Tumor photothermolysis: using carbon nanomaterials for cancer therapy

Solubility

There are various strategies used to solubilize and disperse CNTs in water as well as to make them biologically compatible. Modifications can be grouped into three main categories: 1) covalent attachment of chemical groups onto the CNT skeleton through π-bonds; 2) noncovalent adsorption or wrapping of functional moieties onto the surface of CNTs; and 3) endohedral filling of the inner cavity of CNTs (66). Due to the non-polarity and high van der Waals forces exuded by CNTs, surface functionalization is necessary for increasing nanotube solubility in aqueous environments (19).

PEGylation, functionalization with poly(ethylene glycol) (PEG), has numerous advantages for drug delivery. Its abilities to prevent agglomeration of nanoparticles and shield drug carriers from recognition by the mononuclear phagocyte system (MPS) are attractive properties for the coating of carbon nanomaterials with PEG. Liu et al. demonstrated that PEGylation of SWNTs leads to increased blood circulation times (57). SWNTs were covalently conjugated with phospholipid-PEG, and then intravenously injected into mice. Raman scattering was utilized to track the transport and accumulation of the SWNTs. It was found that as the molecular weight of PEG increased, the concentration of PEGylated SWNTs in the liver and spleen decreased, suggesting that functionalization can be used to prolong SWNT circulation in the bloodstream by preventing MPS phagocytosis. Moreover, no resistance to SWNT clearance was found. Similarly, SWNTs functionalized with poly(ethylene glycol) methyl ether and poly(maleic anhydride-alt-1-octadecene) were found to possess high tumor uptake – roughly 30% of the injected dose – in addition to a blood circulation time of 30 h (67).

Matsumura et al. synthesized a conjugate composed of a comb-shaped PEG and carbon nanohorn-binding peptide (cPEG-NHBP) (58). The oxidized SWNHs (oxSWNHs) conjugated with cPEG-NHBP revealed higher dispersibility than the ones conjugated with 20PEG-NHBP which is made of linear 20 kDa PEG and NHBP. Absorbance readings at 800 nm showed that within 5 h, untreated oxSWNHs began to agglomerate. 20PEG-NHBP inhibited agglomeration to a degree. However, cPEG-NHBP showed only a slight reduction in absorbance and demonstrated good dispersion. Moreover, in vivo evaluation of oxSWNHs treated with cPEG-NHBP showed suppressed agglomeration in the lungs of mice compared to untreated oxSWNHs.

Yang et al. treated mice with nanographene sheets (NGS) coated with PEG, and the behavior of the sheets was studied (59). Due to the efficient passive tumor targeting of their newly synthesized NGS, in vivo PTT studies were carried out. Mice bearing 4T1 tumors were intravenously injected with 200 μL NGS-PEG (2 mg/mL) and exposed to an 808-nm laser at a power of 2 W/cm² for 24 h after injection. One day after laser treatment, tumors disappeared and there was no tumor regrowth for up to 40 days. A combined chemotherapy and photothermal therapy system was developed by Zhang et al. (60). Doxorubicin-loaded PEGylated nanographene oxide (NGO-PEG/DOX) was delivered to murine mammary EMT6 tumor cells both in vitro and in vivo. Cytotoxity of NGO-PEG/DOX compared to free DOX (10 μg/mL) showed an inhibition rate of 82.1% and 57.1%, respectively, when treated with NIR radiation (808 nm, 2 W/cm², 5 min). The trend of a synergistic effect in the inhibition rate of NGO-PEG/DOX compared to free DOX was not obvious at a DOX concentration of 2 and 30 μg/mL, however. In vivo results showed that, free DOX and NGO-PEG and NIR treated tumors began to grow after 10 days, NGO-PEG/DOX and NIR treated tumors however, showed no tumor re-growth up to 30 days. These results suggested that combined chemotherapy and photothermal therapy demonstrated a synergistic effect in vivo.

For photothermal ablation with GO, a high dosage of GO and laser power is needed due to the suboptimal absorption of NIR light by GO. Therefore, Dai’s group developed rGO sheets (61). By chemically reducing covalently PEGylated GO, they were able to partially restore the aromatic and conjugated characteristics of graphene sheets, increasing the NIR absorbance >6-fold. Conjugation with RGD peptides allowed for efficient selection to human glioblastoma cells. Cells were treated with a low concentration of rGO particles (6.6 mg/L) and NIR laser (808 nm, 15 W/cm²) with total cell destruction occurring within 8 min. Liu’s group synthesized a similar PEGylated rGO, and studied how the size and surface coating affects the behavior of graphene in vivo (62). Following treatment with an 808-nm NIR laser at a power density of 0.15 W/cm² for 5 min, complete tumor elimination in mice was observed from ultra-small rGO with non-covalent PEG coating. This research highlighted the importance of optimizing nano-graphene and its derivatives for effective photothermal treatment.

While PEG is commonly thought to be the “gold standard” for enhancing a therapeutic carrier’s delivery, several notable drawbacks have recently emerged (68). These disadvantages have prompted researchers to use alternative solubilizing agents. Mao et al. has synthesized SWNTs functionalized with collagen (51). Collagen not only helped to stabilize the SWNTs for months in solution but increased cellular uptake and retention time. Sobhani et al. synthesized MWNTs functionalized with poly(citric acid) to improve both hydrophilicity and functionality of the MWNTs and to enhance the delivery of paclitaxel (56). These studies demonstrated that modifications to CNTs can improve their dispersion as well as drug biocompatibility, cell penetration efficacy and targeted delivery capabilities, even if PEG is not used.

Immunologically modified SWNTs were developed using glycated chitosan (GC) by Zhou et al. (54, 55). The synergy between immunological and photothermal effects of SWNT-GC was shown to increase cell destruction. Specifically, 980-nm laser light was used to induce cell destruction and also led to secretion of damage associated molecular pattern molecules (i.e., molecules which can perpetuate immune response). In vivo studies on EMT6 cells injected into a mouse model showed that after treatment with SWNT-GC and laser light (980 nm, 0.74 W/cm², 10 min), 89.2% of cells showed apoptosis. Mice were monitored 100 days after tumor inoculation and the mice with CNT treatment and NIR laser showed complete tumor regression. Long-term antitumor effects showed that SWNT-GC inside a viable tumor was a prerequisite for the induction of effective immune response.

Whitney et al. synthesized SWNHs coated with Pluronic, a non-ionic surfactant which improves solubility of biological materials and which acts as a thermal enhancer (64). In this study, kidney cancer cells were treated with 1064-nm laser irradiation at 40 W/cm² for 0–5 min. After 2–3 min of treatment, cell viability showed nearly complete tumor cell death, suggesting SWNHs could be beneficial to treat superficial tumors. Rylander et al. used a fiber optic microneedle device to deliver SWNHs (coated with Pluronic F-127) to the wall of porcine bladders in ex vivo treatment (65). For their study, they wanted to evaluate the distribution of SWNHs as well as if they could control the degree of lateral dispersion and depth in which SWNHs permeate in the tissues. The 1064-nm laser light, at an intensity of 0.95 W/cm², emitted over 40 s irradiated the tissues and increased tumor cell temperatures. For their hyperthermic results, the SWNH-perfused tissue increased temperature by approximately 19°C, falling just short of the 60°C needed for in vivo treatment. The authors acknowledged that there were several limitations to the study. However, they believe that use of a fiber-optic microneedle device to deliver SWNHs is a versatile technology for local delivery of therapeutic agents, not limited to just SWNHs.

Zhang et al. synthesized zinc phthalocyanine (ZnPc, a photodynamic therapy agent) SWNHs conjugated with BSA (to improve dispersion) for a dual photodynamic and photohyperthermia cancer therapy system (50). The effect of ZnPc-SWNHs-BSA was studied in both in vitro and in vivo studies. Interestingly, they found that the double therapy using ZnPc-SWNHs-BSA had greater anticancer efficiency than ZnPc (phytodynamic therapy) and SWNH-BSA (photohyperthermia) treatment alone. One drawback to their study, however, was the use of 670-nm laser irradiation, which does not penetrate biological tissue as deeply as longer wavelengths of NIR light.

In addition to NIR laser adsorption, CNTs have demonstrated conversion of radiofrequency (RF) waves into heat (5). While ablation of malignancies using CNTs subjected to NIR radiation has been studied extensively, use of RF treatment has its merits for further evaluation because RF fields offer significantly deeper tissue penetration in comparison to NIR light (69). Gannon et al. performed in vitro thermal ablation of HepG2, Hep3B, and Panc-1 cancer cells using an RF field (5). SWNTs were functionalized with Kentera, a polyphenylene ethynylene-based polymer. Kentera was bound to the SWNTs through π-π stacking between the phenyl groups of the polymer and the SWNT surface. This modification did not alter the electronic structure of the CNTs appreciably. Moreover, the use of a high-field RF and low concentrations (5–500 mg/L) of aqueous SWNT suspensions produced thermal effects higher than previously reported studies. One explanation for this was attributed to dynamic self-assembly of SWNTs into longer nano-antennae. In vitro and in vivo studies were conducted and showed successful killing of tumor cells. Specifically, rabbits xenografted with VX2 tumors were directly injected with Kentra-functionalized SWNTs and then treated with an RF field at 13.56 MHz for 2 min. Irradiation of all VX2 tumors in mice treated with SWNTs had complete death after 2 days, while control tumors which were not treated with SWNTs were still viable. It was suggested that further studies be conducted with functionalized SWNTs for targeted delivery by linking antibodies, peptides or pharmacological agents to target molecules over expressed on the surface of cancer cells (5).

Targeting

While dispersibility of carbon nanomaterials is important, for clinical use, selective treatment of tumor cells without collateral damage to healthy cells is crucial. Hence, specific targeting to overexpressed markers on the surface of cancer cells must also be achieved. An early demonstration of using CNTs for photothermal cancer therapy was conducted by Kam et al. (52). In the study, SWNTs conjugated with DNA underwent endocytosis by malignant HeLa cells. HeLa cells containing the SWNT complexes were exposed to 808-nm laser radiation at six 10 s on-and-off pulses (1.4 W/cm²). They revealed that SWNTs can be used to deliver DNA into live cells without harming the cells. However, if they increased the exposure time to 2 min, extensive cell death was observed. It was demonstrated that SWNTs could be selectively internalized by cancer cells, and then irradiated to cause cellular destruction. Moreover, since many cancerous cells overexpress folate receptors, they functionalized SWNTs with phospholipid-PEG-folic acid (PL-PEG-FA). It was found that the SWNT-PL-PEG-FAs were internalized by the cells and that NIR pulses can induce local heating of SWNTs for release of therapeutic agents into the cells without harming them. Additionally, selective delivery of SWNTs into cells via receptor-mediated delivery and NIR-triggered death was achieved following NIR laser light administration for 2 min.

Wang et al. conjugated MWNTs with GD2 monoclonal antibodies to selectively target neuroblastoma cells (48). Significant photothermal ablation of the neuroblastoma cells occurred under an 808-nm NIR laser at a power of 6 W/cm² for 15 min. The interaction of MWNTs with the neuroblastoma cells was followed by rhodamine B labeling. It was seen that 6 h incubation with the MWNTs was sufficient for antibody-mediated endocytosis of ligand-tagged MWNTs. Uptake of GD2-targeted MWNTs for 12 h led to oversaturation; laser exposure data confirmed oversaturation of MWNTs generated too much heat, which led to collateral damage of non-GD2-expressing cells. Moreover, MWNTs bound with anti-GD2 antibody could specifically target GD2 expressing neuroblastoma cells, but not cells without GD2 expression, proving the importance of surface functionalization for specific targeting and selective ablating MWNT-laden cancer cells.

Wang et al. obliterated CD133⁺ stem-like cells xenografted to mice using chitosan functionalized SWNTs (49). CD133 has been recognized as a typical stem cell marker which inhabits the surface of cancerous blood, brain, colon, prostate, and liver cells. By conjugating anti-CD133 monoclonal antibody to SWNTs solubilized with chitosan, CD133⁺ cells were selectively targeted by the functionalized SWNT conjugates. Following the preliminary cell culture studies, mice were subcutaneously injected with a mixture of CD133⁺ and CD133-cells which had been pretreated with the anti-CD133-SWNTs. Two days following the treatment, 808-nm NIR laser pulses at 2 W/cm² were directed at the injection sites for 5 min. In addition to complete thermal ablation of the cancerous cells, the treatment prevented the formation of new malignancies.

Synergy between SWNT photothermal therapy and RNA interference was demonstrated by Wang et al. (53). SWNT-polyethylenimine (PEI) was added to siRNA and asparagine-glycine-arginine (NGR) peptides to produce SWNT-PEI-siRNA-NGR. Cell viability studies indicated an absence of cytotoxicity in PC-3 prostate cancer cells at SWNT-PEI concentrations below 30 μg/mL. However, SWNT-PEI-siRNA and SWNT-PEI-siRNA-NGR showed noticeable cytotoxicity in PC-3 cells after 72 h treatment (60 and 55% cell viability, respectively). This study demonstrated that the suppression of PC-3 cells by siRNA is preserved following conjugation of siRNA to SWNT-PEI. In all experimental groups, 3 min of 808-nm NIR laser radiation produced PC-3 cell viabilities below 5%. In vivo trials of SWNT-PEI, SWNT-PEI-siRNA, and SWNT-PEI-siRNA-NGR were conducted in nude mice bearing PC-3 tumors injected subcutaneously. Through molecular marking with quantum dots, it was shown that SWNT-PEI and SWNT-PEI-NGR exhibited higher accumulation in the liver, lung, spleen, and kidney than in the heart and brain which is consistent with localization in organs of the MPS. Importantly, SWNT-PEI-NGR had higher accumulation than SWNT-PEI in PC-3 tumors suggesting that NGR peptide enhanced uptake. These PC-3 tumors, accumulated with SWNT-PEI-siRNA-NGR nanoparticles and concomitantly irradiated with NIR laser, revealed the highest suppression of tumor growth. Without laser treatment, injections of SWNT conjugates containing siRNA led to a decrease in overall tumor volume. SWNT conjugates without siRNA mimicked the behavior of a saline solution and lead to negligible tumor reduction. No difference in tumor size was seen in the saline solutions with and without laser treatment, demonstrating that the conjugated SWNTs photothermally ablated the PC-3 tumor cells. It should be noted that since the NIR laser irradiation was conducted directly on the sites that PC-3 cells were injected subcutaneously, the SWNTs accumulated in organs with low cell toxicity were not exposed to NIR and therefore did not result in photothermolysis of cells.

Cancer stem cells have been proposed to contribute to cancer resistance and tumorigenesis (70). Similar to normal stem cells, cancer stem cells have self-renewal and proliferation capabilities. Due to their resistance to current treatments, cancer stem cells are difficult to kill and may contribute to cancer reoccurrence. For this reason, treatment regimens focused on targeting cancer stems cells are being researched. NIR-stimulated CNTs have been used for the photothermal destruction of breast cancer stem cells (BCSCs) by Torti and coworkers (63). They showed that BCSCs were resistant to traditional hyperthermia treatment and had an increased cancer stem cell fractionation of 1.6–1.9 fold. In contrast, the intense localized heat generated by CNTs treated with an NIR laser (1064 nm, 3 W, 45 s) caused almost complete cell death in both breast cancer cells and BCSCs. Moreover, laser plus CNT treatment diminished the long-term proliferative ability of the BCSCs.