Synergistic Enhancement of Cancer Therapy using a Combination of Carbon Nanotubes and Anti-tumor Drug

Materials and Methods

Nanoparticle Synthesis

Single-wall CNTs (further referred to as CNTs) were grown by radio-frequency chemical vapor deposition (RF-CVD) on a iron:molybdenum/magnesium oxide catalyst with methane as the carbon source. To determine the intrinsic weight loss of the catalyst, a 'blank' experiment, without the hydrocarbon source and with only argon, was performed. The efficiency or the yield (η; %) of the carbonaceous products that resulted after each synthesis was calculated according to the following formula:

where m _ar is the mass of the sample after the synthesis, m _br is the mass of the catalyst before the reaction and m _b is the mass of the catalyst after the blank experiment. After purification, the purity level of the nanotube samples was 98.3%. The produced CNTs were purified in one simple step using diluted hydrochloric acid solution under gentle sonication. To burn the amorphous carbon, the purified samples were heated in air to 400°C for approximately 15 min.

Figure 1 shows a high-magnification transmission-electron microscopy (TEM) image of a single CNT used for this study (Figure 1A), its purity (98.3%) as determined by thermogravimetric analysis (TGA) (Figure 1B) and their corresponding diameter distribution after analyzing 120 CNTs (Figure 1C).

The diameter and the morphology of the CNTs were inspected by TEM by using a field-emission JEM-2100F TEM (JEOL, Inc.) equipped with a charge-coupled device camera. The acceleration voltage was 100 kV for the CNT analysis. The carbon nanostructures were homogeneously dispersed in 2-propanol under ultrasonication for 30 min. Next, a few drops of the suspension were deposited on the TEM grid, dried and evacuated before analysis. To analyze the purity of the nanomaterials, TGA was performed on a Mettler Toledo TGA/scanning differential thermal analysis 851e instrument under airflow (150 ml/min) and with a temperature ramping rate of 5°C/min. The total mass loss in the TGA curve corresponds to the graphitic structures that decomposed thermally and represent the total purity of the CNT sample. A Shimadzu 3600 UV visible and near-infrared spectrophotometer was used to analyze the CNT concentration in solution. The spectrometer was equipped with three detectors: a photomultiplier tube (PMT) detector for the UV and visible regions, and indium gallium arsenide (InGaAs) and lead sulfide (PbS) detectors for the near infrared region. The InGaAs detector bridges the gap between the PMT and PbS switching wavelength, where sensitivity is typically low, to ensure high sensitivity over the entire measurement wavelength range.

The CNTs used in this study had an average diameter of 1.7 nm and a length ranging between 100 and 500 nm.

Cell Cultures

HeLa cells were obtained from the American Type Culture Collection and maintained using established procedures. Cells were normally grown in 75 cm² flasks (density of 10⁶) with F-12K medium containing 10% fetal bovine serum, 1% penicillin (500 units/ml) and streptomycin (500 units/ml) at 37°C in 5% CO₂ atmosphere then subcultured by trypsinization for further experiments. The cells were kept in aseptic conditions and the media was changed every 48 h.

Panc1 human cancer cell lines were obtained from the American Type Culture Collection. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal calf serum, 2% l-glutamine, 1% penicillin and 1% streptomycin stock solutions. The medium was changed every 3 days and the cells were treated by trypsinization before confluence.

Treatment Protocol

The cells were seeded at a density of 1 × 10⁴ cells/well in 48-well plates. Following treatments with CNTs (24 h), the combined action was studied by adding appropriate volumes of etoposide or vehicle as control to obtain the desired final concentrations. Incubations continued for additional 6 h before staining or harvesting of the cells for further analysis. Stock solutions of etoposide (75 × 10⁻⁶ M) and CNTs (10 and 20 µg/ml) were prepared and delivered separately and together to the cell cultures for treatment. All treatments, including controls, were performed in triplicate samples.

Cell Assessment with Light Microscopy

For microscopic studies, cells were grown on 35-mm plates at a density of 25 × 10⁴ cells/dish and supplemented by the CNTs. Following the incubation methods described previously, the cells were thoroughly washed with 10 mM phosphate buffered saline (PBS, pH 7.4) three-times and then fixed with 10% formaldehyde solution for 10 min, washed three-times with PBS and stained with methyl-green dye for 10 min. The cells were monitored by light-transmission microscopy using an Olympus BX 51 microscope.

Cell Viability Analysis

The percentage cell viability was measured by trypan blue dye. First, the cells were cultured as described earlier for 24 h and exposed to various treatments. The cells were then dissociated from the bottom of the plate by trypsinization and transferred to 1.5 eppendorf tubes and centrifuged. Finally, 25 µl of trypan blue dye was added to each sample and incubated for less than 5 min. The number of viable cells was counted using a hemacytometer.

Caspase-3 Assay

Caspase-3 assay was used for the assessment of possible apoptosis and it was performed using the Biovision CaspGLOW™ Red Active Caspase-3 staining kit. The HeLa cells were incubated with and without the CNTs overnight (24 h). The etoposide was then added and incubation continued for additional 6 h. The cells were collected by scrapping and were transferred to 1.5 eppendorf tubes, incubated with 1 µl of the Red-DEVD-FMK for 1 h at 37°C with 5% CO₂, and then centrifuged for 5 min at 3000 r.p.m. The supernatant was removed carefully and the cells were resuspended in 50 µl of buffer and centrifuged again. Finally, the cells were resuspended in 100 µl of the washing buffer and a few drops of the cell suspension were transferred to the microscopic slides in order for the brightness of the red stain to be measured and analyzed. The fluorescence brightness level of the red stain indicates the caspase-3 activation level in the cells.

Flow Cytometry

Flow cytometry was performed following staining of cells with YO-PRO-I and propidium iodide (PI) dyes from a Vybrant kit # 4 (V-13243, Molecular Probes) to assess both apoptotic and necrotic cells. The cells were seeded at a density of 1 × 10⁶ cells/25 cm² culturing flask. Dimethylsiloxide was used as vehicle control while etoposide was used as the positive control for analyzing the combined effect of the etoposide-CNT treatment. After 6 h of additional treatment, cells were trypsined and washed twice with cold PBS, pH 7.4. Aliquots of 1 µl YO-PRO-I stock solution (component A) and 1 µl PI stock solution (component B) were mixed per ml of cell suspension. After 30 min of incubation at 4°C, the cells were analyzed using a BD Bioscience FACSCalibur™ flow cytometer to sort out the cells undergoing apoptosis or necrosis from the normal population, based on labeling with each fluorescence probe. Fluorescence emissions were measured at 515-545 nm for fluorescein isothiocyanate using FL-1 PMT detector and 564-606 nm for PI using FL-2 PMT detector.

Statistical Analysis

All data were expressed as mean ± standard deviation. Independent sample t-test was performed for two group comparisons. P-values of 0.05 or less were considered to indicate significance.

Nanomedicine. 2009;4(8):883-893. © 2009  Future Medicine Ltd.

Cite this: Synergistic Enhancement of Cancer Therapy using a Combination of Carbon Nanotubes and Anti-tumor Drug - Medscape - Dec 01, 2009.