THE AMINOSTEROL ANTIBIOTIC SQUALAMINE PERMEABILIZES LARGE UNILAMELLARPHOSPHOLIPID-VESICLES

The ability of the shark antimicrobial aminosterol squalamine to induc e the leakage of polar fluorescent dyes from large unilamellar phospho lipid vesicles (LUVs) has been measured, Micromolar squalamine causes leakage of carboxyfluorescein (CF) from vesicles prepared front the an ionic phospholipids phosphatidylglycerol (PG), phosphatidylserine (PS) , and cardiolipin. Binding analyses based on the leakage data show tha t squalamine has its highest affinity to phosphatidylglycerol membrane s, followed by phosphatidylserine and cardiolipin membranes. Squalamin e will also induce the leakage or CF from phosphatidylcholine (PC) LUV s at low phospholipid concentrations. At high phospholipid concentrati ons, the leakage of CF from PC LUVs deviates from a simple dose-respon se relationship, and it appears that some of the squalamine can no lon ger cause leakage. Fluorescent dye leakage generated by squalamine is graded, suggesting the formation of a discrete membrane pore rather th an a generalized disruption of vesicular membranes. By using fluoresce ntly labeled dextrans of different molecular weight, material with mol ecular weight less than or equal to 4000 g/mol is released from vesicl es by squalamine, but material with molecular weight greater than or e qual to 10,000 is retained. Negative stain electron microscopy of squa lamine-treated LUVs shows that squalamine decreases the average vesicu lar size in a concentration-dependent manner. Squalamine decreases the size of vesicles containing anionic phospholipid at a lower squalamin e/lipid molar ratio than pure PC LUVs. In a centrifugation assay, squa lamine solubilizes phospholipid, but only at significantly higher squa lamine/phospholipid ratios than required for either dye leakage or ves icle size reduction. Squalamine solubilizes PC at lower squalamine/pho spholipid ratios than PG. We suggest that squalamine complexes with ph ospholipid to form a discrete structure within the bilayers of LUVs, r esulting in the transient leakage of small encapsulated molecules. At higher squalamine/phospholipid ratios, these structures release from t he bilayers and aggregate to form either new vesicles or squalamine/ph ospholipid mixed micelles. (C) 1998 Elsevier Science B.V.