Lipid Scrambling of Asymmetric Liposomes Induced by Membrane Active Substances

Abstract

Membrane leakage assays based on vesicles loaded with self quenching dyes have been widely used for quantifying the activity of membrane active substances that induce membrane pores or leaks. Time-resolved fluorescence decays of calcein-loaded liposomes allow a parallel quantification of the free and entrapped dye fractions and their effective local concentrations.1 Thus, different types and mechanisms of membrane leakage to aqueous solutes can be distinguished by this method. However, much less is known about another way of membrane permeabilization: the enhancement of lipid flip flop that causes a scrambling of the lipids between the leaflets. Since virtually all biological membranes show an asymmetric distribution of lipids between the two leaflets, such a scrambling should have massive impact on the cell even if it proceeds without leakage to aqueous solutes. In order to assess such activity, we utilized a recent protocol2 to produce asymmetric vesicles with anionic diacyl phosphatidyl glycerol (POPG) in the outer layer exclusively. Addition of lipid scrambling agents permits the POPG to flip to the inner leaflet, which can be monitored in terms of a less negative zeta potential of the liposomes. The study compares the activities of nonionic detergents including n-Dodecyl β-D-maltoside, n-Octylglucoside and C12EO8 to induce lipid scrambling and pore formation, respectively. References 1 H. Patel, C. Tscheka, H. Heerklotz; Soft Matter, 2009, 5, 2849-2851. 2 M. Markones, C. Drechsler, M. Kaiser, L. Kalie, H. Heerklotz, S. Fiedler; Langmuir. 2018, 34(5), 1999-2005.