Long-term survival following a single treatment of kidney tumors with multiwalled carbon nanotubes and near-infrared radiation

MWCNT Suspensions Are More Efficient at Producing an NIR-Dependent Increase in Temperature than SWCNTs.

Stable suspensions of MWCNTs or SWCNTs were prepared in physiologic saline with 1%(wt/wt) Pluronic F127, a biocompatible surfactant (see Materials and Methods), and compared for their ability to increase temperature after exposure to a 1,064-nm Nd:YAG (neodymium-doped yttrium aluminum garnet) laser at 3 W/cm² for 30 sec. MWCNTs were more efficient than SWCNTs in inducing a temperature increase (Fig. 1). At low concentrations, where bundling and scattering effects are minimized, MWCNTs performed markedly better than SWCNTs: For example, after a 30-sec exposure to a Nd:YAG laser (3 W/cm², λ = 1,064 nm), the temperature of a 0.1 mg/ml solution of MWCNTs increased from 23 °C to 51 ± 0.7 °C, whereas that of a 0.1 mg/ml solution of SWCNTs increased from 23 °C to 27 ± 0.3 °C. Although SWCNTs were comparable with MWCNTs in inducing a temperature increase at the upper limit of concentrations tested, it required a 20-fold greater concentration of SWCNTs to generate a temperature increase equivalent to that of a 0.1 mg/ml MWCNT suspension (Fig. 1). Varying NIR laser exposure time (range: 15–60 sec) revealed that the thermal ablation temperature threshold (50–55 °C) of a 0.1 mg/ml suspension of MWCNT could be reached within 30 sec of laser irradiation (T₃₀ = 53.1 ± 0.1 °C) (Fig. 2). The ability of MWCNTs to induce temperature increases compatible with thermal ablation at low concentrations and short laser exposure times suggests that MWCNTs may be useful as photothermal mediators.

MWCNTs Efficiently Kill Cancer Cells in Vitro.

We next tested whether we could identify treatment parameters that would enable MWCNTs to be used as thermoablative agents. Cultured RENCA kidney cancer cells were overlaid with growth media containing 0.1 mg/ml MWCNTs and illuminated with the NIR laser at 3 W/cm² for 0, 30, or 45 sec, and then the temperature was measured. As shown in Fig. 2, these parameters generated mean final temperatures of 53 °C and 62.7 °C, respectively, which were in excess of the thermal ablative temperature threshold and significantly higher than control (P < 0.0001). We also assessed the effects on clonogenic survival, a rigorous assay that measures the ability of individual tumor cells to survive, proliferate, and form colonies. MWCNTs themselves had no statistically significant effect on colony-forming ability, and no effect was seen for laser alone (Fig. 2). However, a 62-fold reduction in viability was seen with the combination of MWCNTs and 30 sec of laser exposure (P < 0.0001), and no colonies formed from cells treated with both MWCNTs and 45 sec of NIR laser illumination. Cell-killing depended on reaching thermoablative temperatures and was not a temperature-independent consequence of NIR on MWCNTs, since modifications of the treatment conditions (an increase in media fluid volume) that reduced the temperature increase to below this threshold eliminated the cytotoxic effect even at 10-fold higher concentrations of MWCNTs [supporting information (SI) Fig. S1]. The thermal sensitivity of RENCA cells was similar to that of a number of other cancer cell lines (Fig. S2), indicating that the rapid thermal death observed in this experiment was not due to the inadvertent selection of particularly heat-sensitive cells.

MWCNT Photothermal Therapy Is Compatible with Magnetic Resonance Temperature Mapping and Produces Therapeutically Relevant Temperature Increases in Vivo.

To determine whether MWCNTs could be used to attain thermoablative temperatures in vivo, RENCA tumors were implanted in the flanks of 4 nude mice. Five to 7 days after implantation, 2 mice were injected intratumorally with 50 μl of a MWCNT suspension (100 μg of total MWCNTs), and 2 control mice were injected with 50 μl of diluent. Twenty-four hours later, all tumors were laser-treated (3 W/cm², 30 sec) in the bore of 7T magnetic resonance (MR) magnet to monitor temperature change over the tumor volume in real time. (A diagram of the experimental set-up is shown as Fig. S3.) Proton resonance frequency MR temperature-mapping protocols were used to generate spatially resolved temperature profiles (14, 15). Fig. 3A presents MR images (Fig. 3A Left) and superimposed MR thermometry images (Fig. 3A Right) of a control and MWCNT-containing tumor. MWCNTs induce an increase in temperature measurable by MR thermometry: After 30 seconds of laser illumination, the average temperature of the MWCNT-loaded tumor increased to 74 °C compared with 46 °C in control tumors exposed to laser alone (Fig. 3B). Thus, MWCNTs increased intratumoral temperatures to a level sufficient to induce tumor ablation.

Laser-Stimulated MWCNTs Produce a Temperature Gradient That Extends More Deeply into Tissue than Laser Treatment Alone.

MR temperature maps suggested that the combination of MWCNTs and laser produced a deeper distribution of heat when compared to laser alone (Fig. 3). To confirm this result, we used an independent measure of heat generation: induction of heat shock proteins (HSPs). HSPs are induced by elevated temperatures (typically in excess of 43 °C) and serve as endogenous cellular markers of thermal stress. As seen in Fig. 4, minimal expression exists for all HSPs in untreated tumors. In the tumor treated with laser alone, maximal HSP27, HSP70, and HSP90 expression was induced proximal to the incident laser and then gradually diminished. In contrast, in the tumor treated with laser plus MWCNTs, temperature elevation at the surface was sufficient to induce coagulative necrosis, thus preventing HSP induction. HSP induction was seen at deeper tissue levels, at the interface between tumor and normal tissue (Fig. 4). These results suggest that MWCNTs can be used to extend the depth of thermal therapy.

MWCNT-Mediated Thermal Ablation Reduces Tumor Volume and Enhances Survival.

We next investigated the effectiveness of MWCNT-based therapy in reducing tumor growth and enhancing survival in vivo. For this study, 2-mm³ RENCA tumor fragments were s.c. implanted into the flanks of 60 nude mice. Once tumors measured ≈5.5 mm (5.5 ± 0.5 mm) in greatest dimension (<1 week), mice were randomized into one of 6 treatment groups (n = 10 per group) as illustrated in Fig. 5. There was no statistical difference among group mean tumor volumes at the onset of treatment (P = 0.44). Mice were then injected intratumorally with 50 μl of treatment solution containing 100, 50, or 10 μg of MWCNTs. As assessed by polarized light microscopy and transmission electron microscopy (TEM), MWCNTs introduced into tumors by this method appeared well-distributed (Fig. S4). Control mice were either injected with 50 μl of vehicle (saline with 1% Pluronic) or left untreated. Twenty-four hours after injection, tumors were illuminated with a 1,064-nm NIR laser for 30 sec (3 W/cm²; spot size, 5 mm). The untreated control group received neither MWCNTs nor laser treatment. Tumor growth was monitored, and mice were removed from the study when their tumor burden reached 1,000 mm³ or they were deemed moribund by veterinary consult.

Control tumors (untreated control, laser-only control, and 100 μg of MWCNT with no laser) grew rapidly and uniformly, and all mice were removed from the study within the first 30 days. There was no statistically significant difference in tumor growth rate (P = 0.77) or final tumor size (P = 0.979) in any of the control groups, indicating that tumor growth was not affected by MWCNTs alone or by laser alone. In contrast, a statistically significant dose-dependent attenuation of tumor growth was observed in mice treated with MWCNT plus laser (Fig. 5). Effects were most pronounced at the highest dose of MWCNT (100 μg), where mean tumor volume at 30 days was reduced from 1,472 ± 118 mm³ in controls to 11 ± 11 mm³ (P < 0.0001). A significant reduction in mean tumor volume was also evident at the 50-μg dose (354 ± 176, P < 0.0001). The lowest dose of MWCNTs produced the most modest response, with a mean tumor volume of 590 ± 178 mm³ (P < 0.0001).

The inhibition of tumor growth in mice treated with the combination of MWCNTs and NIR translated into a significant survival advantage (Fig. 6). Relative to control, overall survival was significantly prolonged in each treatment group, with 80% of mice alive and tumor-free >3 months after treatment in the 100-μg treatment group (P < 0.0001) (tumor recurrence accounted for the mice that died in this group). Effect on survival was dose-dependent: 60% remained alive in the 50-μg treatment group (P = 0.0004), and 20%survived in the 10-μg MWCNT treatment group (P = 0.0395). There was no statistical difference between control groups (P = 0.775), which exhibited median survivals of 25 days (95% C.I. 23–27) in untreated controls, 23 days (95% C.I. 21–25) in mice treated with laser alone, and 23 days (95% C.I. 21–25) in mice treated with 100 μg of MWCNTs alone. Median survival in the group treated with 10 μg of MWCNT plus laser was 29.5 days (95% C.I. 25–53). Median survivals could not be calculated for the 2 highest dose treatment groups, because too few mice died after treatment to make the determination; however, it was extended to at least 300 days in these treatment groups.

We performed a limited survey of the long-term effects of MWCNT plus laser treatment on mouse organs. All animals developed a nonpermanent cutaneous surface injury that healed over time. MWCNTs remained evident at the site of injection after tumor regression: In all mice that were treated with MWCNTs and laser, there was visible black material directly under the skin, and this persisted to the conclusion of the study. TEM analysis (performed in a subset of mice) confirmed that this was due to the persistence of MWCNTs at the injection site (Fig. S5). To assess effects on internal organs, 5 mice that had remained in disease remission for 3.5 months after treatment with 100 μg of MWCNT plus laser were randomly selected, and sections of the lungs, liver, spleen, skin (from MWCNT injection site), kidneys, and brain were examined. All were essentially normal, without evidence of nanotube-induced injury or inflammation as assessed by a veterinary pathologist (Fig. S6). Furthermore, the remaining 11 MWCNT-injected mice that were not selected for this analysis continued to survive without evidence of recurrence or notable physical or behavioral abnormalities for >6 months. Thus, the treatment involving MWCNTs produces a durable remission without evident toxicological consequences.

Materials.

MWCNTs were produced by chemical vapor deposition at the Wake Forest University Center for Nanotechnology and Molecular Materials and characterized by TEM (Phillips 400, Phillips) (see Fig. S7). MWCNTs were massed and suspended in saline with 1% (wt/wt) Pluronic F127 (BASF) through sonication. HiPCo-generated SWCNTs were purchased from Carbon Nanotechnologies Inc. (Unidym). Details of this and other methods are presented in SI Materials and Methods.

Cell Culture.

RENCA murine kidney cancer cells were a gift from Robert Wiltrout (National Cancer Institute). MCF-7 breast cancer, PC-3 prostate cancer, Caki-1 kidney cancer, and HeLa cervical cancer cell lines were obtained from the American Type Culture Collection and cultured as described in SI Materials and Methods.

TEM Imaging.

Stock solutions of 2 mg/ml MWCNT and SWCNT were deposited on formvar-coated grids and imaged with a TEM 400 at either 55 or 80 KeV.

Comparison of Heating by SWCNT and MWCNT.

MWCNT and SWCNT were suspended at a range of final concentrations (wt/vol) in saline containing 1% (wt/wt) Pluronic F127. Three hundred microliters of each suspension was illuminated at 3 W/cm² for 30 sec by using a 1,064-nm continuous-wave NIR laser. Temperature was measured by thermocouple.

NIR-Heating and Cell Killing of MWCNTs in Solution.

MWCNT were diluted in growth medium, illuminated with a 1,064-nm continuous-wave NIR laser (power density, 3 W/cm²; spot size, 5 mm; exposure duration, 15–60 sec). Temperatures were measured by thermocouple (Fluke). Effects on clonogenicity were assessed by diluting and replating cells immediately after treatment and measuring colony formation after 13 days.

Thermal Sensitivity of Cultured Cancer Cells.

Cells were trypsinized, suspended in growth medium, and incubated for 30 min in a heat block equilibrated to the desired temperature. Aliquots measuring 100 μl were transferred in triplicate to 96-well plates and incubated overnight at 37 °C in a humidified incubator in a 5% CO₂/95% air atmosphere, and viability was assessed with an MTT assay (Sigma).

Animal Handling.

All animal studies were performed in compliance with the institutional guidelines on animal use and welfare (Animal Care and Use Committee of Wake Forest University Health Sciences) under an approved protocol. Female nu/nu athymic mice were obtained from Charles River Laboratories (5–8 wk old). Mice were housed 5 per cage in standard plastic cages, provided food and water ad libitum, and maintained on a 12-h light/dark cycle.

Tumor Regression and Survival Studies.

RENCA tumor fragments measuring 2 mm³ were transplanted into the flanks of 60 female athymic mice. Once tumors reached a mean diameter of ≈5.5 mm, mice were randomized into one of 6 treatment groups. For groups receiving MWCNTs, 2 mg/ml MWCNT stock solution was diluted with vehicle (1% Pluronic in saline) to provide the appropriate dosages of nanotubes (MWCNT control: 100 μg; MWCNT plus laser groups: 100, 50, and 10 μg, respectively). In all cases, solutions were injected directly into the center of the tumor mass. Twenty-four hours after injection, the following groups were laser treated: vehicle control and all 3 MWCNT plus laser cohorts. Laser treatment consisted of illuminating the tumor with a 1,064-nm continuous-wave NIR laser beam (IPG Photonics) at 3 W/cm² (spot size, 5 mm) for 30 sec. After treatment, tumor volumes were tracked every 2 days by digital caliper, and tumor volumes were calculated according to the formula (4/3)·π·(x/2)·(y/2)·(z/2). For the survival study, mice were removed once their tumor volume reached >1,000 mm³ or when deemed moribund by veterinary consult.

MR Temperature Mapping.

Temperature-mapping experiments were performed in a 7T Bruker Biospin MR system. A high-resolution MR sagittal image was acquired by using a gradient-echo sequence across the mouse tumor followed by a low-resolution sagittal scan to detect the temperature-induced phase difference across the treated mouse tumor. During the first scan the tumor was illuminated with the1,064-nm NIR fiber laser (beam diameter, 1 cm; power density, 3 W/cm²) for 30 sec. All image reconstruction and analysis was performed in Matlab (Mathworks).

HSP Expression Measurement.

Tumors from mice that were untreated (basal control), treated with laser alone, or treated with the combination of laser plus MWCNT were harvested 16 h after laser treatment and embedded in OCT cryomatrix (Sakura Finetek). Sections were prepared at various tumor depths and analyzed by fluorescent immunostaining for expression of HSP27, HSP70, and HSP90. Fluorescence was quantified by using Leica Microsystems AF6000 software.

Statistical Analysis.

All analyses were performed by a statistician (R.D.) in the statistical core facility of the Comprehensive Cancer Center of Wake Forest University with SPSS software (SPSS).