Creating Asymmetric Phospholipid Vesicles via Exchange With Lipid-Coated Silica Nanoparticles.

Recently, effort has been placed into fabricating model free-floating asymmetric lipid membranes, such as asymmetric vesicles. Here, we report on the use of lipid-coated silica nanoparticles to exchange lipids with initially symmetric vesicles to generate composition-controlled asymmetric vesicles. Our method relies on the simple and natural exchange of lipids between membranes through an aqueous medium. Using a selected temperature, time, and ratio of lipid-coated silica nanoparticles to vesicles, we produced a desired highly asymmetric leaflet composition. At this point, the silica nanoparticles were removed by centrifugation, leaving the asymmetric vesicles in solution. In the present work, the asymmetric vesicles were composed of isotopically distinct dipalmitoylphosphatidylcholine lipids. Lipid asymmetry was detected by both small-angle neutron scattering (SANS) and proton nuclear magnetic resonance (1H NMR). The rate at which the membrane homogenizes at 75 °C was also assessed.

Influence of the membrane environment on cholesterol transfer.

Cholesterol, an essential component in biological membranes, is highly unevenly distributed within the cell, with most localized in the plasma membrane while only a small fraction is found in the endoplasmic reticulum, where it is synthesized. Cellular membranes differ in lipid composition and protein content, and these differences can exist across their leaflets too. This thermodynamic landscape that cellular membranes impose on cholesterol is expected to modulate its transport. To uncover the role the membrane environment has on cholesterol inter- and intra-membrane movement, we used time-resolved small angle neutron scattering to study the passive movement of cholesterol between and within membranes with varying degrees of saturation content. We found that cholesterol moves systematically slower as the degree of saturation in the membranes increases, from a palmitoyl oleyl phosphotidylcholine membrane, which is unsaturated, to a dipalmitoylphosphatidylcholine (DPPC) membrane, which is fully saturated. Additionally, we found that the energetic barrier to move cholesterol in these phosphatidylcholine membranes is independent of their relative lipid composition and remains constant for both flip-flop and exchange at ∼100 kJ/mol. Further, by replacing DPPC with the saturated lipid palmitoylsphingomyelin, an abundant saturated lipid of the outer leaflet of the plasma membrane, we found the rates decreased by a factor of two. This finding is in stark contrast with recent molecular dynamic simulations that predict a dramatic slow-down of seven orders of magnitude for cholesterol flipping in membranes with a similar phosphocholine and SM lipid composition.

Anomalous inter-membrane cholesterol transport in fluid phase phosphoserine vesicles driven by headgroup ordered to disordered entropic transition.

POPS is highly enriched in the inner leaflet of the plasma membrane. Here we present measurements of inter-membrane cholesterol transport rates in POPS vesicles. We find that the cholesterol transport kinetics are not only an order of magnitude slower than in POPC lipids at near physiological temperatures, they exhibit a surprising discontinuous Arrhenius behavior around 48 °C. Moreover, thermodynamic analysis suggests that for biologically relevant temperatures, below the discontinuity, the exchange of cholesterol is entropically dominated while it is enthalpically driven, as is the case in POPC vesicles, above that discontinuity. Using the polar fluorescent probe Laurdan we found that POPS fluid membranes retain a large degree of order in the headgroup region for temperatures below the discontinuity but undergo an order-to-disorder transition in the region coinciding with the discontinuity in the transport of cholesterol in POPS membranes providing an explanation not only for the discontinuity but for the entropic dominance at physiological temperatures.