2.6. MTT Assay

HepG2 cells (human hepatocellular carcinoma cells) were cultivated in DMEM with 10% FBS, streptomycin (100 μg) and penicillin (100 units/mL) at 37 °C in a humidified atmosphere containing 5% CO₂. HepG2 cells were seeded in a 96-well plate with a density of 1 × 10⁴ cells/well and cultured with DMEM for 24 h. Blank micelles, DOX-loaded micelles and free DOX were dissolved in DMEM with a series of given concentrations. The fresh medium or pre-prepared sample solution was added in the plate and cultured for 24 h or 48 h. Next, 20 μL of MTT solution (5 mg/mL) was added into each well and subsequently incubated for 4 h. The medium was removed and replaced by DMSO, which was used to dissolve the internalized purple formazan crystals. A microplate reader was used to detect the solution absorbance at 570 nm. The cytotoxicity test was performed in replicates of six wells. The relative cell viability (%) was calculated with the following equation:

where Ablank is the absorbance at 570 nm without cells, and Acontrol and Asample are the absorbance at 570 nm in the absence and in the presence of sample treatment, respectively.