Lipid nanoparticles (LNPs) are essential carrier particles in drug delivery systems, particularly in ribonucleic acid delivery. In preparing lipid-based nanoparticles, microfluidic-based ethanol injection may produce precisely size-controlled nanoparticles. Ethanol is critical in LNP formation and post-treatment processes and affects liposome size, structure, lamellarity, and drug-loading efficiency. However, the effects of time-dependent changes in the ethanol concentration on the structural dynamics of liposomes are not clearly understood. Herein, we investigated ethanol-induced lipid bilayer changes in liposomes on a time scale from microseconds to tens of seconds using a microfluidic-based small-angle X-ray scattering (SAXS) measurement system coupled with molecular dynamics (MD) simulations. The time-resolved SAXS measurement system revealed that single unilamellar liposomes were converted to multilamellar liposomes within 0.8 s of contact with ethanol, and the d-spacing was decreased from 6.1 (w/o ethanol) to 4.4 nm (80% ethanol) with increasing ethanol concentration. We conducted 1 μs MD simulations to understand the molecular-level structural changes in the liposomes. The MD simulations revealed that the changes in the lamellar structure caused by ethanol at the molecular level could explain the structural changes in the liposomes observed via time-resolved SAXS. Therefore, the post-treatment process to remove residual ethanol is critical in liposome formation.
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