CCK-8 cytotoxicity assay for nanocarriers

The cytotoxicity was determined using the CCK-8 (Beyotime, China) assay. Briefly, 4T1 or other cells (HeLa cells, J774A.1 cells, RAW 264.7 cells, HUVECs, and mouse primary macrophages) were seeded in 96-well plate at a density of 1.5 × 10⁴ cells and incubated for 24 hours to allow cell attachment. Then, the cells were cultured with different nanocarrier formulations (equivalent protein concentration ranged from 0 to 100 μg/ml) and corresponding additives for 24, 48, and 72 hours. CCK-8 test solution was added to each well of the plate (the volume of the CCK-8 test solution in each well is ¹/₁₀ of the total volume) and incubated for another 3 hours. After selective reduction by viable cells, the absorption intensity of each well at 450 nm was measured. Percentage of the viability was normalized according to the untreated cells.