Ionizable lipid impact on mRNA vaccine formulation properties and immunogenicity.

The COVID-19 pandemic disrupted global supply chains, causing shortages of critical lipid-based raw materials for mRNA vaccine production. To enhance formulation resilience, we systematically evaluated the impact of five ionizable lipids (ILs) on lipid nanoparticle (LNP) organization, mRNA delivery and immunogenicity. The physiochemical analysis revealed the potential coexistence of liquid crystalline lamellar (Lα) and inverse hexagonal (HII) phases, or a mixture of nucleotide-loaded and non-loaded HII phases within developed LNP formulations. Furthermore, ILs with saturated, multi-tail structures, such as Lipid-5 and SM-102, favored inverse hexagonal (HII) internal organization and fusion intermediates, correlating with enhanced endosomal fusion and higher in vivo protein expression. In contrast, ILs such as cKK-E12, which featured dual ionization headgroups and tended to form lamellar-like structures, showed reduced encapsulation efficiency and transfection potency. The preclinical experiments demonstrated that all developed formulations induced robust binding and neutralizing antibody responses; however, antibody responses were lower in mRNA Lipid 5 LNPs and mRNA SM-102 LNPs than in other formulations. Notably, within the limited panel of ILs in this study, protein expression levels did not show a clear correlation with antibody titers, highlighting that efficient antigen expression alone is not predictive of humoral immunity. These findings underscore how IL structural features influence LNP architecture and function, informing the rational design of next-generation mRNA vaccines.