Unless otherwise mentioned, all chemicals were purchased from Sigma-Aldrich (St Louis, MO). The human breast cancer cell line MCF-7 and Dulbecco’s Modified Eagle Medium were obtained from American Type Cell Culture (Manassas, VA). The p53 plasmids were purchased from Addgene (Cambridge, MA). The caspase assay kit was purchased from BioVision Inc (Mountain View, CA), and the Gentamucin Reagent Solution was from MP Biomedicals, LLC (Illkirch, France).
Carbon nanotube synthesis
Single-walled carbon nanotubes were synthesized on a bimetallic Fe-Co/MgO (2.5:2.5:95 wt%) catalyst system, which was prepared as described previously.30
After a thorough overnight drying, the catalyst system was calcinated at 500°C for a few hours and about 50 mg of the catalyst was placed on a graphite susceptor, which was inserted into a quartz tube. A radio frequency (RF) generator with a frequency of 350 kHz was used in the synthesis of the carbon nanotubes. Argon (Ar) was first introduced at 150 mL/minute into the quartz tube positioned inside the RF coil. After 10 minutes, the RF generator was turned on. Once the temperature of the graphite susceptor reached 800°C, methane was added at 40 mL/minute for 30 minutes. The carbon source and the RF generator were turned off and the sample was cooled to room temperature under Ar. The produced SWCNTs were mixed into a diluted HCl (1:1) solution and the mixture was stirred continuously for 24 hours. The SWCNT mixture was washed with deionized water to a pH of 7 and dried overnight at 100°C. The purified SWCNTs were oxidized at 430°C for 30 minutes to burn off amorphous carbon and to uncover the remaining catalyst nanoparticles. A second purification of SWCNTs was carried out with a nitric acid treatment to further remove any additional metal impurities. The final product contained SWCNTs functionalized with carboxylic groups with enhanced dispersion properties in various solutions.
A Mettler Toledo (Greifensee, Switzerland) TGA/SDTA 851e was used to carry out thermogravimetric analyses (TGA) with an airflow rate of 150 mL/minute. Approximately 3 mg of the sample was heated from 25 to 850°C at a rate of 5°C/minute. Transmission electron microscopy (TEM) images were collected on a field emission JEM-2100F TEM (JEOL Inc, Tokyo, Japan) equipped with a CCD camera and an acceleration voltage of 100 kV. For TEM analysis, SWCNTs were homogeneously dispersed in 2-propanol by ultrasonication for 30 minutes. A few drops of the suspension were deposited on the TEM grid and allowed to air-dry before analysis. A JEOL 7000F high-resolution scanning electron microscope was utilized to obtain scanning electron microscopy (SEM) images of the purified powdered samples. Before microscopy, the samples were mounted on aluminum pins with double-sided carbon tape.
The optical absorption spectra at UV-Vis-NIR range were recorded using a Shimadzu (Kyoto, Japan) UV-3600 double beam spectrophotometer with three detectors. For the optical measurements, SWCNTs were individually dispersed in an aqueous sodium cholate solution. To obtain a homogeneous solution, the mixture was first sonicated for 1 hour and then centrifuged for 2 hours at 15000 × g using a high speed centrifuge and the supernatant was utilized for optical analysis. Raman spectra of the SWCNTs were collected using a Horiba Jobin Yvon (Kyoto, Japan) Model LabRam HR800 system with a He-Ne laser (633 nm) as an excitation source. The Raman shifts were calibrated with a silicon wafer at a peak of 521 cm−1.
Synthesis of amine-functionalized SWCNTs (f-SWCNTs)
In a 100 mL round-bottomed flask SWCNTs (50 mg), dichloromethane (50 mL), and m-chloroperoxybenzoic acid (1 g) were mixed and the solution was refluxed for 48 hours. The solution was then filtered to collect the epoxidized SWCNTs and washed with dichloromethane (2 × 10 mL) and ethanol (2 × 10 mL). The epoxide-functionalized SWCNTs were mixed with lithium chloride (200 mg) and freshly distilled ethylenediamine (20 mL) and the mixture was refluxed for 18 hours under an inert atmosphere. After reflux, the reaction mixture was cooled and filtered to collect the product, which was then washed several times with ethanol (3 × 25 mL) to remove any excess ethylenediamine and lithium chloride. The resulting ethylenediamine f-SWCNTs were used for functionalization with the p53 genes.
Functionalization of f-SWCNT with p53 plasmid
Protein 53 plasmids (20 μg mL−1) were added to a suspension of f-SWCNTs in Dulbecco’s Modified Eagle Medium (DMEM) medium at a ratio (p53 plasmid:f-SWCNTs) of 1:7.4 (w/w). The solution was mixed thoroughly using a pipette to develop the complex of p53 and f-SWCNTs.
Human breast cancer cells (MCF-7) were grown in a 75 cm2 culture flask with DMEM, supplemented with 10% calf serum, 100 U mL−1 penicillin, 100 U mL−1 streptomycin, and 50 μg mL−1 gentamicin sulfate and incubated in a 5% CO2 atmosphere at 37°C. The cells were subcultured by trypsinization and maintained in aseptic conditions with medium changes every 2–3 days.
Cell treatment protocol
The cells were seeded at a density of 25 × 104 cells in each 35 mm cell culture dish and grown for 72 hours in normal growth medium (as detailed above) until they reached 60%–70% confluence. The medium was then removed and cells were supplied with medium containing 20 μg mL−1 of F-SWCNTs, 20 μg mL−1–2.7 μg mL−1 F-SWCNTs-p53. A negative control was prepared by incubating the cells only with normal growth medium. The cells were harvested after 24 hours, 48 hours, and 72 hours of incubation for further analysis. All experiments were carried out in triplicate.
Cell assessment with light microscopy
The cells were plated for microscopy as described above and were washed three times with 10× phosphate buffered saline (PBS) and examined with an Olympus BX51 light microscope.
Analysis of cell death with ethidium bromide and acridine orange staining
To determine the degree of cell death, cells were harvested by trypsinization after an appropriate incubation period, were washed twice with 1× PBS buffer and stained with 17 μL of a solution containing 100 mg mL−1 acridine orange and 100 mg mL−1 ethidium bromide in PBS. The harvested cells were placed on a slide and mounted with a cover slide. The stained cells were immediately visualized with UV light using an Olympus fluorescence microscope with a 10× objective equipped with a digital camera. Images were taken of random fields of view. The percentage of apoptosis was calculated by counting the number of live (green) and apoptotic (red) cells. Acridine orange stained the live cells, thus making them to appear green, whereas the apoptotic cells’ fragmented nuclear DNA was stained by ethidium bromide and appeared red when visualized under UV light using specific light filters. Each experiment was run in triplicate and the results expressed as mean ± standard deviation.
Caspase assays were performed to assess the level of apoptosis using the Biovision CaspGLOW Red Active Caspase-3 staining kit. The cells were incubated following the previously mentioned treatment protocol. After incubation, both the control and treated cells were trypsinized and the cells were resuspended in 1 mL of normal growth medium and incubated with 1 μL of Red-DEVD-FMK for 1 hour at 37°C with 5% CO2 and then centrifuged for 5 minutes at 3000 rpm. The cells were resuspended and washed twice with wash buffer and the final pellet was resuspended in 100 μL of the wash buffer. A few drops of the cell suspension were transferred to a microscope slide, which was examined with a light microscope. The brightness level, which indicates the level of caspase activation in the cells, was analyzed for each sample.
Green fluorescent protein expression
The cells were incubated under the standard conditions using plasmids with green fluorescence protein (GFP) tagged p53. After incubation, both the control and treated cells were harvested, washed twice with 1× PBS buffer, and visualized under UV light by using an Olympus fluorescence microscope at 10× objective with an FTIC filter.
The data were analyzed by one-way ANOVA test and expressed as mean ± SD among three separate samples for each condition, the P-values of 0.05 or less were considered to indicate significance.