Tetrahydropyrimidine\nIonizable Lipids for Efficient\nmRNA Delivery

Lipid nanoparticles (LNPs) have emerged\nas an effective\nand promising\ntechnology for messenger RNA (mRNA) delivery, offering a potential\nsolution to physiological barriers and providing an alternative approach\nto gene therapy without the drawbacks associated with viral delivery.\nHowever, efficiently delivering mRNA remains a significant challenge\nin nucleic acid–based therapies due to the limitations of current\nLNP platforms in achieving optimal endosomal escape and mRNA release,\nwhich largely relies on finding a suitable ionizable lipid. Additionally,\nthe synthesis of these ionizable lipids involves multiple chemical\nreactions, often making the process time-consuming and difficult to\ntranslate. In this study, we employed a facile, catalyst-free, and\nversatile one-pot multicomponent reaction (MCR) to develop a library\nof ionizable lipids featuring a pharmacologically significant tetrahydropyrimidine\n(THP) backbone, tailored for enhanced mRNA delivery. A library of\n26 THP ionizable lipids was systematically synthesized in just 3 h\nand formulated with luciferase mRNA for initial <i>in vitro</i> screening. The THP LNPs exhibited tunable particle sizes, favorable\nζ-potentials, and high encapsulation efficiencies. Among them,\nTHP1 demonstrated the highest transfection efficiency both <i>in vitro</i> and <i>in vivo</i> after intramuscular\nadministration, comparable to DLin-MC3-DMA (MC3), a conventional benchmark.\nFurther optimization of THP1 with phospholipids significantly enhanced\nintramuscular mRNA delivery and showed sustained protein expression <i>in vivo</i> for up to 5 days. More importantly, it demonstrated\nsuccessful intravenous delivery in a dose-dependent manner with minimal\ntoxicity, as indicated by hematological, histopathological, and proinflammatory\ncytokine assessments. Furthermore, THP1 LNPs also demonstrated the\nability to edit genes in specific liver tissues in a tdTomato transgenic\nmouse model, highlighting their precision and utility in targeted\ntherapeutic applications. These findings position THP1 LNPs as promising\ncandidates for advancing mRNA-based therapies, with significant implications\nfor clinical translation in vaccine delivery and CRISPR/Cas9-mediated\ngene editing in the liver.