TRANSPORT STUDIES OF DOXORUBICIN IN MODEL MEMBRANES INDICATE A DIFFERENCE IN PASSIVE DIFFUSION ACROSS AND BINDING AT THE OUTER AND INNER LEAFLETS OF THE PLASMA-MEMBRANE

The kinetics of passive transport of the anticancer drug doxorubicin w ere analyzed in relation to membrane composition in large unilamellar vesicles in which DNA was enclosed. Special attention was paid to lipi ds that are typical for the inner and outer leaflet of the plasma memb rane of mammalian cells: Phosphatidylethanolamine and anionic phosphat idylserine versus phosphatidylcholine, sphingomyelin, and cholesterol, respectively. The presence of anionic phospholipids results in a high ly efficient incorporation of the drug into biological and model membr anes [de Wolf, F. A., et al. (1993) Biochemistry 32, 6688-6695]. There fore, the effect of drug binding on the amount of free, transportable drug was explicitly taken into account. However, even after correction for binding the permeability coefficient was about 35% lower in membr anes containing 50 mol % of the anionic phosphatidylserine than in mem branes consisting only of zwitterionic phospholipids (0.71-0.79 versus 1.18-1.25 mu m s(-1)). This shows that drug binding and insertion als o affect: the intrinsic transport characteristics of the membranes. As compared to pure phosphatidylcholine, binding was not influenced by t he incorporation of sphingomyelin or cholesterol, but equimolar amount s of sphingomyelin and cholesterol in phosphatidylcholine membranes de creased the rate of doxorubicin transport by 60% and 80%, respectively . The inhibitory effect of these two lipids is probably due to a close r packing of the membranes. In accordance, after the acyl chain order was decreased by adding the anaesthetic-like phenethyl alcohol (0.5% v /v), transport was stimulated more than 4-fold. The implications of ou r findings for the functioning and rate of drug pumping by the multidr ug resistance-conferring P-glycoprotein in cancer cells are discussed.