2.5.1 Hemolysis assay

To evaluate the hemolytic activity of the nanobioconjugates, erythrocytes isolated from the freshly drawn blood of a healthy human donor were exposed to the nanostructures. The blood sample was centrifuged at 1800 rpm for 5 min to obtain the erythrocytes, and the plasma (supernatant) was discarded. After the precipitation of the erythrocytes, they were washed thrice by cyclically centrifuging and resuspending them in PBS (1X). A stock containing 1 ml of the isolated erythrocytes and 9 ml of PBS (1X) was prepared upon washing the erythrocytes. The nanobioconjugates were serially diluted in PBS (1X) from 200 to 12.5 μg/ml. Triton X-100 (1% (v/v)) and PBS (1X) were selected as positive and negative controls, respectively. After serially diluting the nanobioconjugates, 100 μl of each treatment were seeded with 100 μl of the erythrocytes and incubated at 37°C, 5% CO₂ for 1 h. After the incubation, samples were centrifuged at 1800 rpm for 5 min, and 100 μl of the supernatant were placed in a 96-well microplate to read their absorbance at 450 nm via a Multiskan FC microplate reader (Thermo Fisher Scientific, Waltham, MA, United States). Hemolysis percentage was calculated as indicated in Eq. (1):