Amphotericin B/Sterol Co-loaded PEG-Phospholipid Micelles: Effects of Sterols on Aggregation State and Hemolytic Activity of Amphotericin B

ABSTRACT Purpose To elucidate the effect of sterols on the aggregation of amphotericin B (AmB) in PEG-phospholipid micelles and its consequences on the hemolytic activity of AmB. Methods AmB-incorporated PEG-phospholipid micelles co-loaded with ergosterol, cholesterol, or 7-dehydrocholesterol were prepared at 4:1:1 and 20:5:1 ratios of polymer-to-sterol-to-AmB. The aggregation state of AmB was elucidated by UV–vis spectroscopy. AmB/sterol co-loaded PEG-phospholipid micelles were incubated with red blood cells and the hemolytic activity of AmB assessed by measurement of free hemoglobin. Results AmB in PEG-phospholipid micelles stayed mostly in a deaggregated state in the absence of sterol or with cholesterol, but aggregated in the presence of ergosterol or 7-dehydrocholesterol. The fraction of aggregated AmB in PEG-phospholipid micelles was lower at the 20:5:1 ratio. In an aggregated state or in the absence of sterol, AmB caused rapid and complete hemolysis. In contrast, deaggregated AmB co-loaded with cholesterol caused slower and incomplete hemolysis, especially at a 20:5:1 ratio. Conclusions The aggregation state of AmB in PEG-phospholipid micelles was sterol dependant. AmB/cholesterol co-loaded PEG-phospholipid micelles caused low in vitro hemolysis due to deaggregation of AmB and micellar stability, presumably owing to cholesterol/phospholipid versus cholesterol/AmB interactions in the interior core region.