2.2.6. Effect on Cell Proliferation

The effect of 5-Fu, CrNPs, and 5-FuCrNPs on the viability of the CACO-2 cell line (Colorectal adenocarcinoma cell line, kindly supplied from VACSERA tissue culture lab, Obour City, Cairo, Egypt) was investigated using the MTT cell proliferation assay (Cell Titer 96^® Non-Radioactive Cell Proliferation Assay, Promega, Dane County, WI, USA) according to the manufacturer’s instructions. In brief, 5000 cells were plated in wells of 96-well plate in triplicates and incubated at 37 °C in a 5% CO₂ incubator (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h. On the next day, different concentrations of 5-Fu, CrNPs, and 5-FuCrNPs were added to the cells and treated cells were incubated for 24 h. Control cells were treated with PBS. After incubation, the MTT reagent was added to the cells and incubated for 4 h in the dark, followed by the addition of the stop solution to dissolve the formed crystals. The absorbance of the solution was measured at wavelengths of 570 and 630 nm (reference filter). Cell viability was expressed as the mean percentage of viability and was calculated by dividing the optical density of the treated sample/optical density of the control samples. The IC50 (the concentration of the sample that caused a 50% inhibition of cell viability) was determined from the nonlinear regression curve obtained by plotting the log concentration of the inhibitor vs. cell viability as a percentage. Each concentration of the inhibitor was tested in triplicate. Data are presented as the average IC50 for the tested inhibitory material. Growth curves and regression analysis were performed using GraphPad Prism 8.4 (GraphPad Software, San Diego, CA, USA).

In addition, the ability of 5-Fu, and 5-FuCrNPs to induce apoptosis of the CACO-2 cells was analyzed by flowcytometry using annexin V and a propidium iodide staining kit (Thermo Fisher Scientific, Suwanee, GA, USA). Cells were acquired on FACSCalibur™ flow cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed by FlowJo software 8.7 (Treestar, Ashland, OR, USA).