From Morphology to Mechanism: Cryo-Electron Microscopy Insights into Lipid Nanoparticles for RNA Delivery.

Lipid nanoparticles (LNPs) have emerged as the leading delivery platform for RNA therapeutics, yet the relationship between their internal structure and biological function remains incompletely understood. Cryogenic electron microscopy (cryo-EM) has revealed a striking diversity of LNP morphologies, but a coherent framework linking structural class to formation mechanism and functional outcome has been lacking. Here, based on cryo-EM evidence, we present a systematic classification of LNP morphologies into monophasic (solid-core, multilamellar, and inverse hexagonal) and biphasic (bleb and liposomal) architectures. For each class, we dissect the mechanistic origins of assembly, explaining how ionizable lipid chemistry, helper lipid geometry, RNA cargo properties, and formulation parameters collectively determine structural outcome, and map the spatial distribution of lipid and nucleic acid components within individual particles. We then evaluate how these structural features affect biological fate, from endosomal membrane fusion and cargo release to protein corona formation and in vivo biodistribution. A central finding across the literature is that equivalent morphology does not always guarantee equivalent biological outcome; the pathway by which a given structure is formed critically shapes its intraparticle molecular organization and, consequently, its transfection efficacy. We further discuss how biological environments, particularly endosomal acidification and protein adsorption, dynamically remodel LNP structure and function. By synthesizing structural, mechanistic, and functional insights, this Review aims to establish the design principles needed to rationally engineer LNP morphology for next-generation RNA therapeutics.