© 2022 Elsevier LtdIn this study, as a new synthesis method, UV light was employed as a type of cross-linking agent to control drug storage and to produce nanoparticles of different sizes and to stabilize the nanoparticles for the first time. We showed that the exposure time of the 5FU albumin solution to UV light produces differences in the size and characterization of the nanoparticles and also produces different cytotoxic effects on MCF-7 breast cancer cells. While the 5FU-A1 nanoparticles we synthesized with 1 h UV storage were approximately 43 nm, the 5FU-A2 nanoparticles we synthesized with UV storage for 3 h increased to an average of 300 nm. 5FU-A1 (IC50 value: 2.5 μg/mL) was approximately 16 times more cytotoxic than free 5FU (IC50 value 39.39 μg/mL) on MCF-7 cancer cells. Moreover, when normal HUVEC cells are treated with 5FU-A1 at a concentration of 2.5 μg/mL, more than 80% of these normal cells remain viable. In addition, we examined the rate of early-to-late apoptosis and necrosis in MCF-7 cancer cells using the Annexin V/PI flow cytometry assay. According to our results, 5FU-A1 promoted the apoptosis pathway. Finally, we examined P-gp activity with MDR1/ABCB1 antibody by flow cytometry and Rhodamine123 with fluorescent dye.