A Cell-Free Kinetic Analysis of Ionizable Lipid Hydrolysis

The prolonged retention of ionizable lipids within the body limits the repeated dosing of lipid nanoparticles (LNPs) for nucleic acid delivery. While most ionizable lipids are primarily metabolized in the liver via the enzymatic hydrolysis of ester bonds, elimination half-lives can range from several hours to days. The development of compounds that undergo rapid biodegradation remains a major engineering challenge in the absence of standardized biodegradability assessments in the early stages of drug discovery. Here, we analyze and compare the hydrolysis kinetics of well-known ionizable lipids (ALC-0315, DLin-MC3-DMA, LP-01, L319, and SM-102) using optimized cell-free reactions monitored by ¹H NMR. Unlike conventional analytical techniques, these NMR-based methods are universal and suitable for high-throughput screening. We demonstrate that enzyme-catalyzed and base hydrolysis reactions can predict whether ionizable lipids undergo fast or slow liver elimination, as our results are in alignment with prior pharmacokinetic studies. Furthermore, we show that the hydrolysis kinetics of ionizable lipids vary by several orders of magnitude depending on steric effects. This study provides a framework to expedite the discovery of rapidly degradable ionizable lipids, with implications for improving the therapeutic index of LNP-based drugs.