4.1 Introduction

Efficient design and fabrication of smart and stimuli-responsive nanosized materials for biomedical applications is among the most important challenges of nanotechnology [1]. Among numerous nanoscale platforms for drug delivery [2, 3], special attention has been paid to nanotubular particles due to their morphological and chemical properties [4]. The paradigm of nanoarchitectonics [5] implies the synergistic use of several types of materials arranged into complex supraparticles, uniting and enhancing the original functionalities of the precursor materials. In this sense, nanosized tubes are an excellent candidate to be a versatile platform for fabrication of smart nanocontainers. In general, nanotubular materials exhibit a similar primary structure (shown in Figure 4.1), where a tubelike particle has an internal cavity (lumen) and external walls along with openings at both ends of the tube. In the simplest case, the tube's walls are not layered; in other words, the tube consists of a single layer of rolled material (i.e., graphene in case of single-walled carbon nanotubes (SWCNTs)). If the tubes are multilayered, the internal structure also contains the interlayer spaces, in addition to the central cavity (lumen). The geometry of nanotubes greatly depends on their chemical nature; certain kinds of nanotubes demonstrate irregular curly geometry and form bundles of varying size and shape, while other nanotubes possess regular rodlike geometry with little or no spatial defects. What makes nanotubes excellent candidates for fabrication of nanoarchitectonic drug delivery vehicles? What makes nanotubes more suitable for drug delivery if compared with other types of nanoscale particles for drug delivery, such as mesoporous silica nanoparticles [6] or graphene-based materials [7]? The major advantages of nanotubular structures are directly associated with the nanoarchitectonic paradigm, that is, nanotubes of any chemical origin possess a similar architecture (a tube having walls, lumen, and open endings), which enables researchers to do the following:

1. 1. Load cargo (drugs, therapeutic nucleic acids, etc.) into the lumen, within the interwall spaces in multiwalled tubes or onto the walls (or using all of these sites simultaneously) [8];
2. 2. Modify the tubes' endings with “smart” stoppers, preferably stimuli-responsive, to modulate the release of the cargo (i.e., designing the stoppers to make them permeable only within certain regions of cells or tissues) [9];
3. 3. Functionalize the tubes' walls with either polymer (multi)layers or nanosized particles (or a combination of both) to enable the external triggering or spatial manipulation with the drug-loaded nanotubes [10].

These features of nanotubes can be hardly reproduced using other non-tubular nanosized particles. Apparently, the best advantage of nanotubes of any kind is the large loading capacity, if compared with nanospheres or nanoporous particles. As a result, numerous reports have been published in the past decades demonstrating the use of nanotubes for drug delivery [11]. In this chapter we overview the use of two types of nanotubular materials, namely, CNTs and halloysite clay nanotubes (HCTs) for fabrication of nanoarchitectonic drug delivery platforms. We also discuss the uptake mechanisms of intracellular uptake of nanocontainers having tubular geometry.

4.2 Carbon Nanotubes for Drug Delivery

4.2.2 Functionalization of CNTs for Drug Delivery

CNTs can be functionalized in a variety of different ways for use in drug delivery applications. They can be functionalized either internally or externally. An example of external functionalization is shown in Figure 4.2a–c. This method of functionalization incorporates the external loading of magnetite nanoparticles, poly(acrylate acid) (PAA), as well as the anticancer drug gemcitabine [16]. Figure 4.2d shows an example of platinum(IV) prodrug inside rather than outside of the tubes [17].

Pristine CNTs are hydrophobic, so surface modification to achieve hydrophilic surfaces is necessary for medical applications. The functionalization techniques can involve covalent as well as non-covalent strategies. The surface of CNTs can be covalently modified; then further functionalization with hydrophilic organic molecules can be employed for increased solubility in aqueous environments [18]. Poly(ethylene glycol) is perhaps the most common species for functionalization in medical applications, which can increase the dispersity of CNTs in aqueous solution as well as enhance biocompatibility of CNTs [19].

4.2.3 Uptake of Carbon Nanotubes

In general, CNTs must be able to enter cells in order to deliver their cargo to cells. It has been proved that various types of functionalized CNTs have the ability to be taken up by many different types of cells and can migrate intracellularly through different cellular barriers [20]. Figure 4.3 shows the uptake of CNTs by human promyelocytic leukemia (HL60) cells and human T-cells (Jurkat). It has been stipulated that the pathway of cellular uptake is endocytosis. Fluorescently labeled functionalized nanotubes were incubated with HL60 cells for 1 h in order to determine if the CNTs can enter the cells. The cells were washed twice, collected by centrifugation, and resuspended in growth medium. Confocal microscopy showed CNTs aggregating around the cell membranes as well as in the interior of the cells Figure 4.3 [21].

Functionalized MWNTs have also been proved to be able to enter cells. Surface functionalization generally increases the uptake of CNTs [22]. There are different theories as to how CNTs enter cells. An interesting theory is that MWNTs enter cells in different ways depending on aggregation. The theory suggests that bundles of MWNTs enter cells via endocytosis, while single tubes enter by directly penetrating the cell membrane [23].

4.3 Halloysite-Nanotube-Based Carriers for Drug Delivery

4.3.2 Modified Halloysite Nanotubes with a Time-Extended Effect on the Drug Release

Typically, pristine nanotubes exposed to aqueous media exhibit a complete release of water-soluble drugs within 4–12 h [47], which is not appropriate for some applications that needed a longer time efficiency of the entrapped active molecules. Both the formation of tube end stoppers and the HNT coating with polymeric layers were proposed for designing modified nanotubes with slower release kinetics [42, 63, 66, 67]. Figure 4.4a shows the benzotriazole release curves from pristine halloysite, HNTs coated with urea–formaldehyde (UF), and with copper tube end stoppers.

A time-extended effect on the drug release was observed for both modified nanotubes. In particular, the slowest kinetics release was observed for the nanotubes capped with copper-inhibitor clogs because of the chelation of Cu(II) ions by released inhibitors, which are predominantly at the tube ends (Figure 4.4b). Similarly, the formation of dextrin (or glycogen) tube end stoppers (Figure 4.5a) was successfully employed to control the release of brilliant green (BG) inside human lung carcinoma cells (A549). Specifically, the BG release was triggered by the enzymatic dextrin decomposition (mediated trough the intercellular glycosyl hydrolases). Consequently, the destruction of the malignant cells was enhanced with respect to that observed for pure HNTs loaded with BG (Figure 4.5b).

4.3.3 Covalently Functionalized Halloysite Nanotubes as Drug Delivery Systems Sensitive to Specific External Stimuli

Recently, covalent functionalization of halloysite surfaces was investigated with the aim of obtaining hybrid nanostructures with controlled drug release capability. These hybrid nanostructures are sensitive to external stimuli, such as temperature [35], pH [42], and intracellular enzymes [41]. The release kinetics of curcumin (an anticancer agent) from HNTs functionalized with chitosan is much faster at cell lysate than that at pH 7.4 [43]. Accordingly, the curcumin loaded in the modified nanotubes exhibited a specific toxicity toward cancer cells. The grafting of poly(N-isopropylacrylamide) (PNIPAAM) onto the HNT external surface allowed the generation of an efficient nanocarrier for thermoresponsive curcumin release [68]. Temperature-sensitive features were observed due to the coil-to-globule transition of the polymer at the LCST (lower critical solution temperature) occurring around 32 °C. In addition, the curcumin release was affected by the pH conditions. The in vitro kinetics studies conducted at 37 °C highlighted that the curcumin release was larger at pH = 6.8 (about 10 wt%) with respect to that estimated at pH = 1 (about 2.5 wt%). Interestingly, the thermoresponsive PNIPAAM/HNT carrier was complementary to the triazole/HNT hybrid [69], where a full release was observed only in acidic medium. Similarly, the release of cardanol (an anticancer agent) from the clay nanotubes functionalized with triazolium salt was favored by a decrease in pH [70]. Covalently functionalized HNTs were successfully employed as dual-responsive nanocarriers [71]. Figure 4.6 illustrates the scheme for HNT-Cur prodrug with a controlled curcumin release on dependence of both intracellular glutathione (GSH) and pH conditions.

4.3.4 Hybrids Based on Halloysite Nanotubes as Dual Drug Delivery Systems

Multicavity nanostructures for co-delivery of drugs can be successfully obtained by the selective modification of the HNT external surface. The grafting of the HNT shell with amphiphilic cyclodextrin (CD) generated hybrid nanomaterials with loading abilities toward silibinin (Sil) and quercetin (Que), which possess different affinities toward the CD cavity and the halloysite lumen [40]. The amounts of flavonoids encapsulated in the hybrid nanomaterial were 6.1 and 2.2 wt% for silibinin and quercitin, respectively [40]. Accordingly, the CD/HNT system is a proper nanocarrier for the loading/delivery of both drugs that exhibit synergic effects in anticancer therapy as demonstrated by in vitro cytotoxicity assays (Figure 4.7) [40]. Similar composite nanocarriers were obtained by the covalent modification of the HNT outer surface with CD glycocluster [72]. The preparation of the dual drug delivery system was based on a green protocol using absolvent-free microwave irradiation [72]. The CD glycocluster/HNTs showed efficient carrier ability toward silibinin and curcumin, which present a pH-dependent affinity toward the functionalized nanotubes. Slower kinetics release was observed for both natural drugs. Furthermore, supramolecular interactions were exploited to design composite nanostructures with tunable loading capacity. Composite materials based on HNT and cucurbit[6]uril (CB[6]) molecules were effective for the entrapment of essential oils because of the formation of hydrophobic cavities onto the halloysite surfaces [73]. The CB[6]/HNT hybrid was successfully employed as a nanofiller for pectin-based biofilms with antioxidant and antimicrobial properties.

4.4 Tubular Nanosized Drug Carriers: Uptake Mechanisms

Both CNTs and HNTs are considered as transporters for various cargos including medical formulations for target drug delivery. Mechanisms of anisotropic particle uptake (including CNTs, HNTs, and nanorods) in mammalian and non-mammalian cells were studied by numerous authors over the past few decades, but still this task remains not fully understood. In general, there are four main pathways of nanoparticle internalization (Figure 4.8): energy-independent membrane piercing by passive diffusion (a), calveolae-mediated endocytosis (b), phagocytosis (c), and clathrin-mediated endocytosis (d).

Most of the studies based on the use of endocytosis inhibitors have demonstrated that the mechanism of CNT uptake is the energy-dependent endocytosis. For example, Kam et al. [21] have demonstrated that SWCNTs) functionalized with small molecules and proteins can enter both nonadherent and adherent cell lines (CHO and 3T3). Based on early publications [74, 75] they have proposed that the nanotubes nonspecifically associate with hydrophobic regions of the cell surface and are internalized via endocytosis. Fluorescence microscopy has been employed for detection of nanotubes inside the cell cytoplasm using green fluorescent conjugate for visualization of SWCNTs. Incubation at 4 °C completely inhibited the endocytosis [74, 75]. Red FM 4-64 marker was used to stain endosomes which formed around nanotubes during endocytosis; yellow color was observed due to overlapping of green fluorescence from nanotubes and red-stained endosomes. This observation is the direct evidence for endocytosis of nanotubes which accumulate in the cytoplasm of the cells after internalization, and the uptake pathway is consistent with endocytosis.

Haniu et al. have reported that CNT internalization was suppressed by cytochalasin D, an endocytosis inhibitor, in different types of cells [76]. In the other study, this group has used two types of endocytosis inhibitors with different targets: chlorpromazine as a clathrin-mediated endocytosis inhibitor and indomethacin as a caveolae-mediated endocytosis inhibitor [77, 78]. It was proved that internalization of MWCNTs was suppressed by both types of endocytosis inhibitors. In contrast, Kostarelos et al. [20] reported that the cellular uptake of functionalized CNT is not affected by sodium azide, which is also known as an endocytosis inhibitor. They also have revealed that the process of CNT internalization is independent of cell type including bacterial and non-mammalian eukaryotic cells such as Saccharomyces cerevisiae and Cryptococcus neoformans. However, the studies performed by Haniu et al. demonstrated that the cellular uptake changes in response to cell differentiation and is inhibited by endocytosis inhibitors [77, 78]. However, the comparison of these studies is not correct enough because the MWCNTs used were not functionalized or labeled with fluorescein isothiocyanate. Moreover, Tabet et al. demonstrated that the mechanism of MWCNT uptake may depend on their modification or its absence [79] whereas Kostarelos et al. postulated that the internalization process is independent of the nature of functional groups [20].

Maruyama et al. [80] have demonstrated the MWCNT uptake into nonphagocytic cell lines (human bronchial epithelial cells (HBECs) and human mesothelial cells (HMCs)) as well as their accumulation in the lysosomes of the cells. They used a standard technique with corresponding endocytosis inhibitors and determined that clathrin-mediated endocytosis inhibitors significantly suppressed MWCNT uptake, whereas caveolae-mediated endocytosis and macropinocytosis were also found to be involved in MWCNT uptake. Apparently, the mechanism of CNT endocytosis represents the combination of three pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis; wherein clathrin-mediated endocytosis plays a key role and other pathways may be also involved in varying degrees.

As was demonstrated recently, the uptake of CNTs into cells is a size-dependent process [81]. Kang et al. [82] showed that the large MWCNTs were not taken up by cells through endocytosis due to size limitations, whereas the single-walled CNTs can enter human HepG2 hepatocellular carcinoma cells via endocytosis. Surface characteristics are also critical in CNT uptake: oxidized nanotubes are more likely to enter cells via endocytosis with the help of specific ligands [83–85] because the oxidation of nanotubes introduces carboxyl groups, which provides a negative charge to their surface. This in turn results in repulsion from the plasma membrane unless the CNT has ligands that are recognized with a high affinity by the cell. Some ligands (folic acid [83], albumin [84], and epidermal growth factor [85]) have been reported to facilitate uptake by specific cell types.

The size and possibility of nanotubes to form clusters and aggregates must be taken into account when it comes to the rational design of CNT-based carriers for cell therapy. A recent review by Raffa et al. [86] summarized the works that described the mechanism of cellular uptake of nanotubes and demonstrated that the degree of dispersion, the formation of supramolecular complexes, and the nanotube length play a crucial role in the process of uptake. Relatively big aggregates – bundles, clusters, or single dispersed nanotubes 1 µm or more in length – enter the cells by phagocytosis. Endocytosis appears to be the internalization pathway for CNT-forming supramolecular structures; wherein passive diffusion is the internalization mechanism for submicron CNTs that do not form supramolecular complexes.

In addition, Yaron et al. [87] have demonstrated that relatively small single-wall CNTs enter the mammalian cells by energy-dependent endocytosis, which was determined as suppression of nanoparticle transport at 4 °C compared with 37 °C. The authors also examined the possibility for physical penetration of SWCNTs through the plasma membrane. Using electrochemical impedance spectroscopy and Langmuir monolayer film balance measurements, they demonstrated that Pluronic-stabilized SWCNTs associated with membranes but did not penetrate through the membrane. They described the stages of SWCNT uptake by cells:

1. 1. Adsorption of SWCNTs onto the cell surface and penetration into the outer leaflet of the bilayer;
2. 2. Increasing of membrane tension and induction of imbalance between the outer leaflet and the inner leaflet;
3. 3. Local disturbances in membrane tension, which formed from the previous stage, stimulating endocytosis as the membrane attempts to regulate tension;
4. 4. Formation of endosomes, which shrink during processing;
5. 5. Distribution of SWCNTs inside the cytoplasm by disruption of the endosomes or lack of lysosomal processing.

Earlier, Jin et al. [3] have observed that SWCNTs incorporated into and expelled from NIH-3T3 cells using a perfusion microscope cage. This study represents the first conclusive evidence of SWCNT exocytosis. The rate of excluded nanotubes closely matches the endocytosis rate with negligible temporal offset. Therefore, in this study, the authors identify the exocytosis pathway that leads to the previously observed aggregation and accumulation of SWCNT within the cells.

Hong et al. also demonstrated the reverse process of endocytosis, exocytosis of single nanotubes or molecules on plasmonic substrates [88]. In this work, cellular uptake and transmembrane displacements show clear correlation with temperature and clathrin assembly on cell membranes. This strongly suggests that the cellular entry mechanism for a nanotube molecule is clathrin-dependent endocytosis through the formation of clathrin-coated pits on the cell membrane (Figure 4.9).

Halloysite is a nontoxic material which is a promising candidate for the formation of effective and safe drug delivery systems. The mechanism of HNT internalization has been studied in some works, and most of them proposed that clay nanotubes enter the mammalian cells via energy-dependent endocytosis. For example, Massaro et al. and Zhang et al. postulated in their works that halloysite-drug complexes' internalization mechanism presumably involves an endocytosis process and nanotubular containers could be taken up into the cytosol by endosomes (Figure 4.10) [72, 89].

Liu et al. have observed under confocal microscope the internalization and localization of HNTs inside A549 cells [42]. The results of this work show that HNTs can readily internalize into live cells and aggregate together around the nucleus region (Figure 4.11). This phenomenon has also been demonstrated in our previous work [57] using a combination of enhanced dark-field and fluorescent microscopy techniques.

Liu et al. [90], using the autofluorescence properties of curcumin, demonstrated the intracellular uptake of curcumin-loaded HNT-g-CS nanoparticles by fluorescence microscopy. They showed that HNTs began to enter the cell membrane at 2 h, entered into the cells at 4 h, and the cytoplasm was filled at 8 h. It was also observed that the cells treated with curcumin-functionalized HNTs showed profound fluorescence intensity in the cytoplasm at 12 h. Therefore, the authors speculated that the observed higher uptake of HNTs might be due to lysosome-mediated endocytosis, which has already been found in other systems.

Yang et al. [91] proposed that the drug-loaded nanotubes could penetrate the plasma membrane directly and/or via endocytosis mechanism due to their unique needle-like morphology similar to CNTs [87]. They observed the doxorubicin-loaded HNT internalization, but the authors suggest that the exact mechanism is yet to be decyphered, and they revealed that nanotubes can remain in both the cell cytoplasm and nuclei with a prolonged residue time. This provides the possibility of inducing cell apoptosis via the mitochondrial injury pathway. The authors hypothesized that HNT-g-COS would promote the anticancer efficiency of DOX through synergistic mechanism of both the mitochondria and nuclei injury (Figure 4.12).

4.5 Conclusions

Nanotubes are excellent candidates for fabrication of nanoarchitectonic drug delivery vehicles due to their structural and chemical properties. CNTs are theoretically useful in a variety of medical applications; however, there are many concerns on their possible toxicity. Loaded CNTs can be used for vaccine treatment, gene therapy, and cancer treatment due to their efficient penetration into cells. CNT composites are prospective materials that can be used as tissue scaffolds and artificial implants. They have been used as electro-reactive components in model biosensors, but no real medical applications were reported. There may be exceptional cases where CNTs themselves did not show toxic effects; however, CNTs are still not safe to use indiscriminately in vivo. CNTs are not biodegradable and exact mechanisms of their removal from organisms have not been determined [12–15, 32, 92]. HCTs are a natural nanosized material currently used in fabrication of drug delivery vehicles. The exceptional biocompatibility of halloysite facilitates its use in a number of biomedical applications. The mechanisms of cellular internalization of nanotubes have been briefly overviewed, although there is still need for additional studies. The future research in fabrication of drug delivery vehicles using nanotubes is focused on designing novel functional (bio)macromolecules to render the nanotubular drug containers with the ability for targeted delivery and triggered slow release inside the target cells.

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