Carbon nanotubes as nanocarriers in medicine

Current Opinion in Colloid & Interface Science 17 (2012) 360–368

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Current Opinion in Colloid & Interface Science journal homepage: www.elsevier.com/locate/cocis

Carbon nanotubes as nanocarriers in medicine Sivan Peretz a,⁎, Oren Regev a, b,⁎⁎ a b

Department of Chemical Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel The Ilse Katz Institute for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

a r t i c l e

i n f o

Article history: Received 27 June 2012 Received in revised form 31 August 2012 Accepted 5 September 2012 Available online 12 September 2012 Keywords: Carbon nanotubes Liposome Nano-carrier Drug delivery Photothermal therapy Gene delivery Toxicity

a b s t r a c t Carbon nanotubes (CNTs) possess outstanding properties and a unique physicochemical architecture, which may serve as an alternative platform for the delivery of various therapeutic molecules. This review focuses on recent progress in the field of CNTs for biomedical applications. After a short, general physico-chemical introduction to CNTs, we introduce different methods for CNT surface modification, facilitating their dispersions in physiological solutions, on the one hand, and binding a wide range of molecules or drug-loaded liposomes, on the other. We summarize imaging evidences on the structure of CNT-drug conjugates and their relevant uptake mechanisms by the cell. Lastly, we review current repots on CNT toxicity and new developments in CNT-based medical applications: photo-thermal therapy, drug delivery and gene therapy. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Carbon nanotubes (CNTs) were first discovered in 1991 by Sumio Iijima in Japan [1] and can be described as rolled graphene sheets held together by van der Waals interactions [2]. These strong interactions dictate CNT bundling together and the formation of large aggregates. A single rolled layer of graphene forms a single-walled carbon nanotube (SWCNT) with a diameter between 0.4 and 2 nm [3], while a few coaxial cylinders form a multi-walled carbon nanotube (MWCNT) with a diameter between 2 and 100 nm. Having a typical length of a few microns, CNTs have an aspect ratio (L/D) of 1:1000 (Fig. 1). CNTs possess high tensile strength, are ultra-light weight and have excellent transport conductivity, as well as thermal and chemical stability [4,5]. Owing to their large surface area, CNTs may be conjugated with various biological molecules, such as proteins, enzymes, nucleic acids and drugs. These molecules can be bound to the CNT covalently or non-covalently. CNTs are produced by three major methods: electric arc discharge (EAD) [6], laser ablation (LAB) [7] and chemical vapor deposition (CVD) [8]. CNTs are generated from the vaporization of graphite targets (EAD, LAB) or by passing a carbon containing vapor (e.g., CO) over supported metal catalyst nanoparticles in a furnace (CVD) [9].

⁎ Corresponding author. Fax: +972 86472916. ⁎⁎ Correspondence to: O. Regev, The Ilse Katz Institute for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. Fax: + 972 86472916. E-mail addresses: [email protected] (S. Peretz), [email protected] (O. Regev). 1359-0294/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cocis.2012.09.001

Most synthesis procedures yield CNT aggregates with a narrow distribution of diameters, lengths and defect concentrations, although their variability of length and diameter may be tuned by controlling the growth conditions [10]. Thanks to their unique properties, CNTs have become one of the most studied materials over the last two decades. Their applications range from energy and gas storage devices [11] and sensors[12] through enhanced thermal [13] and electrical [11] conductivity or strength reinforcement in composites [14], to a wide range of biomedical applications, such as drug-delivery carriers [15], photo-thermal therapy and gene delivery systems [16]. In this review, we will focus on the medical applications of CNTs.

2. Cell uptake of CNTs The development of novel biomedical therapies is greatly dependent on the ability of drugs and other extracellular species to cross the cellular barrier. As such, many drug-delivery systems rely on a ‘transporter,’ i.e., a platform able to be loaded with a cargo of choice and having the ability to carry that cargo through the cellular membrane without any detrimental effect on its biological functionality [17•]. CNTs have been proposed and actively explored as multipurpose, innovative nano-carriers for drug-delivery systems. Thanks to their extremely high aspect ratio, CNTs can penetrate the cell membrane and be uptaken by cells. After entering the cell, CNTs are mainly located inside cell endosomes and lysosomes. Individualized CNTs are able to travel through various cellular barriers and even enter the nucleus [18].

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Fig. 1. Carbon nanotubes: a. single-walled carbon nanotubes (SWCNT) b. multi-walled carbon nanotubes (MWCNT) (reprinted with permission from Ref. [3]).

The relevant cell-internalization mechanisms for CNTs are the endocytosis- phagocytosis pathway and passive diffusion. Briefly, endocytosis represents the engulfing of an extracellular particle by the cell (for example, viruses, ∼100 nm in size) through the formation of a vesicle that is then integrated into the cell. Phagocytosis is similar to endocytosis, but usually involves an uptake of larger particles, such as bacteria (∼1 μm). These two processes are energy-dependent and are hindered at low temperatures [19,20•]. In the passive-diffusion pathway, CNTs cross the lipid bilayer in a needle-like manner [2,21] (Fig. 2). Several studies were performed in this field in order to understand the exact uptake mechanism of different types of CNTs [22]. Energy-dependent endocytosis was reported as the main internalization mechanism of SWCNTs bound to various types of proteins [23]. In addition, the imaging of SWCNTs within the phagosomes and lysosomes of healthy cells also suggests uptake by phagocytosis [24]. Uptake by diffusion was reported for MWCNTs [25]. Here, shorter (i.e., b 1 μm) MWCNTs were readily internalized by cells, while longer ones were not. Short CNTs can act as straight ‘nano-needles,’ able to penetrate the cell membrane more efficiently than the longer CNTs, which are often arranged in a coiled or bundled shape, hindering their efficient uptake.

3. Direct attachment of drugs 3.1. Covalent attachment Pristine (as-prepared) CNTs readily aggregate in bundles due to van der Waals forces and, therefore, cannot be dispersed effectively in physiological aqueous environments. One way to exfoliate the bundles is chemical modification on the CNT surface, also termed ‘functionalization’ or f-CNT [26]. Covalent functionalization may be described as a chemical grafting of molecules onto the sp 2 carbon atoms of the π-conjugated skeleton of the CNTs [27]. The basic reaction for CNT functionalization is oxidation [28–33 ••] (Fig. 3A), performed under strong acidic conditions. During this process, carboxyl groups are initially formed at the tube's ends and then near defects on the CNT's side walls, also shortening the CNTs. Furthermore, the acid functionalities may react with alcohols or amines, yielding ester or amide linkages, respectively [33 ••].

However, although oxidized CNTs are soluble in water, they aggregate in the presence of salts, due to charge screening and, thus, cannot be directly used in biological applications because of the high salt content of most physiological media. Therefore, further modification is needed, e.g., attaching hydrophilic polymers, such as polyethylene glycol (PEG), forming CNT-polymer conjugates, stable in physiological environments [34]. The disadvantage of this method is that it results in the partial loss of electronic structure and optical properties of the CNTs and a loss of material, due to the oxidative process [4,35]. Nonetheless, these issues are of less importance in drug-delivery applications. Tumor-targeted drug delivery system is also possible via covalent conjugation of specific ligand to oxidized SWCNT. Studies performed in vitro demonstrated rapid decrease of tumor size in comparison to non targeted SWCNT thus insuring maximum drug efficiency with minimum side effects [36]. Another abundant covalent modification is based on the 1,3-dipolar cycloaddition of azomethine yields (Fig. 3b), generated by the condensation of an R-amino acid and an aldehyde; this reaction is widely applied to the organic modification of CNTs [37]. 3.2. Non-covalent attachment Another method for dispersing CNT in aqueous solutions can be carried out by coating CNTs with amphiphilic surfactant molecules or polymers, which are absorbed at the interfaces between the immiscible bulk phases and act to reduce the surface tension [39–41•]. This approach is rather popular since, unlike covalent functionalization (see Section 3.1), here, CNT structure and properties are preserved, due to the fact that the preparation procedure includes only sonication and centrifugation. Non-covalent coating on CNTs for biological applications should be sufficiently stable to resist detachment from the CNT surface, especially in serum having a high salt content. Several biocompatible molecules have been suggested for pristine CNT dispersion [42–44] as well as some proteins [45–47]. 3.3. Covalent attachment via Click chemistry 'Click chemistry', invented by K. Barry Sharpless [48•], defines a series of chemical reactions respecting several criteria: modular reaction, wide in scope, producing a very high yield, generating only inoffensive

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Fig. 2. Possible mechanisms for CNT interactions with cells (Reprinted with permission from Ref. [19]).

byproducts that can be removed by non-chromatographic methods, and stereospecificity. “Click chemistry” cannot be performed directly on CNTs, due to the poor reactivity of the sp 2 framework and requires one or more chemical steps before the key reaction, in order to introduce the reactive groups onto the CNTs. Cu(I)-catalyzed azide-alkyne [3 + 2] cycloaddition (CuAAC ) is one of the most popular reactions in this field and leads to the formation of 1,2,3-triazoles. The catalyst (copper, in most cases) permits selective access to the 1,4-regioisomer of the triazole and allows the reaction to be performed at room temperature. This reaction is an efficient tool for the surface modification and functionalization of nanomaterials and biomolecules [2,49–51]. The toxicity of the copper catalyst, hindering biomedicine application, led to the development of a copper-free cyclo-addition employing difluorinated cyclooctyne reagent, that rapidly reacts with azides in living cells [52]. These highly-strained alkynes react selectively with azides to form regioisomeric mixtures of triazoles at ambient temperatures and pressures, without the need for metal catalysis and with no apparent cytotoxicity [53]. 3.4. Biocompatible surface modification of CNTs One of the main problems in using CNTs as drug nanocarriers is their recognition as foreign particles in the bloodstream. Opsonins,

blood serum components, interact with CNTs mostly through hydrophobic and electrostatic interactions. Macrophages of the reticuloendothelial system (RES) recognize these components and remove the CNTs from the bloodstream within seconds [34,54,55]. A widely used approach for overcoming this problem is by means of the surface modification of CNTs, by neutrally-charged chemical moieties, such as hydrophilic polymers and nonionic surfactants. Polyethylene glycol (PEG) is one of the most popular CNT surface modifiers, due to its hydrophility, flexibility and biocompatibility [34]. PEG-modified CNTs may also be dispersed in relatively higher salt-concentrated aqueous solutions [56]. The effect of polymer surface coating on the in vivo behavior of CNT was explored. At first, PEG with different molecular weights (PEGylation degree) is bound to CNTs. The blood circulation of the CNT-PEG in mice-bearing breast cancer tumor was studied by Raman spectroscopy [57,58] method. A fine tuning of the PEGylation degree on the CNT is required in order to control the blood circulation half-life of CNT conjugate (13 h). The optimized CNT-PEG conjugate afforded relatively low RES accumulation, high tumor uptake and low skin retention, thus making CNT an ideal choice for in vivo cancer treatment [59]. The cellular internalization of the CNTs depends, among other factors, on the PEG-chain length; the longer PEG chains reduce cellular uptake [60]. In vivo studies indicate that PEGylation decreases SWCNT toxicity. Raman analysis of solid excreted suggested rapid clearance of

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Fig. 3. Chemical functionalization of carbon nanotubes: (A) oxidation by strong acids (B) 1,3-dipolar cycloadditions (R = CH3O(CH2CH2O)2CH2CH2). (Reprinted with permission from Ref. [38]).

PEG-SWCNT via the renal pathway in comparison non-PEG-SWCNT which lodges in various organs [61].

to

4. Photo-thermal cancer therapy Laser-induced thermal cancer therapy can provide a minimally invasive and potentially more effective alternative compared to conventional treatment. The goal of this therapy is to supply a lethal dose of heat to the prescribed tissue volume while causing minimal damage to the surrounding normal tissue. Nanoparticles have significant potential as selective photo-absorbing agents for laser-based cancer treatment, due to their unique physical properties. There are several nanoparticles acting as photo-thermal transducers: gold nanoparticles [62], quantum dots [63], graphene [64,65], carbon nanohorns [66] and CNTs. CNTs exhibit strong optical absorption in the near infrared (NIR) regions (NIR I: 700-900 nm, NIR II: 1–1.4 μm). NIR optical radiation has a penetration depth of 1.6 mm into biological tissue. CNTs generate heat by light absorption and induce the thermal destruction of those cancer cells containing sufficient CNT concentrations. To avoid damage to normal tissues, targeted CNTs have been prepared by the covalent attachment of tumor-specific ligands to the CNTs. The conjugated CNTs show good stability under physiological conditions and generate a highly specific photo-thermal killing of the targeted cells. Internalized CNTs are more sensitive to NIR-mediated photo-thermal damage than cells with CNTs on their surface [67,68]. Mitochondria-targeted PEG-SWCNTs selectively destroy cancerous cells by laser irradiation. Dispersed PEG-SWCNTs efficiently accumulate in the mitochondria of the cancer cells, due to higher mitochondrial trans-membrane potential, and induce high levels of tumor cell death in mice with significant apoptosis characteristics [69]. The effect of intra-tumorally injection of PEG-SWCNT, followed by NIR irradiation, was studied in vivo on solid malignant tumors. Complete destruction of the tumors was obtained without harmful side effects. Moreover,

most of the SWCNTs were removed from the body within two months [70]. Other studies supporting these results were performed with different types of CNTs, like DNA-coated MWNTs [71] and phospholipids-PEG-folic acid–SWCNT [72]. The path of the SWCNTs through the mice anatomy may be observed by means of the inherent fluorescence (NIR II range) of the CNTs. Video-rate fluorescent imaging enables one to track the circulation pathways of tail-vein‐injected SWCNTs in organs belonging to the RES system. This may enhance various future applications, such as the identification of tumors according to their differential blood-flow rates in leaky tumor vessels and organ imaging without the need for radioactive tags or MRI [73].

5. Gene therapy Gene therapy is a novel form of molecular medicine, which involves identifying DNA sequences and then developing suitable ways to deliver this genetic material into cells [74]. Gene therapy may be performed via viral vectors, where the genes are carried by viruses into the cells. Such viral vectors can be immunogenic, oncogenic, induce inflammations and be difficult to functionalize by ligand targeting [34,75]. An alternative is non-viral vectors, in which synthetic liposomes and polymers host naked DNA. Most of these vectors cannot, however, cross the nuclear membrane [2]. In order to overcome this problem and others, CNTs are explored as a non-viral delivery system, due both to their ability to cross the nucleus membrane and to their favorable pharmacokinetics. The attachment of DNA molecules to CNT surfaces is performed via various functional, structural DNA groups, available for interaction with CNTs [16]. The CNT-DNA conjugate increases CNT solubility in aqueous and organic media. Due to DNA degradation by cellular enzymes, there is a need to protect the DNA cargo during the prolonged transport. Indeed, SWCNT-modified DNA has increased delivery capability into cells and intracellular biostability when compared to free DNA, thus having great potential in this area [76].

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Ammonium-functionalized CNTs associate with plasmid DNA via electrostatic interactions. This DNA-conjugated CNT was tested in-vitro and demonstrated a superb cell uptake, 10 times higher gene expression when compared to free DNA [77]. Ammonium-functionalized CNTs have also been studied for siRNA delivery. This type of SWCNT can electrostatically bind siRNA and can effectively downregulate a type of oncogene overexpression [78]. SWCNTs for siRNA delivery have also showed superior silencing effects over conventional liposome-based non-viral agents in T cells [60].

growth suppressing efficiency than that of the Taxol®, due to the higher tumor uptake of the SWCNT-PTX. An alternative, novel nano-carrier for PTX delivery is the PEGylated hydrophobic carbon clusters (ultra-short SWCNTs) termed PEG-HCCs. PTX is non-covalently loaded onto the PEG-HCCs. This PTX/PEG-HCC conjugate shows low toxicity both in vitro and in vivo. Upon mixing the PTX/PEG-HCCs with an antibody, such as Cetuximab, the antibody non-covalently associates with the nano-carrier to produce a formulation capable of targeted drug delivery [94,95].

6. Drug delivery — anticancer drugs

6.3. Liposome-CNT conjugation

6.1. Doxorubicin (DOX) delivery

Low drug-to-CNT ratio is one of the major problems in efficient CNT-assisted drug-delivery. Therefore, to deliver a sufficient amount of drug, one needs to increase the CNT concentration, which may result in toxicity [19,97 •]. To overcome this problem, Karchemski and coworkers have developed a new drug-delivery system [98 ••], in which drug-loaded liposomes (see 6.1, DOXIL®) are covalently attached to CNTs to form CNT-liposome conjugates (CLCs). Here, the advantages of both are combined: high drug loading of the liposomes and high uptake efficiency of the CNTs. Oxidized MWCNTs (f-MWCNTs), decorated by carboxyl groups, were covalently bound to liposomes by an amidation reaction [99]. CLC formation was evidenced by cryogenic transmission electronmicroscopy (cryo-TEM) imaging (Fig. 5), in which the CLC is imaged in solution after a vitrification process. It is found that the average liposome-to-liposome distance (bound on the f-MWCNT) is 100–300 nm. This observation, along with the average f-MWCNT length of 400–600 nm, indicates an average of 2–6 liposomes per single f-MWCNT, i.e. , about 50,000 drug molecules per CLC [100] in comparison to about 80 drug molecules when directly attached to the CNTs [101]. Cell uptake by CLC was studied by in-vitro fluorescence microscopy of human embryonic kidney cells (HEK 293) including high CLC uptake by the cells, as opposed to free liposomes (unattached to f-MWCNTs). The new and efficient CLC platform for drug delivery combines the efficient cell uptake of CNTs with the well-known high drug-loading capacity of the liposomes. Another advantage of this method is the possibility of binding different liposomes, loaded with different drug contents, onto the same CNT [102]. Furthermore, active targeting [103] may be achieved by the covalent attachment of targeting agents to binding sites, either on the CNTs or on the liposomes, or both, thus enhancing the delivery of drugs to diseased cells while reducing drug interaction with healthy tissues.

Doxorubicin (DOX) is a well-known anticancer, chemotherapeutic drug, usually administered intravenously in the form of salt; however, it is unable to cross cellular barriers and has several other toxic effects [79]. Liposomal delivery, DOXIL® [80], reduces DOX toxicity. Liposomes are artificial vesicles consisting of lipid bilayers. Their inner, hollow, aqueous volume may be filled with a broad spectrum of molecules, including drugs. They are biologically inert, biocompatible and have a low cytotoxicity effect and rarely cause antigenic reactions [81]. Liposomes have been approved by the U.S. Food and Drug Administration (FDA) for use as an anticancer drug [82]. The main disadvantage in using liposomes is their rapid elimination from the blood stream due to their negative surface charge. Also, their large diameter results in low cell uptake efficiency [83]. The aromatic rings in DOX enhance both adsorption on CNTs via π–π stacking (see Section 3.2) and hydrophobic interactions [84]. DOX was successfully loaded (non-covalently) onto the surface of PEGylated SWCNTs to create a new drug formulation, namely, SWCNT–DOX (Fig. 4A), characterized by a high drug-loading efficiency and a remarkable reduction in toxicity compared to free DOX and DOXIL ®. The DOX-SWCNTs complex is stable at neutral pH, but dissociates in acidic environments [85]. Therefore, the dissociation of DOX from its SWCNT nano-carrier occurs only at the tumor sites, which are slightly acidic [86]. A triple functionalization of oxidized SWCNTs was also performed with DOX, a monoclonal antibody and a fluorescent marker. In order to bind all of the above to the SWCNT backbone (Fig. 4B), two binding methods were employed: the non-covalent attachment of DOX to the SWCNT, triggering drug release based on pH differences, and covalent attachment to attach the fluorescent marker and the antibodies to the SWCNTs. In-vitro study of the resultant conjugated SWCNT indicates complete penetration into the cancer cells, followed by the release of DOX to the nucleus, while the SWCNTs remain in the cytoplasm [87]. Similar results were observed for SWCNTs modified by polysaccharides, folic acid and the anticancer drug DOX (Fig. 4C), when tested on human cervical carcinoma cells. The conjugated SWCNTs selectively accumulate in the diseased tissues and release their toxic payload in a controlled manner [88,89]. 6.2. Paclitaxel (PTX) delivery Paclitaxel (PTX) is an anticancer drug with high therapeutic efficacy, but with low water solubility. In one of the commercial drugs, Taxol®, PTX is dissolved in a solvent, Cremphor-EL®, which contributes to some of the toxicities commonly associated with PTX-based therapy [90]. In Abraxane®, PTX is bound to albumin; this formulation has significant advantages over Taxol®, but is primarily used to treat breast cancer [91]. Therefore, CNTs are being explored as an alternative platform for PTX delivery. The Dai Group [92,93] conjugated PTX to branched PEG-coated SWCNTs (Fig. 4D). This PTX-SWCNT conjugate was found to reduce tumor volume in breast-cancer-bearing mice with a higher tumor

7. Toxicity of CNT The toxicity of nanomaterials, in general, and of CNTs, in particular, has been deeply investigated over the past decade, due to increased interest in their bio-applications. The toxicity and biocompatibility of CNTs depend on several factors including: • CNT structure (diameter, length, and shape). • Production methods — possibly requiring toxic metal catalysts (Co, Fe, Ni, Mo), organic materials (amorphous or micro-structured, residual organic carbon) or supporting materials (alumina, magnesium oxide or silica) [97•]. • CNT functionalization – covalent and non-covalent, with various moieties. Although a wide range of nano-toxicity studies have been done, the results are still controversial and await a consensus. 7.1. CNT structures When the CNT length is comparable to or longer than the diameter of a phagocytic cell (> 15 μm), it becomes difficult for the

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A) Poly ethylene glycol (PEG) DOX

B)

C)

D)

Fig. 4. CNT conjugation with anticancer drugs: A. PEGylated SWCNTs loaded by DOX anticancer drug via supramolecular π-π stacking (Reprinted with permission from Ref. [86]). B. DOX– fluorescein–BSA – antibody conjugate – SWCNT (red=doxorubicin, green=fluorescein, light blue=BSA, dark blue=antibodies) (reprinted with permission from Ref. [87]). C. SWCNTs (derivatized with –CO2H groups) conjugate with ALG, CHI and DOX (Reprinted with permission from Ref. [88]) D. PTX conjugated to PEG-SWCNT (Reprinted with permission from Ref. [96]).

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higher than 100 μg/ml, due to the high negative charge of acidfunctionalized SWCNTs. It was also reported that the molecular weight of the functional group might also increase toxicity above 60 kDa [2,19]. 8. Prospects and future challenges

Fig. 5. Cryo-TEM micrograph of the CNT-liposome conjugate complex: The spherical liposomes (~100 nm in diameter) are covalently attached to oxidized MWCNTs (see arrow). The TEM grid polymer support is starred.

CNTs are nanomaterials with exceptional potential in the biomedical field, due to their unique properties. This review attempts to highlight the most recent biomedical applications of CNTs, while considering possible hurdles, such as toxicity issues. There are a few more bio-related applications which have not been thoroughly reviewed in this article, such as biosensors [113,114], vaccine nano-carriers [115], soft tissue substitutes and other tissue engineering applications [116]. We have presented the versatility of CNTs as manifested by their rich structural (e.g., length, diameter), chemical (e.g., attached functionalization) and physical characteristics, indicating their successful use as nano-carriers for targeted drug transport; multiple drugs or targeting agents may be attached to the CNTs in many different ways. The toxicity issue still awaits a consensus, as demonstrated by inconclusive or contradictory studies. These issues should be resolved before the first practical applications of CNT-based medicine are approved. Acknowledgements

macrophages to phagocytose it. This invokes the chronic release of inflammatory mediators and contributes to fibrosis. Longer MWCNTs cause Inflammation similar to long asbestos [104], while shorter ones were reported to be easily phagocytosed and cleared from the body [105]. 7.2. Production method Catalysts, such as Cr, Ni or Fe, organic materials (amorphous or micro-structured residual organic carbon) or supporting materials (alumina, magnesium oxide or silica) [97•] are thought to substantially contribute to CNT toxicity. These metals interact with oxidative species in cells, causing oxidative damage to healthy tissue. Only when it has a residual metal content, CNT demonstrates redox catalytic ability, causes cell activation, generates free radicals and reduces mitochondrial membrane integrity [106,107]. The level of metal contamination required to generate these effects varies from 0.26 wt.% to 26 wt.% [108]. Mice subcutaneously implanted with iron-contaminated MWCNTs exhibited severe hair loss and inflammation, as opposed to highly pure and clean MWCNTs implanted mice [109]. 7.3. Functionalization The addition of functional groups changes the chemistry of the CNTs. Raw CNTs (without functionalization) have a highly hydrophobic surface that may cause aggregation and interactions with cells inducing apoptosis. Functionalization makes the nanotubes more hydrophilic and, as such, more water soluble and biocompatible. The functionalization process, especially in the case of acid-treated CNTs, reduces their length and the degree of impurity. Several studies [85,110,111] were performed in-vitro with different types of functionalized SWCNTs having different charges (positive, negative or neutral). As a consequence of CNT implantation, these cells did not exhibit enhanced apoptosis/ neurosis. Therefore, it is expected that increased functionalization will diminish the CNTs cytotoxicity. Nevertheless, controversial studies exist. It has been reported that acid-functionalized SWCNTs are noticeably more toxic than raw SWCNTs [2,112], mainly at concentrations

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Glossary CLC: carbon nanotubes liposome conjugated CNT: carbon nanotubes Cryo: TEM-cryogenic transmission electron microscopy CuAAC: Cu(I)-catalysed azide-alkyne [3 + 2] cycloaddition CVD: chemical vapor deposition DNA: deoxyribonucleic acid DOX: doxorubicin EAD: electric arc discharge f-CNT: functionalized carbon nanotubes FDA: food and drug administration HEK: human embryonic kidney cells LAB: laser ablation MW: molecular weight MWCNT: multi-walled carbon nanotubes NIR: Near infrared PEG: poly ethylene glycol PTX: paclitaxel RES: reticuloendothelial system siRNA: small interfering ribonucleic acid SWCNT: single-walled carbon nanotubes