2.6. Cytotoxicity assay and uptake test

The in vitro cytotoxicity of HES-10-HCPT-SS-Ly micelles was evaluated by MTT assay and Hep-G2 cell lines were used as model object. The Hep-G2 cells were seeded by using 96-well plates and the cell density was 6 × 10³. The temperature of cell culture was 37 °C and the concentration of CO₂ was 5%. The culture medium was removed after 24 h and the micelle solutions with different concentrations are added. The incubation time was 48 h and 72 h respectively to fully evaluate the relationship between cytotoxicity and culture time. Then, 50 μL MTT (0.25 mg/mL) was added to each well and the supernatant was removed. The precipitated formazan crystals were dissolved by DMSO solution after 4 h. The OD values were determined by using an ELIASA reader and the detection wavelength was 492 nm. In addition, cellular uptake of HES-10-HCPT-SS-Ly micelles was also assessed in Hep-G2 cell line. Similarly, the culture medium was removed and different 10-HCPT preparations were added with concentrations of 1.0 mg/mL 24 h after incubation. The 96-well plate was incubated at room temperature on a plate shaker for 24 h, the cells were then washed three times with PBS solution. Briefly, cells were collected and lysed using a French Pressure Cell, the lysate was clarified by centrifugation, and the supernatant liquid was processed by applying liquid–liquid extraction. Then, the concentration of 10-HCPT in Hep-G2 cells was determined by HPLC–MS/MS method.