Tailoring Alkyl Side Chains of Ionizable Amino-Polyesters for Enhanced In Vivo mRNA Delivery

Lipid nanoparticles (LNPs) containing ionizable lipids are the most clinically advanced platform for mRNA delivery, but their application beyond the liver remains challenging. Polymer-lipid hybrid nanoparticles offer a promising alternative, combining the synthetic versatility and unique properties of polymers with the biocompatibility of lipid excipients. While the significance of alkyl tail design is well-recognized for ionizable lipids, the impact of the polymer side chain composition on interactions with lipid excipients, mRNA delivery efficacy, and tissue specificity remains poorly understood. Here, we focus on a class of ionizable amino-polyesters (APEs) that exhibit features desired for potential clinical applications, including narrow molecular weight distribution and a good safety profile, and investigate the effect of polymer side chain composition on the formulation of APE lipid nanoparticles (APE-LNPs) for mRNA delivery. A library of 36 APEs was synthesized via ring-opening polymerization of chemically diverse tertiary amino-alcohols and lactone monomers with distinct alkyl side chain compositions, including variations in length and unsaturation. We show that optimal alkyl side chain length is critical for the assembly of stable mRNA nanoparticles and efficient mRNA delivery both <i>in vitro</i> and <i>in vivo</i>. Top-performing APE-LNPs display superior delivery efficacy <i>in vitro</i> and in extrahepatic tissues compared to benchmark LNPs, including DLin-MC3-DMA ionizable lipid. The polymer chain composition affects the tissue selectivity of APE-LNPs, with shorter side chains (4-5 carbons) effectively targeting the spleen and lungs, while longer chains (7-9 carbons) show enhanced liver delivery. We also explored the relevance of lipid excipients in APE-LNPs, demonstrating the essential role of unsaturated phospholipids in enhancing cellular uptake and mRNA delivery, and the limited relevance of cholesterol. These findings provide valuable insights into the design of polymers for use in the LNP context, which could aid the development of polymeric alternatives to ionizable lipids and expand the utility of mRNA LNP technology to nonliver tissues.