Chemicals, materials and media
Dimethylformamide (DMF), hydrochloric acid, Sodium hydroxide, silane a174, and Phosphate-buffered saline (PBS) were bought from Merck Company (Germany). Fetal Bovine Serum (FBS) and RPMI medium were purchased from Gibco Company (USA), MTT dye [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide], dimethyl sulfoxide (DMSO), Graphene quantum dots, Azobisisobutyronitrile(AIBN), and Methacrylic acid 99% were procured from Sigma-Aldrich (USA). 4T1 a breast cancer cell line was prepared from the National Cell Bank of Iran Pasteur Institute (Iran). Diallyldimethylammonium Chloride (60% in Water) was purchased from Exir GmbH Company (Austria). Doxorubicin HCl was prepared from Actoverco Pharmaceutical Company (Iran). Tris buffer was prepared from AppliChem (Germany). Dialysis Tubing 28.6 mm inflated (Molecular Weight Cut-Off is 12000 to 14000 daltons) was obtained from scientific laboratories Company. All solutions were made with Deionized Water
Synthesis of reduced GQD (RGQD)
First, 100 mg of GQD was added to 5 ml of water and 2.5 ml of sodium hydroxide solution (0.1 M) and mixed at 300 rpm for 30 minutes, 20 mg of NABH4 (0.528mmol) was added, The pH was adjusted to 10 with a solution of hydrochloric acid or sodium hydroxide (0.1M) The above solution was mixed for 3 hours at 80 ° C under reflux conditions. The temperature of the solution was reduced to room temperature and the pH was adjusted to 7 with hydrochloric acid (0.1M) .
Synthesis of silane-RGQD
200 mg of silane a174 (0.805mmol) was added to the RGQD solution under nitrogen gas and mixed at room temperature at 300 rpm for 24 hours, then 50 ml of 96% ethanol was added to the solution. The mixture was centrifuged and the resulting precipitate was dried at room temperature.
Synthesis of PMA-DDA-DOX
Finally, silane-RGQD was added to 100 ml of DMF and sonicated for 15 minutes. Then 500 mg methacrylic acid (5.80 mmol), 70 mg DOX (0.120 mmol), 5.5 g diallyl dimethyl ammonium chloride (60% in water) (20.41 mmol anhydrous) and, 200 mg AIBN (1.21 mmol) was added to the above solution under nitrogen gas at the temperature of 70 to 75 Cº and reflux conditions. After 24 h, the temperature was reduced and the nitrogen gas was cut off, the resulting mixture was centrifuged and kept in 20 mL of 20 mmol TRIS buffer. Also, to evaluate the biological properties of PMA-DDA-DOX, a nanocomposite without DOX was once again synthesized, which was labeled PMA-DDA. Preparation of PMA-DDA-DOX route was shown in figure 1.
Figure 1. Preparation of PMA-DDA-DOX.
Animals ethics statement
Mice with the following characteristics were prepared from Pasteur Institute (Iran). Female adult BALB /C to 8 weeks, Storage conditions: 12h light/12h dark and 24º C with enough water and food.
4T1 cancer cells
4T1 cancer cells were obtained from the tumor cell bank of Pasteur Institute (Iran) which were maintained according to supplier instructions. 4T1 cells were harvested at exponential growth phase from culture, rinsed with sterile PBS two times, counted, and resuspended in PBS before running subcutaneously on the female BALB/C mice’s right flank.
In vivo study
First, Mice were anesthetized with ketamine and xylazine (100 and 10 mg/kg/BW, respectively) by intraperitoneal injection. Then 200 μl of the viable cells with concentration (4×107/mL) were injected subcutaneously into BALB/C mice’s flank area. A 29 gauge syringe needle was used for injection
The structure of (PMA-DDA-DOX) was evaluated using FT-IR (FTIR; 6700 Thermo Nicolet) spectroscopy. After drying PMA-DDA-DOX at room temperature, PMA-DDA-DOX spectra were obtained using KBr pellets in the range of 400-4000 cm-1
The morphology and particle size of PMA-DDA-DOX were analyzed by FESEM (TESCAN Model: MIRA III, Czech Republic) with a field emission gun based on the standard protocol.
Evaluation of DOX release kinetics from PMA-DDA-DOX
To evaluate the release kinetics of DOX from PMA-DDA-DOX, a UV-Vis spectrometer (Varian-Cary100, Australia) was used. 50 mg DOX was exposed to 20 ml PMA-DDA-DOX for 5 hours, the solution was transferred to a dialysis tube, and every 4 hours; Adsorption of the solution around the dialysis tube was taken at a wavelength of 520 nm. (520nm was the λ maximum DOX determined by UV-Vis spectrometer) .
In vivo fluorescence imaging
A BALB / C mouse with a tumor size of 100 mm3 was randomly selected. After anesthesia, 200 μl of PMA-DDA-DOX was injected intraperitoneally. After two days, in vivo imaging (KODAK In-Vivo Imaging System FX Pro (Carestream Health Inc., New Haven, CT, USA)) was used to monitor the release of PMA-DDA-DOX. These images were taken at the excitation wavelength of 400 nm and the emission wavelength at 535 nm.
To assess the toxicity of (PMA-DDA, PMA-DDA-DOX, and DOX), 4T1 cell line was cultured at 1 105 cell/well in 96-well plates for 24h under optimal conditions (37 , 5% CO2 in humidified incubator). In the next step, the growth media (10% FBS) was removed and the cells were washed two times with PBS. A new maintenance RPMI medium (10% FBS) containing 0.05, 0.5, 5, 50, and 500µg/mL was added to all samples, and the cells were incubated for 48h. Triple wells were analyzed for each concentration and column elution buffer was selected as the control. A 10μL solution of freshly prepared 5mg/mL MTT in PBS was added to each well and allowed to incubate for an additional 4 hours. The media was removed and DMSO was added at 100µL/well. Plates were shaken gently to facilitate formazan crystal solubilization. The absorbance was determined at 545 nm with a microplate reader (STAT FAX 2100, BioTek, Winooski, USA). The percentages of cell toxicity and half-maximal inhibitory concentration (IC50) were calculated . The percentage of cell viability was calculated as follows:
Fresh red blood cells (RBC) in a healthy person were used for this analysis. Then, 15% (vol/vol) suspension of RBCs was prepared and diluted 1: 20 in PBS. Next 100μL of a 2-fold serial dilution series of the agent (PMA-DDA) was added in triplicate to 100μL of RBCs suspension in a 96-well plate and the plate was incubated for 1 h at 37°C. After centrifugation at 3,000 rpm for 10 min, the percentage of hemolysis was determined by measuring the absorbance at the wavelength of 414 nm of the 150μL of the supernatant. Negative control and 100% hemolysis were determined in PBS buffer and the presence of 1% Triton X-100, respectively. Finally, the percentage of hemolysis was calculated as follows: