2.5. Cytotoxicity Assay

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess the cytotoxicity of ACA and the NLC formulations on PC-3 prostate cancer cells and RWPE-1 normal prostate cells. Into 96-well culture plates, 1×10⁴ cells/well were seeded, and the cells were incubated at 37 °C with 5% CO₂ for 24 h. After incubation, the cells treated with different doses of ACA standalone, blank NLC, ACA-NLC, AMD-NLC, and AMD-ACA-NLC, were incubated for varying durations to observe the effects of the treatment. Next, 20 µL of (5 mg/mL) MTT (Calbiochem, San Diego, CA, USA) was added to each well, and after 90 min of incubation, all the media were removed completely before 200 µL of dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) was added to each well. The plate was kept on the shaker for 10 min. Finally, the absorbance intensity was measured via a micro-titer plate reader (Tecan Sunrise, Männedorf, Switzerland) at an absorbance wavelength of 570 nm, and a reference wavelength of 650 nm. Cell viability was evaluated using the following equation, with Int_S referring to the absorbance intensity of the treated cells and Int_Control referring to the absorbance intensity of the untreated cells: