Thioamides Adjacent to the Ionizable Amine Headgroup in Ionizable Lipids Reduce the p <i>K</i> _a of Lipid Nanoparticles and Enhance mRNA Transfection Efficiency in Vitro and in Vivo

Abstract Lipid nanoparticles (LNPs) are currently the most clinically advanced mRNA delivery vectors. However, optimizing LNPs for in vivo applications remains largely empirical. The apparent p K a of LNPs is a predictive factor for in vivo performance, with p K a values between 6 and 7 showing the highest efficacy. Despite this critical role of ionizable lipids in LNPs, the relationship between lipid structure and its influence on LNP p K a remains poorly studied. In this study, we report the design and the synthesis of a novel class of ionizable lipids featuring a thioamide moiety, enabling direct comparison between thioamide‐containing (SAM) LNPs and amide‐containing (OAM) LNPs. We find that substituting oxygen with sulfur in the amide group significantly decreases the apparent p K a of LNPs, increasing the likelihood of identifying lipids in combinatorial libraries that yield LNPs with a p K a in the desired 6–7 range. The reduction in p K a in LNPs containing SAM lipids, compared with OAM lipids, is attributed to the increased hydrophobicity of the thioamide group. Furthermore, by synthesizing multiple libraries of SAM lipids and varying the ionizable head group, alkyl chains, and linker length, we discovered thioamide lipids with distinct tissue tropism, including lipids that mediate splenic targeting by LNPs.