In vitro hemolysis test

For hemolysis test of FA-ALNBs, 1 mL whole blood was collected from a healthy mouse. Through centrifugation, the red blood cells (RBCs) were separated. FA-ALNBs at different concentrations in PBS were incubated with RBCs. After 1 hour incubation at 37°C, the treated RBCs were centrifuged at 10,000 rpm for 1 minute. The absorbance of the supernatants was detected by UV-vis spectrophotometer at 541 nm. The hemolytic percent (HP) was calculated according to the following equation:

where A_t, A_pc, and A_nc are the absorbance value of the supernatant of the test sample, water-treated sample (positive control), and PBS-treated sample (negative control), respectively.

First, FA-LNBs with the concentrations of 0, 50, 100, 250, and 500 µg/mL were cultured with 4T1 cells. After 24 hours treatment, the cells were treated with RPMI-1640 media containing 10% CCK-8 for 20 minutes in an incubator. The absorbance of the cells at 450 nm wavelength was detected by a Multimode Plate Reader (EnVision; PerkinElmer) to characterize the cell viability. In addition, Arte, ALNBs, and FA-ALNBs with the same concentrations of Arte (0, 10, 20, 30, and 40 µg/mL) with or without 1 minute US irradiation were treated with 4T1 cells for 6 hours. After that, the cells were incubated for further 24 hours. The cell viability was also evaluated by CCK-8 assay.