Coarse-Grained Molecular Dynamics Simulations of Lipid Nanodroplets and Endosomal Membranes: Focusing on the Fusion Mechanisms

Lipid nanoparticles (LNPs) have received significant attention as effective RNA carriers in RNA-based therapeutics and vaccines. Particularly, ionizable lipids (ILs) of LNPs play a crucial role in endosomal escape and lipid-mediated RNA delivery owing to their pH-dependent molecular characteristics. Therefore, it is essential to enhance the endosomal escape efficiency of ILs, which is primary bottleneck in the successful cytoplasmic delivery of RNA. However, the molecular-level understanding of the roles and dynamics of ILs during the endosomal escape stage remains unclear. To elucidate this, we utilized coarse-grained (CG) molecular dynamics (MD) simulations. In this simulation, we designed lipid nanodroplets (LNDs) containing D-Lin-MC3-DMA (MC3) and ALC-0315, which have proven effective as LNPs in RNA-based therapeutics and vaccines, respectively, while accounting for the pH environments of early and late endosomes. Also, we formulated lipid bilayers reflecting the composition of early and late endosomal membranes to investigate the fusion process between LNDs and endosomal membranes. Our findings reveal that, irrespective of endosomal membrane composition and LNP types, ILs are the first lipids to enter the endosomal membrane during the fusion, and the flip-flop process of ILs from the inner leaflet to the outer leaflet of the endosomal membrane is a critical step for LNP endosomal escape. More specifically, we observed that protonated ILs predominantly participate in the flip-flop process, while many deprotonated ILs remain clustered and disordered within the intermediate layer of the endosomal membrane. Furthermore, we found that the extent of IL flip-flop varies with the cholesterol content of the endosomal membrane. Additionally, under identical pH conditions, MC3-containing LNDs exhibited a more active IL flip-flop process toward the outer leaflet than ALC-0315-containing LNDs. This observation supports experimental findings that MC3-containing LNPs manifest higher endosomal escape efficiency than ALC-0315-containing LNPs in mRNA delivery studies. The mechanistic insights into the endosomal escape mechanism demonstrated by our simulations could aid in the development of effective ILs.