2.8. Hemolysis Assay

For the hemolysis test, blood samples were collected from the rats; then, erythrocytes were obtained by 10 min centrifugation at 3500 rpm and 4 °C, and washed with sterile isotonic PBS three times. One hundred microliters of erythrocytes were incubated with PTX-NaHCO₃-PLGA NPs containing different drug concentrations (0, 8, 16, 32, 64, 128, and 256 μg/mL) for 4 h at 37 °C. The cells were then centrifuged for 3 min at 3500 rpm and 4 °C, the supernatant was collected, and the absorbance at 541 nm was read by UV-visible spectrophotometry. Erythrocytes dispersed in PBS were used as positive controls and erythrocytes dispersed in deionized water served as a negative control. The hemolysis rate was calculated using the following formula: