*2.6. Cytotoxicity Studies*

For the cytotoxicity study, human colon carcinoma cell lines (Caco2) were used. Cells were cultured in Eagle's minimum essential medium supplemented with 2 mM of glutamine, 20% fetal bovine serum (FBS), 1.5 g/L of sodium bicarbonate, 1 mM of sodium pyruvate, and 0.1 mM of nonessential amino acid. Cells were equilibrated with 5% CO2. Growing temperature was set at 37 ◦C in an incubator allowing humidified air to pass through. Cytotoxicity studies of the 5-FU solution, CS-5-FU-NPs, and FA-CS-5-FU-NPs were performed on the Caco-2 using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. First, 5 × <sup>10</sup><sup>3</sup> cells were seeded in 96-well plates and incubated for 24 h without drug/formulations. The cells were then treated with the 5-FU solution, CS-5FU-NPS, and FA-CS-5FU-NPS, and were then incubated for 24 h. The cells in the absence the 5-FU solution, CS-5FU-NPS, and FA-CS-5FU-NPSNPs were considered as the control group. The MTT solution was added to assess the cytotoxicity of drug and nanoparticles. Cells were incubated for 4 h in MTT solution followed by the addition of DMSO to dissolve formazan and quantified spectrophotometrically using a microplate reader (Thermo Varioscan Multiplate Reader).
