Conventional mRNA-LNPs typically self-assemble in acidic solutions through electrostatic interactions, necessitating long-term dialysis prior to use, which complicates the applications. In this study, a series of peptide ionizable lipids (PILs) were synthesized using solid-phase supported synthesis (SPSS) technology, aiming to utilize the amide bonds in PILs as hydrogen bond donors to form hydrogen bond-assisted mRNA-LNPs in RNase-free water, achieve extrahepatic targeted mRNA delivery, and simplify the preparation process. Through multiple rounds of optimization, a hydrogen bond-assisted two-component (2C) LNP platform composed of PILs and cholesterol was established. Among the 25 well-formulated 2C-PIL LNPs tested, 20 of them showed significant mRNA expression in the spleen, with a12K1 (2 amide bonds) and a12Orn4 (5 amide bonds) demonstrating the best performance. In addition, the two lead 2C-LNPs exhibited distinct transfection behaviors in different cell types in the spleen, indicating that suitable delivery vehicles can be tailored to meet the particular needs of specific disease indications. Notably, we found that among all K1-series (fewer hydrogen bond donors) LNPs, the efficacy of 2C-LNPs assembled based on hydrogen bond interactions was significantly higher than that of conventional four-component (4C) LNPs prepared via electrostatic interactions, suggesting that the 2C-LNPs platform may be a promising strategy to convert "inactive" LNPs into "active" LNPs. All together, these results suggest that the hydrogen bond-assisted 2C-LNPs can serve as an alternative and effective platform for spleen-targeted mRNA delivery and LNP optimization. in vivo
