AFM and SPR combined studies of Cholesterol extraction from model biomembranes mediated by methyl--cyclodextrin

VAZQUEZ, ROMINA F.; VELA, MARÍA E.; DAZA MILLONE, MARÍA A.; MATÉ, SABINA

Linz

Workshop; XXI. Annual Linz Winter Workshop; 2019

We have studied the interaction of methyl-β-cyclodextrin (MβCD), a Cho-extracting drug with a ternary lipid mixture (DOPC/SM 16:0/Cho) that exhibits phase coexistence, i.e. a liquid-ordered (Lo) phase enriched in sphingomyelin (SM) and cholesterol (Cho) which is segregated from the liquid-disordered (Ld) phase composed mainly of DOPC. This ternary lipid mixture with lipid raft-like domains mimics the outer leaflet of human erythrocytes. MβCD has a characteristic cylindrical structure with a hydrophilic exterior and hydrophobic central cavity and can form inclusion complexes with Cho but the mechanism of its extraction from cellular membranes is not completely understood.In this work, we present results from Surface Plasmon Resonance (SPR) and Atomic Force Microscopy (AFM) of the interaction of MβCD with ternary supported lipid bilayers (SLBs) of DOPC/SM 16:0/Cho on mica and on polycrystalline Au surfaces. SLBs were formed by vesicle fusion either on mica or on dithiothreitol (DTT) self-assembled monolayers (SAMs) on Au following previous procedures1. The presence of different lipid phases was characterized by means of atomic force microscopy (AFM) topographic images on mica substrates and by force spectroscopy (FS) on Au/DTT surfaces. FS gives information about the nanomechanical properties and distribution of lipid phases in SLBs2 and is used in this work to confirm the lipid bilayer formation on a non-flat surface like polycrystalline Au. The nanomechanical properties of SLBs formed on polycrystalline Au were comparable to the ones obtained on a flat mica substrate. The combination of these techniques corroborated the adequate formation of SLBs to study their interactions with solutions of MβCD by SPR. The amount of Cho extracted reached 24 % for 3 mM MβCD, which is consistent with reported values for human erythrocytes in isotonic solution. By means of this simple model, the observed changes in red blood cell membranes and their consequences on cellular function could be correlated.