Affinity of amphotericin B for phosphatidylcholine vesicles as a determinant of the in vitro cellular toxicity of liposomal preparations.

Candida albicans and human erythrocytes were treated with liposomal amphotericin B (AmB) obtained by incubation of free AmB with small unilamellar vesicles (SUV) composed of unsaturated fatty acyl chains phosphatidylcholine (egg-yolk PC). Cellular effects were determined by changes in the K+ internal content of cells and in the number of colonies formed by fungal cells or as hemolysis, measured as a decrease in haemoglobin retention by erythrocytes. Dose-response curves were obtained by two procedures: either the ratio of AmB to phospholipids was kept constant over the AmB concentration range used (R = 10(-2] or the phospholipid concentration was kept constant (C = 0.2 mM) and the concentration of AmB varied. The liposomal preparations of AmB were as active against fungi as AmB in dimethylsulfoxide but less active (internal K+ changes) or inactive (hemolysis) against erythrocytes. On the other hand the binding of AmB to the SUV, as a function of the AmB concentration, was monitored by circular dichroism, fluorescence and UV absorption, in the two conditions used for the cellular studies. The amount of AmB bound when the total concentration of antibiotic was 2.10(-7) M was very low but increased with concentration and reached 90% at 10(-5) M. In all the assays we used, the anticellular effects could be attributed to the levels of AmB remaining free (unbound to the lipids). The variations of these levels with total AmB concentration could therefore explain the increased selectivity of liposomal AmB in toxicity against fungi and erythrocytes as compared to that of AmB added as a solution in dimethylsulfoxide. Indeed fungal cells are sensitive to low concentrations of AmB in dimethylsulfoxide; at these concentrations, in liposomal preparations, AmB is not bound to phospholipids and therefore as active as if added in dimethylsulfoxide. By contrast erythrocytes are only sensitive to much higher concentrations of AmB in dimethylsulfoxide; at these concentrations AmB is almost totally bound to phospholipids and therefore much less active.