A Cell-Free\nKinetic Analysis of Ionizable Lipid Hydrolysis

The prolonged retention of ionizable\nlipids within the body limits\nthe repeated dosing of lipid nanoparticles (LNPs) for nucleic acid\ndelivery. While most ionizable lipids are primarily metabolized in\nthe liver via the enzymatic hydrolysis of ester bonds, elimination\nhalf-lives can range from several hours to days. The development of\ncompounds that undergo rapid biodegradation remains a major engineering\nchallenge in the absence of standardized biodegradability assessments\nin the early stages of drug discovery. Here, we analyze and compare\nthe hydrolysis kinetics of well-known ionizable lipids (ALC-0315,\nDLin-MC3-DMA, LP-01, L319, and SM-102) using optimized cell-free reactions\nmonitored by <sup>1</sup>H NMR. Unlike conventional analytical techniques,\nthese NMR-based methods are universal and suitable for high-throughput\nscreening. We demonstrate that enzyme-catalyzed and base hydrolysis\nreactions can predict whether ionizable lipids undergo fast or slow\nliver elimination, as our results are in alignment with prior pharmacokinetic\nstudies. Furthermore, we show that the hydrolysis kinetics of ionizable\nlipids vary by several orders of magnitude depending on steric effects.\nThis study provides a framework to expedite the discovery of rapidly\ndegradable ionizable lipids, with implications for improving the therapeutic\nindex of LNP-based drugs.