The in vitro hemocompatibility study assesses the impact of the test substances on the architecture and viability of erythrocytes by determining the release of hemoglobin into the plasma as a result of erythrocyte lysis after contact with a potentially toxic substance [33 (link),34 (link),35 (link)]. Evaluation of the hemolytic properties of lipid vesicles was performed through spectrophotometric quantification of hemoglobin release after exposure to them.
Fresh blood was collected from the lateral vein of the rat’s tail in heparin vacutainers under local anesthesia with 1% benzocaine. The erythrocytes were separated by centrifuging the blood at 5 °C for 5 min at 1500 RCF (relative centrifugal force) and then washed 3 times with saline. As negative control, a 2% (v/v) erythrocyte suspension was used, obtained after immersion in saline and incubated for 45 min at 37 °C. Triton X-100 10% (v/v), known to possess hemolytic activity [36 (link)], incubated for 45 min at 37 °C, was considered a positive control in the experiment. The erythrocyte suspension was incubated with the test substances for 45 min at 37 °C and centrifuged for 10 min at 1000 RCF to remove cells, and the absorbance of the supernatant (including plasma and lysed erythrocytes) was measured at 540 nm using a Hewlett Packard 8453 UV–vis spectrophotometer (Waldbronn, Germany).
The percentage of hemolysis (% hemolysis) was calculated according to the formula [37 (link)]:
Fresh blood was collected from the lateral vein of the rat’s tail in heparin vacutainers under local anesthesia with 1% benzocaine. The erythrocytes were separated by centrifuging the blood at 5 °C for 5 min at 1500 RCF (relative centrifugal force) and then washed 3 times with saline. As negative control, a 2% (v/v) erythrocyte suspension was used, obtained after immersion in saline and incubated for 45 min at 37 °C. Triton X-100 10% (v/v), known to possess hemolytic activity [36 (link)], incubated for 45 min at 37 °C, was considered a positive control in the experiment. The erythrocyte suspension was incubated with the test substances for 45 min at 37 °C and centrifuged for 10 min at 1000 RCF to remove cells, and the absorbance of the supernatant (including plasma and lysed erythrocytes) was measured at 540 nm using a Hewlett Packard 8453 UV–vis spectrophotometer (Waldbronn, Germany).
The percentage of hemolysis (% hemolysis) was calculated according to the formula [37 (link)]:
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