Lipid nanoparticles (LNPs) represent the most clinically advanced delivery platform for mRNA therapeutics and vaccines, yet currently approved formulations may not be broadly applicable for next-generation utility due to the double-edged sword of immunogenicity. While prior studies have primarily evaluated the contribution of ionizable lipids to immune stimulation, the effects of other components remain underappreciated. Here, we investigate the role of phospholipids in modulating LNP physicochemical characteristics, delivery efficiency, and immunogenicity. Among seven commonly used phospholipids, unsaturated and zwitterionic phospholipids exhibit moderate membrane fluidity and enhanced cellular uptake compared to the saturated counterparts. Notably, LNPs formulated with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) demonstrate significantly enhanced mRNA delivery efficiency following intravenous administration in vivo. In contrast, intramuscular delivery results in relatively consistent mRNA expression across different phospholipids, particularly in lymph nodes. Furthermore, DOPE-containing LNPs mediate relatively low immunogenicity in vivo, endowing non-immunogenic therapy potential. While incorporation of the immunostimulatory phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) amplifies immune responses by promoting cytokine and chemokine secretion as well as immune cell infiltration, highlighting its promise for mRNA vaccine applications. Our findings demonstrate that the immunogenic profile of LNPs can be finely tuned through rational phospholipid optimization, underscoring the importance of tailoring LNP formulations to optimize performance across diverse therapeutic applications.
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