Lipid nanoparticles (LNPs) have emerged as effective carriers for mRNA delivery in vaccine and therapeutic applications, attracting substantial attention since the COVID-19 pandemic. Continued efforts are crucial to optimize LNP composition for improved delivery efficacy and to elucidate the underlying mechanisms driving differences in protein expression. This study systematically screened PEGylated lipids for intramuscular mRNA delivery, followed by optimization of the formulation composition, physicochemical characterization, and investigation of the structure-activity relationship (SAR). Using a model ionizable lipid, we initially evaluated twenty-nine PEGylated lipids from four lipid families (glyceride, phosphoethanolamine (PE), cholesterol, and ceramide), each varying in linker chemistries, tail structures, or PEG molecular weights. 1,2-Dimyristoyl-rac--3-methoxypolyethylene glycol - 5000 (DMG-PEG5k) was identified as a promising candidate from this screening. Using a design of experiments (DoE) approach, we further optimized the formulation to increasetransfection efficacy, achieving an increase in protein expression over the DMG-PEG2k benchmark. To explore the SAR of the DoE formulations, advanced physicochemical characterization was conducted including Laurdan assay, SAXS, Cryo-TEM, and QCM-D, alongside standard LNP analysis. Among the key factors examined, high mRNA encapsulation efficiency, LNP membrane integrity (especially under acidic conditions), and ordered internal structures were identified as the critical parameters for transfection efficiency. mRNA encapsulation efficiency increased with a lower PEG-lipid fraction. LNP membrane integrity, assessed by the generalized polarization (GP) ratio at pH 7.5 and 4.5 from the Laurdan assay, was strongly affected by the ionizable lipid ratio and, to a lesser extent, the cholesterol ratio. A lower GP/GPratio correlated with enhanced protein expression, primarily driven by a higher GPobserved with lower ionizable lipid and higher cholesterol fractions. Overall, balancing the ratios of all LNP components is critical for maximizing LNP functionality. This study presents a systematic evaluation and characterization of LNPs with different PEG-lipid moieties, deepens SAR understanding, and provides valuable guidelines for rationally designing more effective next-generation LNPs. glycero in vivo7.5 4.5 4.5
