MTT Assay and Combination Index Calculation

HT-29 cells, SW480 cells, and CCD-18 cells were seeded in 24-well plates (1×10⁴ cells/well) in 300 μL of supplemented DMEM. MTT assay was used to determine the cytotoxic effects of nano-systems against cancer or normal cells.38 Briefly, after 24 hours of culture, the cells were treated with drug-loaded nano-systems and free drugs at certain concentrations for 24 hours. Subsequently, MTT was added to the treated cells for 3 hours, with added DMSO, and then placed in the cells for 15 minutes. Finally, the absorbance at 570 nm was measured. The half maximal inhibitory concentration (IC₅₀) values of all the formulations were calculated.

The combination index (CI) values were determined by IC₅₀ and calculated by the median-effect equation as described by Chou and Talaly39,40 and Palko-Łabuz et al. The value of CI<1 represents drug synergy, CI>1 indicates drug antagonism, while an additive effect is observed when CI=1.