The hemolysis test was carried out according to the standard methods described in the Chinese Pharmacopoeia (2020). Briefly, fresh blood was collected from a New Zealand rabbit through a marginal ear vein. A 2% red blood cells (RBCs) suspension was obtained after fibrinogens were removed by slowly stirring using a glass rod and diluting with 0.9% NaCl solution. Then, 0.15 mL of DOX-BSA/MnO2 NPs at various doses (25 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and 500 μg/mL) and 1.10 mL of 0.9% NaCl were added into 1.25 mL of 2% RBCs suspension followed by 3 h, 37 °C water bath incubation. Purified water and 0.9% NaCl incubated with RBCs presented the positive and negative controls, respectively. The entire sample was then subjected to 300 rpm centrifugation for 10 min. Therefore, 200 µL of the supernatant was taken on a 96-well plate, and its absorbance (A) was assessed at a wavelength of 540 nm with a microplate reader (Model 680, Bio-Rad, United States). The hemolysis percentage was calculated as follows:
Asample, A0, and A100 refer to the sample absorbance values, negative control, and positive control groups, respectively.
Asample, A0, and A100 refer to the sample absorbance values, negative control, and positive control groups, respectively.
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