Tuning Membrane Asymmetry: Controlled Uptake Of Negatively Charged Lipids Into The Outer Leaflet Of Liposomes

The presence of negatively charged lipids in the outer leaflets of cellular membranes is a key component of many biological membranes. For example, eukaryotic organisms accumulate negatively charged lipids on their extracellular membrane leaflets during programmed cell death through apoptosis. As a general feature, the outer leaflets of bacterial cellular membranes exhibit high contents of anionic phosphatidylglycerol (PG). Recently, a cyclodextrin-based lipid exchange assay has been introduced that transports phospholipids from the outer leaflet of donor liposomes to that of acceptor liposomes, thereby generating model membranes of asymmetric lipid composition. In an effort to master and further develop the cyclodextrin method, we utilize the zeta potential of mixed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes of symmetric and asymmetric composition to quantify the incorporation of negatively charged POPG into POPC liposomes. The adjustment of cyclodextrin and lipid concentrations below the maximum lipid-binding capacity of cyclodextrins allows a controlled POPG transfer without the need of donor liposomes. Furthermore, the zeta potential clearly differentiates between asymmetric and symmetric POPG incorporation, quantifies the degree of lipid asymmetry, and determines the total yield of lipid exchange. Our approach represents an easy to use method for the efficient incorporation of negatively charged lipids into the outer membrane leaflet of liposomes at physiologically relevant lipid contents.