Characterization of mRNA-LNP structural features and mechanisms for enhanced mRNA vaccine immunogenicity

Lipid nanoparticles (LNPs) used for nonviral gene delivery have achieved significant success, particularly in COVID-19 mRNA vaccines. LNPs are routinely characterized by their particle size, polydispersity, and mRNA loading efficiency. However, the internal structure of these particles has not been specified, despite evidence showing that LNPs can be highly heterogeneous, with variations in lipid composition and preparation methods. How these structural features contributed to mRNA LNP vaccine activities is also unclear. In this study, we prepared LNPs with distinctly different internal structures. They were named the emulsion-like LNPs (eLNPs) and membrane-rich LNPs (mLNPs) respectively and compared with the classic "bleb" structure LNPs (cLNPs). The eLNPs contained higher molar percent of the ionizable lipid and lower molar percent of DSPC and cholesterol. The different lipid organization structures lead to varying mRNA delivery activities in vitro and in vivo. After intramuscular injection, eLNPs remained at the injection site and expressed antigens locally. The resulted immune responses had a very fast onset (higher titer at week 2) and lasted longer and stronger (higher titers at week 8) than other LNPs (cLNPs and mLNPs). We hypothesize that the rapid onset and local expression of antigens by muscle cells in the eLNP groups may be favored by the antigen recognition and presentation process, despite the overall mRNA expression activities was not as high especially in liver and other organ. Our data support that eLNPs are potentially the more suitable delivery system for mRNA vaccine due to their high immunogenicity and low systemic toxicity.