNTs in mice mainly seem to empty in urine, with far less found in liver, spleen and lungs [41]. However, some studies indicate liver and spleen to be the main site of CNT accumulation. Liver may be a preferred site for CNT accumulation due to its greater vascularity. Studies have also found that CNTs deposit mostly in the excretory systems like bladder, kidney and intestines. Studies indicate that f-CNTs interact differently with cells depending on the conjugated moiety. In fact, the bio-distribution of most functional compounds can be modified due to their attachment with CNTs, for example, paclitaxel conjugates with SWNTs seem to localize more in intestine and liver, whereas when they conjugate with PEG, localization occurs more frequently in lungs [42]. A superficial Lung is regarded as one of the main portals of entry for nanoparticles, which emphasizes the importance of pulmonary toxicity evaluation. Unprocessed nanotubes are very light, and could become airborne and potentially reach the lungs. But it has been reported that adequately functionalized nanotubes are non-toxic. In fact it has been demonstrated that highly water soluble functionalized CNTs are taken up by immune cells without being toxic and preserving their activity [43],and it has been found that agglomerates of CNTs are more toxic than their dispersions. Importantly, numerous studies have published reports on how surfactant modified nanotubes are well tolerated in-vivo [44]. In general, they have blood clearance half-life in the order of hours. Moreover, tissue distribution studies show they are distributed into urine, via glomerular filteration or into faeces with low residual amount into body. Furthermore, some recent work has shown invitro enzymatic degradation of CNTs, thus presenting another possibility for the elimination of this carrier from the body once the therapeutic function has been exerted [45]. Also it has been shown that functionalized CNTs are easily degraded than pristine nanotubes, indicating that defects through functionalization probably facilitate the attack through enzymes [46]. 9. Measurement of Anticancer Activity of CNT-drug Conjugate One of the most recent topics that have been added to the list of issues in nano-toxicology of CNTs is the bioassays to study anticancer activity. The most commonly used bioassays which have been employed in the current study; MTT assay and detection of apoptotic cells by flow cytometry are discussed below.TheMTT{3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole} Cell Proliferation Assay Kit is a laboratory test and standard colorimetric assay (an assay which measures changes in color) for measuring the activity of enzyme (tetrazolium succinate reductase system) present in the mitochondria that reduces MTT to formazan, giving a purple color.

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It can also be used to determine cytotoxicity of potential medicinal agents and other toxic materials, since those agents would result in cell toxicity which will lead to metabolic dysfunction and, therefore, can be used as a measure for anticancer activity [47]. Flow cytometry has been used successfully for analysis of apoptosis. Annexin V/Propidium iodide assay in this technique can differentiate between dead and live cells.This assay has been shown to provide a single-cell-based, rapid, quantitative, and sensitive assay to detect lymphocyte-mediated target cell killing in various animal models. Unlike conventional chromium release assays, the FC assay enables monitoring of cellular immune responses in real time and at the single-cell level using diverse fluorescence detection methods such as flow cytometry and fluorescence and CLSM [48]. Cellular uptake and cytotoxicity of drug conjugate (DTX-MWNTs) has been studied on human breast cancer cells (MCF-7 and MDA-mb-231). Cytotoxicity studies were conducted through MTT 3-(4,5 Dimethylthiazol-2 yl) 2,5–diphenyltetrazolium bromide, a cytotoxicity assay and flow cytometry on MCF-7 cells {Fig. 5(A)}.

Fig. 5: Cytotoxicity study by (A) MTT assay, and (B) flow cytometry [48] The study further emphasizes on cellular viability under conjugated and non-conjugated drug.In Fig. 5(A), less toxicity has been found for DTX as compared to the drug conjugate at all the concentrations. The conjugate shows maximum toxicity of 32.4% at 40 µg/mL, after which there has been no significant increase in toxicity. For DTX also the cytotoxicity increases gradually and has been found to be concentration dependent with maximum toxicity of 30.3% at 100 µg/mL. In case of flow cytometry, the maximum cell death observed for drug conjugate was 26.5% at 40 µg/mL. Almost similar results were found in both the assays (Fig. 5 B). Maximum toxicity of drug conjugate was found to be at concentration of 40 µg/mL as assessed by MTT and flow cytometry assay on MCF-7 cells {Fig. 5 (A) & (B)}, respectively. The results indicated that the number of viable cells after treatment with drug conjugate (DTX-MWNTs) were lower than that with the “drug alone” treatment at different concentrations. Conclusively, the MWNTs conjugated drug holds more promising drug formulation in terms of more cytotoxicity and high therapeutic index of cytotoxic drug. The cytotoxicity was found to be dose dependent but more for the drug conjugate over the naive drug. The rapid internalization of DTX by MWNTs in a nontoxic manner coupled with its anticancer property leads to an improved efficiency of the drug action [48].Confocal microscopy was used to establish efficient internalization and localization of the conjugates inside the cells. And the images for the internalization of the conjugate are given below:

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Fig. 6:(A) Cells treated with FITC labeled drug conjugated MWNTs,(B) Cells treated with DAPI dye, and(C) & (D) Bright field images of the cells [48] The cellular uptake study of the conjugate has been performed by CLSM by labeling the conjugate with FITC, which gives a green color after staining. DAPI is used to stain the nucleus blue as it is a nuclear specific dye. Green fluorescence inside the cells gives the information regarding the presence of conjugate in the cytoplasm and the blue region has shown the region of nucleus of the cell. The green and blue regions overlap each other indicating that fluorescence from drug conjugate is not only in the cytoplasm but also in the nucleus of the cell, where the drug has been reported to show its mechanism of action (Fig. 6 A-D). 10. Conclusions The development of nanomedicines depends on toxicological and pharmacological profile of nanomaterials. CNTs are such materials that have been poised to revolutionize a variety of biomedical applications. The in vivo toxicological and pharmacological studies undertaken so far indicate that functionalized carbon nanotubes can be developed as nanomedicines, contrary to nonfunctionalized, pristine carbon nanotubes. Functionalization renders the surface of carbon nanotubes water-soluble, compatible with biological fluids and leads to their rapid excretion through the renal route, minimizing unwanted tissue accumulation. The door of opportunity for the development of carbon nanotubes as diagnostic and therapeutic nanomedicines has opened, and systematic study of their therapeutic efficacy is anticipated. Acknowledgements The authors acknowledge the funding support from Indian Council of Medical Research (ICMR), Council of Scientific and Industrial Research- Senior Research Fellowship (CSIR-SRF), Nanotechnology: Impact on Safety, Health and Environment Programme (CSIR-NANOSHE), and academic support from Central Scientific Instruments Organization (CSIR-CSIO), Banasthali Vidyapeeth University, Academy of Scientific and Innovative Research (AcSIR). References [1] D. Hanahan, R.A. Weinberg, The Hallmarks of Cancer, Cell 100 (2000) 57-70. [2] A. Kamb, S. Wee, C. Lengauer, Why is cancer drug discovery so difficult?, Nat. Rev. Drug Discov. 6 (2007) 115-120. [3] A.F. Chambers, A.C. Groom, I.C. MacDonald, Dissemination and growth of cancer cells in metastatic sites, Nat. Rev. Cancer 2 (2002) 563-572. [4] D.K. Chang, C.T. Lin, C.H. Wu, H.C. Wu, A novel peptide enhances therapeutic efficacy of liposomal anti-cancer drugs in mice models of human lung cancer, PLOS ONE 4 (2009) 1-11.

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