Chemicals
Tetrachlorauric acid (HAuCl4.3H2O), Trisodium Citrate salt, Dimethylsulphoxide (DMSO),RPMI-1640 medium,Sodium bicarbonate,Trypan blue, Fetal Bovine Serum, Penicillin/Streptomycin,Trypsin,Acetic acid, Sulphorhodamine-B (SRB),Trichloroacetic acid (TCA), Tris base 10 mM (PH 10.5) are obtained from Sigma Aldrich Chemical Co., St.Louis, Mo, U.S.A.
Preparation of AuNPs
AuNPs were synthesized according to the standard wet chemical method [14, 15]. Trisodium citrate (38.8 mM, 10 mL) was added to a boiling HAuCl4 solution (1 mM, 100 mL). In addition, the yellow-colored solution of gold chloride turned the wine red in color. The formed particle size and shape were investigated using a TEM and UV–Visible spectrophotometer. In order to make sure that the cytotoxic effect is due to a laser-induced modification to AuNPs rather than other influences from the surrounding medium, absorption spectra were measured on AuNPs in culture media RPMI containing fetal bovine serum. The photostability of 1mMAuNPs has been studied by irradiation with DPSS (Diod Pumped Solid State) laser 532 nm and 250mW. The absorption spectra of the solution have been measured before exposure, and then it is irradiated with a laser source for 2 min, 4 min, 6 min, 8 min and10min. The absorption spectra have been measured after irradiation to monitor any change in the absorption spectra.
Preparation of Dox@AuNPs nanocomposite
Dox-AuNPs nanocomposite was made according to Vaithilingam method [16]. 1 ml of different concentrations of Dox (10, 20, 30, 40 μM) were mixed drop-wise with 1 ml of 0.125 mM of AuNPs with continues stirring and sonicate for 10 min until deep red becomes blue.
Characterization of AuNPs and Dox@AuNPs nanocomposite
The prepared AuNPs and Dox@AuNPs nanocomposite were characterized by UV–visible absorbance spectra using a double beam spectrophotometer (PG instrument, T80+, UK.). 200 μl from AuNPs and Dox@AuNPs nanocomposite were diluted to 2 ml with distilled water then placed in 1 cm UV-quartz and the absorption was recorded within the appropriate scan range (200 to 800 nm). The spectra were taken against distilled water as a pure solvent reference for each sample. The morphology of the prepared AuNPs and Dox@AuNPs nanocomposite carried out using TEM—Nanotechnology& Advanced Material Central Lab. (NAMCL), Agriculture Research Center (ARC). Company name: FEI, Netherland. Model: Tecnai G20, Super twin, double tilt, and Applied voltage: 200 kV, Magnification Range: up to 1,000,000 X and Gun type: LaB6 Gun. A drop from very dilute solutions were deposited on an amorphous carbon-coated copper grid and left to evaporate at room temperature forming a monolayer then detected by TEM.IR measurements were carried out using FT-IR spectrometer (Shimadzu FT-IR 8400) in the range (500–4500 cm−1). Prepared samples (free DOX and DOX-AuNPs) were dried using a lyophilizer.IR spectra of powdered samples were diluted with a potassium bromide (KBr) pellet. Where 4–8 mg from the dried AuNPs were added to 200 mg KBr then careful grinding of the sample was of great importance for the elimination of errors caused by scattering. then the particle size and surface charges of the prepared AuNPs were analyzed through DLS with Zeta sizer 300 HAS (Malvern Instruments, Malvern, UK) based on photon correlation spectroscopy. Analysis time was 60 s and the average zeta potential was determined. The zeta potential of nanoparticulate dispersion was determined as such without dilution.
Photothermal therapy on breast cell line
This method was carried out according to that of Skehan et al. (1990) [17, 18]. Cells were incubated with different concentrations of AuNPs (0.125, 0.25, 0.375, 0.5 mM) and then completed to a total of 200 μl volume/well using a fresh medium, and incubation was continued for 24 h. Cells was were obtained from the American Type Culture Collection (ATCC, Minnesota, USA). The tumor cell line maintained at National Cancer Institute (NCI), Cairo, Egypt. For each drug concentration, three wells were used. For the laser irradiation experiment, the DPSS (Diod Pumped Solid State laser) with wavelengths 532 nm and 250 mW was chosen (due to wavelength overlapping with the absorption band of the AuNPs). The microplate was divided as follows: Cells were seeded in 96-well microtiter plates at a concentration of 5 × 103 cell /well in a fresh medium and left to attach to the plates for 24 h. The prepared 0.125 mM AuNPs were added leaving definite numbers of wells as control. The cells were left to grow overnight in the presence of AuNPs. In this way, the nanoparticles are incorporated inside cells by the endocytosis process. Cells were incubated with 1 mM AuNPs and then exposed to laser light at 250 mW for different times: 2, 4, 6, 8, and 10 min. Cells without nanoparticles have been exposed to laser light at 250 mW for the same different times to test the photothermal stability of the cell themselves. Following 24 h treatments, the cells were fixed with 50 μl cold 50% trichloroacetic acid for 1 h at 4 °C. Wells were washed 5 times with distilled water and stained for 30 min at room temperature with 50 μl 0.4% SRB dissolved in 1% acetic acid. The wells were then washed 4 times with 1% acetic acid. The plates were air-dried and the dye was solubilized with 100 μl/well of 10 mM tris base (ph 10.5) for 5 min on a shaker (Orbital shaker OS 20, Boeco, Germany) at 1600 rpm. To test for the potential cytotoxicity of Dox and Dox@AuNPs on the MCF7 cell line, cells were incubated with different concentrations of free DOX ((10, 20, 30, and 40 µM)) and Dox@AuNPs nanocomposite with the same concentrations then completed to a total of 200 μl volume/well using fresh medium and incubation was continued for 48 h. For each drug concentration, three wells were used. Following 48 h treatment, the cells were treated as previously described. The optical density (O.D.) of each well was measured spectrophotometrically at 564 nm with an ELIZA microplate reader (Meter tech. Σ 960, U.S.A.). The mean background absorbance was automatically subtracted and means values of each nano concentration were calculated. The percentage of cell survival was calculated as follows:
$$ {\text{Survival fraction }} = {\text{ O}}.{\text{D}}. \, \left( {\text{treated cells}} \right)/{\text{ O}}.{\text{D}}. \, \left( {\text{control cells}} \right). $$
The IC50 values (the concentrations of thymoquinone required to produce 50% inhibition of cell growth) “the experiment was repeated 3 times for each cell line.”.
Statistical analysis
Data are expressed as the arithmetic mean ± SD. Statistical analysis was carried out using GraphPad Software Prism v5 (San Diego, USA). The statistical analysis of the transfection assay data was done using Tukey multiple comparison test with a single pooled variance. Differences were considered statistically significant when p ≤ 0.05.