Modular lipid nanoparticles (LNPs) are a promising platform to deliver mRNA to various tissues and cells. Optimization of LNPs for hepatic and extrahepatic tissues often involves substitution of helper lipids or addition of novel lipids not found in conventional four-component LNPs. Among the lipids that comprise LNPs, the functional contributions of phospholipids (PLs) in selective organ targeting (SORT) LNPs remain poorly understood. In this study, we systematically evaluate the roles of PLs within SORT LNPs. Our results demonstrate that PL enrichment enhances cellular transfection efficiency by increasing membrane fusion and endosomal escape. In vivo, we observe that PL-containing SORT LNPs significantly increase protein expression following intramuscular administration in mice, whereas moderate PL inclusion is optimal for intravenous delivery. Cryo-electron microscopy reveals that PL modulation induces distinct morphological rearrangements in LNP structure, which may influence the selective adsorption of plasma proteins, an essential factor in endogenous targeting mechanisms. These findings highlight the fundamental role of PLs in supporting intracellular delivery and guiding organ-specific biodistribution through protein corona formation. A deeper understanding of the structural and functional impact of lipid components, especially PLs, will be crucial for the rational design of next-generation mRNA delivery systems with improved efficacy and precision.
