In vitro Cytotoxicity Assay

Briefly, 100 μL of DMEM was added to the peripheral wells of a 96-well tissue culture microtiter plate, and 100 μL of HepG2 cell suspension (1 × 10⁴ cells/mL) added to the remaining wells. After incubating for 24 h, the medium was removed and 100 μL of the different concentrations of stock solutions diluted with fresh medium added to each well, with 100% DMEM as a negative control (n = 3). After incubation for 72 h, 20 μL MTT solution (5 mg/m L) was added to each well and the cells incubated at 37°C for an additional 4 h. Subsequently, the MTT solution was removed, replaced with 100 μL DMSO, and the mixture shaken at a low speed for 10 min on a shaker to fully dissolve the crystalline formazan. The optical density (OD) at 540 nm of each well was measured using a microplate reader. To express the in vitro hemolytic properties of our samples, cell viability was calculated using the following formula:

Cell viability (%) = OD_570S/OD_570N × 100

where OD_570S represents the average optical density of each tested sample group, and the OD_570N represents the average optical density of the negative control group. According to the ISO standard (GB/T 16886.5–2017/ISO 10993–5:2009), cell viability was then classified according to percentage of viable cells, to reflect the degree of toxicity of the material as follows: 0, 100%; 1, 80–100%; 2, 50–80%; 3, 30–50%; 4, 0–30%.