In vitro Hemolytic Assay

The in vitro analysis of the inhibition of erythrocyte hemolysis was performed as reported by Wang et al.³⁹ Blood samples (10 mL) were obtained from healthy volunteers in prefilled K₂-EDTA tubes. The collected blood samples were centrifuged for 10 min at 1500 rpm to separate the erythrocytes (RBCs). The RBCs were then washed three times with phosphate-buffered saline (PBS, pH 7.4) to eliminate serum proteins and debris. The transparent supernatant after each centrifugation was removed carefully and discarded. A 50% v/v suspension of RBCs was prepared in PBS and stored at 4 °C for ≤ 48 h. Fifty microliters of RBC suspension was added to PBS (950 µL) containing different concentrations of HePC-NLCs or HePC alone, for the purpose of assessing hemolysis. Triton X-100 (1% v/v in water) and PBS were used as the positive control (100% lysis) and negative control (0% lysis), respectively. All samples were then incubated in an Eppendorf thermomixer for 1 h. The mixing frequency and temperature were adjusted to 450 rpm and 37 °C, respectively. Intact erythrocytes were isolated by centrifugation at 10,000 rpm for 5 min. Finally, the absorbance at 540 nm of the supernatant was measured. The following equation was used to determine the percent hemolysis: