On the Mechanism of Bilayer Separation by Extrusion, or Why Your LUVs Are Not Really Unilamellar

Abstract

Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Submicroscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would impact interpretations from many model membrane experiments. Using three probe-free methods-small angle X-ray and neutron scattering and cryogenic electron microscopy-we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100-nm diameter pores. In such preparations, only $35% of lipids are externally accessible and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein the formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity.

NACore Amyloid Formation in the Presence of Phospholipids

“…This allows some features of the bilayers to be determined, such as the thickness and overall internal structure. We can see from the scattering pattern of POPC:POPS vesicles that it fits well with the scattering pattern of a single bilayer with a headgroup-to-headgroup distance of about 3.8 nm ( Figure 4B), consistent with expectations from the literature (Kucerka et al, 2011;Eicher et al, 2017;Scott et al, 2019). In the case of vesicles composed of POPC only, we can fit them with the scattering of bilayers with the same headgroup-to-headgroup thickness as for POPC:POPS ( Figure 5B).…”

“…While the distorted vesicles in both types of samples are associated with the fibrils, the bilayers of these distorted vesicles can still be rather clearly distinguished from the fibrils. In the samples containing POPC vesicles, some of them are multilamellar ( Figure 1B) which is probably due to lack of electrostatic repulsion between the bilayers due to the zwitterionic nature of the glycero-3-phosphocholine (PC) head group (Scott et al, 2019). Multilamellar vesicles were observed also for POPC without NACore present ( Supplementary Figure 4).…”

Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states

“…SANS data were analyzed with the multilamellar form factor in the SasView application. The data showed a broad shoulder at q ≈ 0.06 Å -1 due to the presence of a mixture of unilamellar and multilamellar vesicles (Scott et al, 2019). SANS data were fit with an array distribution of N, where N is the number of lamellar shells and the reported results are for the distribution that gave the best fit to the data, defined as the minimum χ 2 value.…”

Exploring the utility of hybrid siloxane-phosphocholine (SiPC) liposomes as drug delivery vehicles

“…In some instances, the number of bilayers present is larger owing to smaller vesicles nested inside of larger vesicles, potentially even aer extrusion. 55 However, in spite of releasing a lower percentage of the entrapped calcein, the non-extruded liposomes, in all cases, both entrapped and subsequently released a greater overall amount of calcein than the liposomes that had not been subjected to extrusion (Table 4). This suggests that calcein is lost during the extrusion process and is therefore not incorporated into the liposomes.…”