2.5. Antiproliferative (cell viability) assay

HeLa Cells were grown in 96-well tissue culture plates (100 μl/well) at a concentration of about 15,000 cells/well using complete tissue culture medium. The media were removed after 24 h and the attached cells were treated in triplicates with 200 μl of 0.05 mg/ml of five different samples of suspended NPs as well as free drugs combination and incubated for 24 h. The amount of free drugs was equivalent to the amount encapsulated into NPs. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was then performed to measure the cell viability. From each well, 100 μl of culture media was removed and 10 μl of thiazolyl blue tetrazolium solution was added and the plates were then kept in the incubator for 3 h. The reaction was then stopped by adding 100 μl/well of MTT solubilization solution and incubated for another one hour. Absorbance was measured at 550 nm by microplate reader and the cell viability (% survival) was calculated and used to get the percentage of cell toxicity.