Haemolytic activity

Haemolytic activity is a parameter suggestive of cytotoxic activity in vivo [21]. The assay was performed following previously described procedures [22,23] with minor modifications. Briefly, the test samples were suspended in sodium chloride 0.9% at a stock concentration of 6 mg/mL and prepared as previously described. An aliquot of sheep blood in Alsever’s solution liquid (Fisher Scientific, Loughborough, UK) was centrifuged at 250g for 10 min and then washed 3 times in sodium chloride 0.9%. After centrifugation, the supernatant was discarded, and 200 μL of packed erythrocytes were diluted into 7.2 mL of sodium chloride. One hundred and fifty μL per well of saline with or without the materials were added in a clear 96-well plate. Then 75 μL per well of the erythrocyte suspension were added giving a final concentrations of each material of 4, 2, 1 mg/mL. The plates were incubated for 15 min at room temperature protected from light on a plate shaker and then centrifuged at 250g for 15 min to pellet erythrocytes and materials. 100 μL of supernatant were then transferred into a new clear 96-well plate, and the absorbance was read at 540 nm with a plate reader (Fluostar Optima, BMG Labtech, Aylesbury, UK). Results were expressed as percentage haemolysis, with 0% being set for the saline control and 100% set for Triton X100 0.1% in sodium chloride, used as a positive control.