pH-Sensitive Amino Lipid-Driven Pore Formation Enables Endosomal Escape of Lipid Nanoparticles

ABSTRACT Lipid nanoparticles (LNPs) have transformed nucleic acid delivery for vaccines and gene therapies, yet their efficiency remains limited by incomplete endosomal escape. While experimental studies have revealed endosomal membrane disruption facilitating cytosolic release, the molecular basis of this process remains poorly understood. Here, we employ molecular simulations to elucidate how LNPs fuse with the endosomal membrane and release their payloads. We identify multiple fusion pathways, with a dominant stalk-pore mechanism. Our simulations reproduce and rationalize key experimental observations, including the transfer of ionizable lipids from LNPs to the membrane, which promotes nucleic acid reorientation and facilitates stalk formation and expansion. Furthermore, lipid shape, pH sensitivity, membrane tension, and nucleic acid encapsulation emerge as critical molecular determinants of endosomal escape efficiency. These findings advance our mechanistic understanding of intracellular delivery and provide a framework for the rational design of LNPs with improved performance for gene therapies and RNA vaccines.