Ionizable lipids play a crucial role in mRNA-lipid nanoparticle (LNP) formulations by facilitating mRNA encapsulation, promoting cell uptake, and enhancing endosomal escape of mRNA-LNPs. Despite their importance in mRNA delivery, the specific effects of ionizable lipids on mRNA-LNP in vivo pharmacokinetics (PK) and biodistribution remain underexplored. This study examines the effect of SM-102, ALC-0315, DLin-MC3-DMA (MC3), and 113-O12B ionizable lipids in mRNA-LNP formulations on plasma PK of lipid and mRNA, and biodistribution of expressed protein following subcutaneous (SC) and intravenous (IV) administration in mice. Our findings highlight that altering ionizable lipids significantly influences both plasma PK of mRNA and LNP lipids, and tissue biodistribution profiles of expressed protein. The SM-102 LNP formulation demonstrated superior mRNA protection in plasma, resulting in the highest bioavailability - approximately three-fold higher than other lipids following SC injection. Conversely, ALC-0315 LNPs resulted in prolonged lipid exposure but reduced mRNA plasma concentrations relative to SM-102 LNPs across both administration routes. Despite these PK differences, SM-102 and ALC-0315 LNPs achieved comparable overall tissue protein expression with both injection routes which our data suggest may be because the mRNA in LNP in plasma at early timepoints is more available for expression. MC3 ionizable lipid exhibited the longest terminal half-life, accompanied by delayed mRNA expression after both administrations. Following IV administration, protein expression was predominantly observed in the liver across all formulations, with biodistribution patterns shifting over time. In contrast, SC injections led to higher localized expression, particularly in the skin and dosing-side lymph nodes. These findings provide valuable insights into the impact of the ionizable lipid on mRNA-LNP PK and biodistribution, suggesting that the choice of ionizable lipid significantly impacts mRNA-LNP PK and biodistribution of expressed protein, and can be strategically modified to optimize the PK and biodistribution of mRNA-LNP formulations for specific indications to provide more effective therapies while reducing off-target effects.
