Super-swelling behavior of stacked lipid bilayer systems

Abstract

Bilayer systems comprising lipid mixtures are the most well-studied model of biological membranes. While the plasma membrane of the cell is a single bilayer, many intra- and extra-cellular biomembranes comprise stacks of bilayers. Most bilayer stacks in nature are periodic, maintaining a precise water layer separation between bilayers. That equilibrium water separation is governed by multiple inter-bilayer forces and is highly responsive. Biomembranes re-configure inter-bilayer spacing in response to temperature, composition, or mass transport cues. In synthetic bilayer systems for applications in cosmetics or topical treatments, control of the hydration level is a critical design handle. Herein we investigate a binary lipid system that leverages key inter-bilayer forces leading to unprecedented levels of aqueous swelling while maintaining a coherent multilamellar form. We found that combining cationic lipids with bicontinuous cubic phase-forming lipids (lipids with positive Gaussian modulus), results in the stabilization of multilamellar phases against repulsive steric forces that typically lead to bilayer delamination at high degrees of swelling. Using ultra-small-angle X-ray scattering alongside confocal laser scanning microscopy, we characterized various super-swelled states of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and glycerol monooleate (GMO) lipids, as well as other analogous systems, at varied concentration and molar ratios. Through these experiments we established swelling profiles of various binary lipid systems that were near-linear with decreasing lipid volume fraction, showing maximum swelling with periodicity well above 200 nanometers. Confocal fluorescence micrograph of super-swelled multilamellar structures in 90GMOD sample at 25 mM concentration. Inset plot shows intensity profile of orange line, with pink triangles indicating maxima.

Fig. 1

A Confocal fluorescence micrograph of super-swelled multilamellar structures in 90GMOD sample at 25 mM concentration. Inset plot shows intensity profile of orange line, with pink triangles indicating maxima. Overlaid image on right shows visible light diffraction from swelled structures. B Calculated lamellar repeat distances of 90GMOD samples, plotted against concentration. “+” symbols indicate individual data points from each vertical scan, and blue dots are averages of said scans. 95% confidence interval is shown in light purple. C A replotting of the data in (B), but with the lamellar repeat distances plotted against the inverse of a calculated volume fraction. A linear trend line is imposed onto the plotted data to show possible linear dependence. D An example dataset showing integrated USAXS data from a vertical scan of a 90GMOD sample at 130 mM concentration. Each scan was taken 4.5 mm apart, moving up the capillary towards the sample meniscus (represented by gradient of pink to purple)

Fig. 2

Dilution behavior of xGMOD system. A Concentration-dependent swelling of lipid systems with 0 mol%, 20 mol%, 40 mol%, 60 mol%, and 80 mol% GMO, the remainder being DOTAP (0GMOD, 20GMOD, 40GMOD, 60GMOD, and 80GMOD, respectively). “+” symbols indicate d-spacing calculated from single USAXS scan in vertical scan set. Open circles indicates average of said dataset. Translucent polygons indicate 95% confidence interval. B Averages from dataset shown in A, plotted against calculated inverse volume fraction

Fig. 3

Lamellar repeat distance analysis of comparative and contrasting binary lipid systems. A Depictions of phytantriol (cubic lipid) and DOTAP (charged lipid), as well as lamellar repeat distance of the mixture plotted against total lipid concentration. B Depictions of GMO (cubic lipid) and 18:1 EthylPC (charged lipid), as well as lamellar repeat distance of the mixture plotted against total lipid concentration. C Depictions of DOPC (lamellar lipid) and DOTAP (charged lipid), as well as lamellar repeat distance of the mixture plotted against total lipid concentration. In Lewis diagrams, purple represents the hydrophobic region, and blue represents the hydrophilic region of the lipids used. In d-spacing plots, light gray polygons indicate 95% confidence intervals, “+” symbols indicate unaveraged d-spacings from vertical scans, and open circles indicate averaged vertical scan data

Fig. 4

Effect of sodium chloride concentration on 90GMOD phase and lamellar repeat distance. Plot (bottom) shows example SAXS plots from 125 mM 90GMOD with increasing NaCl concentration. The peaks are indexed to plane families corresponding to the depicted liquid crystalline phases displayed (top)