Spatial Tail Design in Ionizable Lipids Enhances the Safety and Efficacy of mRNA Delivery

Messenger RNA (mRNA) delivery critically depends on the molecular architecture of ionizable lipids within lipid nanoparticles (LNPs). We report a modular platform of sterol‐containing ionizable lipids designed to probe how sterol motifs and branched dialkyl tails govern in vivo performance. Each lipid incorporates a sterol‐derived (e.g., cholesterol) tail and a dialkyl tail with tunable branching and spatial arrangement, enabling systematic control over packing behavior, protonation characteristics, and nanoparticle assembly. The resulting LNPs exhibited favorable apparent pKa values (6.2–6.8), encapsulation efficiencies higher than 90%, and strong transfection potency. Our lead LNP (L1‐aCho‐e3 LNP) exhibited higher transfection efficiency than the benchmark SM‐102 LNP. Incorporation of cationic (DOTAP) or anionic (DOPA) lipids as fifth components further demonstrated reorienting biodistribution from liver to lung or spleen. Finally, mRNA H1N1 @L1‐aCho‐e3 LNPs elicited robust antibody responses and full protection against lethal H1N1 challenge. These results reveal how rational sterol tail design can be applied to create ionizable lipids with tunable structural functional properties for next‐generation mRNA therapeutics.