When lipids embrace RNA: pH-driven dynamics and mechanisms of LNP-mediated siRNA delivery

We present a comprehensive analysis of an efficient lipid nanoparticle (LNP) formulation that exhibits strong nucleic-acid delivery and potent inhibition of targeted RNAs. Using a combination of coarse-grained and atomistic molecular dynamics simulations, we characterized the pH-dependent structure of both unloaded and RNA-loaded LNPs, elucidated the mechanism of RNA encapsulation, and used these models to propose a plausible mechanism of endosomal escape. Consistent with prior experimental and computational studies, our simulations reproduce an inverted-hexagonal-type morphology in RNA-loaded LNPs, in which a hydrated core provides a polar environment suitable for accommodating RNA, whose charge is neutralized mainly by ionizable lipids that remain protonated near the RNA even at high pH, thereby bridging the RNA with the surrounding lipid environment. This structural picture is consistently observed across our multiscale simulations, with smaller self-assembled LNP mimetics reproducing the same local organization at both coarse-grained and atomistic resolution. A potential mechanism of endosomal escape emerges spontaneously from the simulations, involving stalk formation between the LNP and the endosomal membrane, followed by the opening of a water-filled pore that permits slow RNA diffusion, in line with the low efficiency and slow kinetics reported for endosomal escape. The rate-limiting step of endosomal escape arises from persistent electrostatic coupling between RNA and protonated ionizable lipids maintained by the immediate RNA-lipid environment, hindering cargo disengagement even after pore formation. This delayed release is consistent with the experimentally observed time-dependent inhibitory activity of the loaded LNP. Together, these results highlight the importance of local pK and protonation effects near the RNA, which are not captured by global apparent pK measurements.