Transient interactions between cationic ionizable lipids and anionic lipids foster lamellar to hexagonal phase transition

RNA-based therapeutics have demonstrated remarkable efficacy and hold great promise for future applications in personalized medicine. The most common delivery systems for these drugs are lipid-based nanoparticles (LNPs), which incorporate ionizable cationic lipids (ICLs) as key components. Among other, ICLs are believed to facilitate endosomal escape of the cargo by interacting with anionic lipids in the endosomal membrane, although the underlying molecular mechanism remains unclear. One proposed model suggests that the membrane is destabilized by cone-shaped complexes formed between ICLs and endosomal anionic lipids. However, no clear evidence of stable co-localization of anionic and cationic lipids has been presented, so far. Here, we re-examine the problem through equilibrium and nonequilibrium simulations of model membrane systems containing DODMA (ICL), DOPS (anionic lipid) and DOPE (helper lipid). Our results confirm absence of co-localization at equilibrium, but reveal a transient formation of cone-shaped complexes during lamellar-to-inverted-hexagonal phase transitions, which considerably accelerates the transition process. These findings may open new ways for controlling endosomal escape through the rational design of ICLs optimized to interact with cell- or stage-specific endosomal anionic lipids.