Decoding the FunctionalRoles of Individual Componentsin mRNA Lipid Nanoparticles

Lipid nanoparticles (LNPs) have represented a leading platform for mRNA delivery and therapeutics. While extensive studies have focused on optimizing ionizable lipids, the specific roles of helper lipids such as cholesterol, phospholipid, and polyethylene glycol lipid (PEG-lipid) remain relatively poorly understood. To elucidate the distinct contributions of individual lipid components, we reengineer LNP formulations by selectively removing each lipid component, especially helper lipids, to systematically analyze their effects on physicochemical properties, in vivo mRNA delivery, and inflammatory responses. Results reveal that ionizable lipid plays a critical role in mRNA delivery efficacy. Cholesterol is essential for efficient liver-targeted delivery but dispensable for extrahepatic delivery. Phospholipid is not directly associated with organ tropism, whereas phospholipid-free LNPs significantly alleviate inflammation. PEG-lipid influences particle size, with PEG-lipid-free LNPs exhibiting preferential spleen tropism. This study comprehensively demonstrates the important roles of each lipid component in determining the in vivo fate of LNPs, guiding the rational design of next-generation LNP-based mRNA delivery systems.