Mechanistic insights into mRNA–LNP interactions: role of ionizable lipid content in regulating mRNA intracellular release and translation

Messenger RNA (mRNA) therapeutics rely on lipid nanoparticles (LNPs) for effective intracellular delivery, yet the intracellular release of mRNA from these carriers remains an underexplored barrier to translation. In this study, we investigate how the ionizable lipids within clinically relevant LNP formulations influence mRNA delivery and translation. By systematically varying the molar ratio of ionizable lipids (SM-102 and ALC-0315) while maintaining other lipid components constant, we found that LNPs containing lower ionizable lipid levels (~ 30 mol%) achieved significantly higher in vitro transfection efficiency and in vivo mRNA expression compared to standard clinical formulations (50 mol% for Moderna; 46.3 mol% for Pfizer/BioNTech). Notably, this enhancement occurred despite similar cellular uptake and endosomal escape, implicating intracellular mRNA-LNP dissociation as a previously underappreciated determinant of translational efficiency. Fluorescence colocalization analyses confirmed greater cytosolic mRNA release from LNPs with reduced ionizable lipid content. Moreover, these optimized formulations elicited stronger antigen-specific humoral and cellular immune responses in mice, highlighting their potential for improved mRNA performance. Collectively, these findings identify intracellular mRNA dissociation-rather than endosomal escape alone-as a key bottleneck in LNP-mediated delivery and demonstrate that fine-tuning ionizable lipid composition can substantially enhance mRNA performance.