Doxorubicin dox

A variety of cell lines including HeLa, B16-F10 and LX-2 cells were used to examine the cytotoxicity of CY-1-4 free drug and CY-1-4 NPs via the Cell Counting Kit-8 (CCK-8) test kit (Beyotime Biotech, Shanghai, China). CY-1-4 free drug was prepared by stepwisely diluting the stock solution of CY-1-4 (dissolved in DMSO at a concentration of 1 mg/ml) with a blank culture medium. Moreover, classical chemotherapeutic drugs such as doxorubicin (Dox; Macklin, Shanghai, China), daunorubicin (Dau; Pharmabiology, USA) and 5-fluorouracil (5-FU; Macklin, Shanghai, China) were used on B16-F10 cells to give a comparison with CY-1-4 free drug regarding cytotoxicity. In brief, 5 × 10 3 cells per well were seeded in 96-well plates overnight. Then, the cell culture medium was removed and various concentrations of CY-1-4 free drug or CY-1-4 NPs were added to the cells. After 24 h of incubation and removing the remaining culture medium, 10 μl of CCK-8 solution and 100 μl of DMEM were added to each well of the plate and further incubated for another 2 h. Absorbance was measured with a microplate reader at 492 nm (Multiskan FC; Thermo Scientific, MA, USA). Untreated wells were used as controls. Cell viability was calculated as follows: (OD treated -OD blank )/(OD control -OD blank ) × 100%.