2.8. Cytotoxicity Assay

CrFK cells at a density of 2 × 10⁴ were incubated in 96-well culture plates for 24 h at 37°C. Then, the cells were washed with PBS. Twofold serial dilutions of curcumin, Cur-CS nanoparticles, and chitosan nanoparticles were added to the semiconfluent cells and incubated for 24, 48, and 72 h. A colorimetric MTT assay was performed. Briefly, 20 μL 5 mg/mL MTT solution was added to each well and incubated for 2 h at 37°C. Then, 170 μL solution was removed from each well; subsequently, 100 μL DMSO was added to dissolve the formazan crystals. The plate was then incubated for 1 h before optical density (OD) measurement at 570 nm using a plate reader (BioTek EL800, BioTek, Winooski, VT, USA).

The relative cell viability (%) of CrFK cells after treatment was calculated using the formula (OD_{570 (treated cells)}/OD_{570 (control cells)}) × 100. The median cytotoxic concentration (CC₅₀) is the concentration that reduces the absorbance of treated cells to 50% with respect to the cell controls. The CC₅₀ of the compounds was calculated by regression analysis in SPSS software version 22.0 (IBM, Chicago, IL, USA).