Lipid-siRNAOrganization Modulates the IntracellularDynamics of Lipid Nanoparticles

Lipid nanoparticles (LNPs) are widely used for delivering therapeutic nucleic acids, yet the relationship between their internal structure and intracellular behavior, particularly before RNA release, remains unclear. Here, we elucidate how lipid-siRNA organization within LNPs can modulate their intracellular delivery dynamics. We use cryo-electron microscopy and photochemical assays to reveal that increased siRNA loading can reduce helper lipids’ distribution to the LNP surface, while siRNA consistently localizes near the surface. These alterations in lipid-siRNA organization affect LNP membrane fluidity, enhancing LNP fusion with cellular membranes and promoting cytosolic siRNA delivery, primarily via macropinocytosis. Using photosensitive lipids and live cell imaging, we demonstrate that lipid-siRNA organization regulates LNP responsiveness to external stimuli, significantly affecting siRNA endosomal escape efficiency upon light activation. We further confirm this observation using convex lens-induced confinement microscopy and single-particle imaging. Overall, our findings provide critical insights into how lipid-siRNA organization shapes LNP intracellular dynamics, offering rational design principles for optimizing LNP-based RNA therapeutics.