In vitro cytotoxicity and transfection assay
The in vitro cytotoxicity assay for blank nanoparticles (NPsA, NPsB and NPsAB) and siRNA-FAM/PTX encapsulated nanoparticles (NPsA/siRNA/PTX, NPsB/siRNA/PTX and NPsAB/siRNA/PTX) was evaluated on MCF-7 cell lines. The cells were seeded in 96-well plates at a density of approximately 7000 cells per well, in 100 µL of complete growth medium (RPMI 1640 culture medium supplemented with 1% Penicillin/Streptomycin and 10% FBS) and were incubated at 37°C in 5% CO2 for 24 hours. Then the cells were exposed to various concentrations of the blank nanoparticles (100, 200, 400, 600, 800 and 1000 µg/ml) for 48 hours at 37°C. Next, each well was supplemented with 20 µL of MTT solution (5 μg/mL in culture medium) for 5 hours at 37°C. The supernatant was removed and 200 µl of dimethyl sulfoxide (DMSO) was added per well to dissolve the Formazan crystals. Following this step, the plates were incubated at 37°C for 30 minutes. Next, the absorbance level at 570 nm was recorded by a multiwall plate reader (BioTek Instruments; Winooski, VT, USA) (Qian Wang et al., 2017). Finally, the percentage for the cell viability was determined according to the following Eq. (3):
Cell viability (%) =\(\ \frac{OD\ of\ each\ sample\ at\ 570\ nm\ }{OD\ of\ negative\ control\ at\ 570\ nm}\)×100 (3)
The in vitro cytotoxicity assay for the nanoparticles encapsulated with siRNA-FAM/PTX (NPsA/siRNA/PTX, NPsB/siRNA/PTX and NPsAB/siRNA/PTX) was evaluated with equal amount of PTX (equivalent PTX concentration of 40 nM for each sample) in each sample on the MCF-7 cell lines as described above. To investigate the transfection ability of nanoparticles, the cells were incubated with NPsA/siRNA/PTX, NPsB/siRNA/PTX and NPsAB/siRNA/PTX, then were washed twice using PBS, and the siRNA-FAM transfection efficiency was determined using an inverted-fluorescent microscope (Nikon TE200).