2.6. In vitro cytotoxicity assay

MTT was used to evaluate the cytotoxicity of the different drug-loaded nanoparticles (Hao et al., 2021). Briefly, after treating the cells with blank nanoparticles, free DOX, DOX@NP, DOX@HNP, and DOX@BHNP at different DOX concentrations, 20 µL MTT solution was added, and the resulting formazan product was dissolved in DMSO solution. The optical density (OD) value at 570 nm was measured using a microplate reader (Bio-Rad 550, Hercules, CA). The OD value of the cells incubated in culture medium containing 10% fetal bovine serum (FBS) was also measured as a control. In addition, after the tumor cells were incubated with DOX@BHNP for 4 h, mitochondrial morphology was observed via bio-TEM. Specific details about this experiment are provided in the SI.