In vitro hemolysis assay

Hemolysis studies were carried out for empty mMic, along with different Brb-containing samples, according to previous report.30 The release of hemoglobin from the erythrocytes was used for toxicity measurements of these carriers. Briefly, defibrinated rabbit blood (Thermo Fisher Scientific) was diluted ten times with PBS and centrifuged at 2,000 rpm for 15 minutes. The supernatant was decanted, and the precipitate was rinsed three times with PBS, followed by centrifugation at 2,000 rpm for 15 minutes. The concentration of the resulting blood cells was adjusted to 2% (v/v). At a physiologically relevant dilution scenario of IV bolus administration (10 μg/mL of Brb-equivalent concentration), 50 μL of test samples were mixed with 500 μL of blood cells, and the resulting suspensions were incubated at 37°C for 3 hours. The samples were then centrifuged at 2,000 rpm for 15 minutes. The absorbance of the supernatant was measured at 540 nm to determine the amount of hemoglobin release. Zero hemolysis and 100% hemolysis consisted of red blood cells suspended in physiological saline (−ve ctrl) and distilled water (+ve ctrl), respectively.

The percentage of hemolysis was determined using the following equation:

where A_ts is the absorbance of the test sample, A₁₀₀ is the absorbance of completely lysed red blood cells in distilled water, and A₀ is the absorbance of zero hemolysis.30