In vitro cytotoxicity test

The cytotoxicity of Caco-2 and SW480 cells treated with NCs were evaluated by using MTT assay.30 Cells were seeded into 96-well microplates at a concentration of 10⁵ cells/mL and allowed to grow for 24 hours to a subconfluent state (80% confluence). The culture medium was replaced with fresh medium supplemented with 10% FBS having different concentrations of free 5-FU (5-FU) or different kinds of NCs, and samples were incubated for 72 hours with 100 µL of tested suspensions. Then suspensions were removed and 50 µL of MTT solution (1 mg/mL in culture medium) was added and cells were incubated for 4 hours at 37°C in 5% CO₂ prior to the analysis. Thereafter, the medium was removed and 100 µL of dimethyl sulfoxide was added to dissolve blue formazan crystals. The absorbance of the formed dye was measured at 570 nm using a microplate reader. Absorbance values for untreated cells were taken as control (100% survival). Cell viability was calculated according to the equation: (absorbance of test cells)/(absorbance of control)×100.