Biodegradable polymers with tertiary amines enhance mRNA delivery of lipid nanoparticles via improved endosomal escape.

Abstract

Messenger RNA (mRNA)-based therapy has become a promising and scalable approach for treating various diseases, and lipid nanoparticles (LNPs) have recently gained prominence as a safe and effective delivery vehicle. LNPs not only protect mRNA from degradation during systemic circulation but also facilitate its intracellular uptake and endosomal release. However, the endosomal release efficiency of standard LNPs has been shown to be poor, limiting the transfection efficiency. Here, we explored incorporating a biodegradable polymer which only contains tertiary amines as a pH-sensitive functional group into LNPs, aiming to introduce the proton sponge effect to facilitate the endosomal release. We developed a series of novel LNP formulations by spiking the polymers with different molecular weights into LNPs at a range of ratios. Our results demonstrated that the polymer-modified LNPs (p-LNPs) maintained a particle size of approximately 80 nm, a neutral surface charge, and an mRNA encapsulation efficiency >90 %, along with increased pH buffering capacity. The optimal p-LNP formulation tripled the cellular uptake and enhanced the endosomal escape efficiency from 20 % to 80 % compared to the standard LNPs. Furthermore, cells treated with the p-LNP formulation at 1 mg/mL showed no cytotoxicity. Upon intravenous administration, the optimal p-LNP formulation loaded with luciferase mRNA significantly increased the transgene expression evidenced by a 100-fold increase in luciferin bioluminescence from the liver compared to the standard LNPs. Moreover, p-LNPs did not elevate inflammatory cytokines in the treated mice, including IFN-gamma, IL1β, TNFα, and IL6.