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

Abstract

Multiwalled carbon nanotubes (MWCNTs) exhibit physical properties that render them ideal candidates for application as noninvasive mediators of photothermal cancer ablation. Here, we demonstrate that use of MWCNTs to generate heat in response to near-infrared radiation (NIR) results in thermal destruction of kidney cancer in vitro and in vivo. We document the thermal effects of the therapy through magnetic resonance temperature-mapping and heat shock protein-reactive immunohistochemistry. Our results demonstrate that use of MWCNTs enables ablation of tumors with low laser powers (3 W/cm(2)) and very short treatment times (a single 30-sec treatment) with minimal local toxicity and no evident systemic toxicity. These treatment parameters resulted in complete ablation of tumors and a >3.5-month durable remission in 80% of mice treated with 100 microg of MWCNT. Use of MWCNTs with NIR may be effective in anticancer therapy.

Fig. 1.

MWCNTs produce a greater temperature increase than SWCNTs in response to NIR illumination. MWCNT and SWCNT were suspended at a range of final concentrations in saline containing 1% (wt/wt) Pluronic F127 and illuminated at 3 W/cm² for 30 sec with a 1,064-nm continuous-wave NIR laser. Temperature was measured by thermocouple. Shown are mean and standard deviations of triplicate measurements. (Inset) Detail on the dilution range from 1 to 100 μg/ml.

Fig. 2.

NIR stimulation of MWCNTs induces thermoablative temperatures that inhibit clonogenic survival of cultured kidney cancer cells. RENCA cells in a final volume of 300 μl of medium were either treated with 100 μg/ml MWCNT or left untreated. Approximately 15 min after the addition of MWCNT, cells were exposed to 30 sec or 45 sec of NIR laser illumination (3 W/cm²). Temperature (two curves, right-hand scale) and clonogenic survival (bars, left-hand scale) were measured. Shown are means and standard deviations of triplicate measurements. (No colonies formed from cells treated with MWCNTs and 45 sec of laser exposure.) The combination of MWCNTs and 30 sec of laser exposure reduced viability 62-fold, and 0 colonies formed from cells treated with both MWCNTs and 45 sec of NIR laser illumination (P ≤ 0.02).

Fig. 3.

Intratumoral temperature distribution during MWCNT-mediated photothermal therapy resolved by MR temperature imaging. (A) High-resolution sagittal MR images across s.c. RENCA tumors in mice after injection with either 100 μg of MWCNT (i) or vehicle (iii). Bright dots in each image (indicated by white arrows in i) correspond to tubes of Magnevist (a gadolinium-based contrast agent) used in alignment of the laser aperture. Tumors were exposed to 30 sec of NIR laser, and temperature maps were obtained by MR temperature imaging. False-colored images depicting maximal temperatures are overlaid on the MR images (ii and iv). Temperature increase after 30 sec of NIR laser exposure in a saline-injected tumor is lower in magnitude and more superficial than that seen in the MWCNT-containing tumor. (B) Quantification of temperature changes in the center of the tumors.

Fig. 4.

Characterization of the HSP response to MWCNT-mediated photothermal therapy. Tumors that had been either untreated, treated with 30 sec of NIR laser alone, or treated with the combination of MWCNT plus laser were serially sectioned, and HSP expression was detected by immunofluorescent staining as described in Materials and Methods. (A) The top row of images depicts basal level of expression of HSP27, HSP70, and HSP90 in an untreated tumor. The middle two rows of images depict HSP expression in tumor sections taken at increasing tissue depths in a tumor treated with laser alone. Induction of HSPs proximate to the skin surface after 30 sec of NIR laser exposure is consistent with the superficial heating detected by MR thermometry (see Fig. 3). The lower two rows of images depict HSP expression in tumor sections taken at increasing tissue depths in a tumor treated with the combination of MWCNT plus laser. Thermoablative temperatures reached with the combination of MWCNTS and 30 sec of NIR laser exposure prevent HSP induction at the tumor surface. (B) HSP levels were quantified as described in Materials and Methods and expressed relative to untreated tumors (HSP expression in untreated tumors was the same throughout the tumor). Due to changes in tissue size imposed by processing, distances from the surface are presented as relative units: 1 is closest to the tumor surface (indicated as “surface” in A) and 4 is furthest from the surface (labeled “tumor base” in A). Means and standard deviations of 3 replicate 5-μm sections from 5 independently treated tumors in each group are shown.

Fig. 5.

MWCNT-based photothermal therapy reduces tumor volume. Nu/nu mice were implanted s.c. with RENCA tumors and divided into groups of 10. Mice were either left untreated, treated with MWCNT alone, treated with laser alone, or treated with the combination of MWCNT and laser. (A) Photographs at day 21 of representative mice from groups treated with laser only, untreated controls, or mice treated with 100 μg of MWCNT plus laser. (B) Mice treated with the combination of MWCNTs and laser were injected with a range of MWCNT doses. Tumor sizes were measured every 2 days. Means and standard errors are shown. Control groups (untreated, treated with MWCNTs alone, or treated with laser alone) were statistically identical. There is a dose-dependent attenuation in tumor growth after 30 sec of NIR laser treatment of MWCNT-loaded tumors (P < 0.0001).

Fig. 6.

MWCNT-based photothermal therapy increases long-term survival of tumor-bearing mice. Survival of mice treated as described in Fig. 5 was assessed for ≈10 months after treatment. Kaplan–Meier curves demonstrate a significant increase in survival in mice treated with all doses of MWCNTs plus laser (P < 0.0001). Mice were removed from the study (considered dead) when their tumor burden exceeded 1,000 mm³ or they were deemed moribund by veterinary consult. Survival curves for all control groups were statistically identical (P = 0.775).