Engineering Asymmetric Lipid Vesicles: Accurate and Convenient Control of the Outer Leaflet Lipid Composition

Abstract

The asymmetric distribution of lipids between the two bilayer leaflets represents a typical feature of biological membranes. The loss of this asymmetry, for example the exposure of negatively charged lipids on the extracellular membrane leaflet of mammalian cells, is involved in apoptosis and occurs in tumor cells. Thus, the controlled production of asymmetric liposomes helps to better understand such crucial cellular processes. Here, we present an approach that allows us to design asymmetric model-membrane experiments on a rational basis and predict the fraction of exchanged lipid. In addition, we developed a label-free and nondestructive assay to quantify the asymmetric uptake of negatively charged lipids in terms of the zeta potential. This significantly enhances the applicability, impact, and predictive power of model membranes.

Gramicidin increases lipid flip-flop in symmetric and asymmetric lipid vesicles

“…One prominent example is the transverse lipid distribution in cell membranes: whereas a self-assembled lipid bilayer has the same lipid composition in its two leaflets (i.e., it is symmetric), the leaflet compositions in the plasma membranes of eukaryotic cells differ (i.e., the bilayer is asymmetric), and this difference is actively maintained by the cell. Not surprisingly, the biophysical mechanisms underlying membrane asymmetry are the subject of intense studies [1][2][3][4][5] which are rapidly increasing in number as a result of recent advances that have enabled the preparation and biophysical characterization of asymmetric lipid-only model membranes in vitro [6][7][8]. Because such model membranes are not at chemical equilibrium and their asymmetry is not actively maintained, the time window for examining their properties is limited by the gradual redistribution of the lipids between the two leaflets until a symmetric lipid composition is achieved.…”

Extracavity Effect in Cyclodextrin/Surfactant Complexation

“…Thus, good global fits were obtained for all data, indicating that DIMEB formed 1:1 complexes with all SB3-x tested. It is well known that, in addition to 1:1 associations, some CDs form higher-order complexes with acyl chains of phospholipids 36,37 or long-chain surfactants. 38 However, this phenomenon is not observed for all CDs, as demonstrated in our previous study for HPβCD 9 or here for DIMEB/SB3-x complexes.…”

Preparation of Asymmetric Liposomes Using a Phosphatidylserine Decarboxylase

“…z potential z potential measurements were performed with the Zetasizer Nano ZS (Malvern) using a flow-through, high-concentration z potential cell (high concentration cell [HCC]) (Malvern), as described previously (14). Briefly, the kinetics of PS decarboxylation by PSD were recorded at 28 C. A new measurement was started every 3-5 min for 80-120 min.…”

“…It joins separate monolayers without a need for subsequent lipid exchange, but its yield is limited, and some oil typically remains in the membrane. Cyclodextrins (13)(14)(15) or lipid transport proteins (16) are used as lipid carriers or complexing agents allowing for the exchange of outer-leaflet lipids after liposome formation. Cyclodextrins are specific for sterols compared to diacyl phospholipids, with a-cyclodextrin excluding cholesterol (17) and methyl-b-cyclodextrin preferring to form complexes with cholesterol as compared to with phospholipids (18)(19)(20)(21).…”

“…Overall, our strategy was to first prepare symmetric liposomes that have PS in both membrane leaflets and then treat the outer leaflet with PSD to render them asymmetric. The decreasing local PS content in the outer leaflet was monitored in a label-free manner in terms of a less negative z potential (14). The decrease of PS and the increase of PE were quantified by high-performance thin-layer chromatography (HPTLC), which yields the overall lipid composition of the bilayer.…”