Unraveling the Molecular Mechanism of Transient Multilamellar Formation in Ethanol-Modified Vesicle Solutions

A recent microfluidic-based small-angle X-ray scattering (SAXS) measurement intriguingly suggested the transient formation of multilamellar structures during the mixing of unilamellar vesicles with ethanol in an aqueous solution. This study explores a possible molecular mechanism underlying this phenomenon, primarily through coarse-grained molecular dynamics (CG-MD) simulations. We first examined lipid aggregate morphology as a function of ethanol concentration in an aqueous solution. Even though vesicles were observed in pure aqueous solution, increasing ethanol concentrations led to more frequent pore formation in vesicular membranes. At ethanol concentrations above 52%, vesicles destabilized and transformed into worm-like micelles. We hypothesized that the transient multilamellar structures might arise from vesicle stacking due to variations in the effective interactions between vesicles. However, a series of potential of mean force (PMF) calculations consistently showed repulsive interactions between vesicles, regardless of ethanol concentration, ruling out this possibility. In contrast, once lipid aggregates transformed into worm-like micelles, the PMF barrier between them dropped (∼5<i>k</i>_B<i>T</i>), promoting fusion. Our CG-MD simulations further demonstrated that lipid aggregates (micelles) readily fused and grew in high ethanol concentrations. Upon subsequent exposure to lower ethanol levels, these enlarged aggregates reorganized into vesicles with internal lamellar structure─multilamellar vesicles. These findings suggest that the heterogeneous mixing of unilamellar vesicular solutions with ethanol in a microfluidic device plays a key role in the emergence of transient multilamellar structures.