Cholesterol-Containing Ionizable Lipids for Safe and Efficient mRNA Delivery via Lipid Nanoparticles

Abstract

Efficient and safe delivery systems are essential for advancing mRNA-based therapeutics and vaccines. Here, we designed and synthesized a series of ionizable lipids featuring a cholesterol-bearing hydrophobic tail and ester linkages to enhance delivery efficiency and safety. Six ionizable lipids were incorporated into four-component LNP formulations using microfluidic mixing. The resulting LNPs exhibited desirable physicochemical properties, including approximately 200 nm particle sizes, low polydispersity, mild negative surface charge, high mRNA encapsulation efficiencies and suitable apparent pKa values (6.4-6.7). In vivo imaging revealed that LNPs containing ionizable lipids A13, B12, and C12 delivered mRNA efficiently, with A13 demonstrating the highest luciferase expression. These lead formulations also showed excellent storage stability. In vitro, they promoted good protein expression while maintaining low cytotoxicity. Biosafety studies confirmed substantially reduced tissue damage, muscle pain, and inflammatory cytokine responses compared to an SM-102 benchmark. This study verifies the potential of natural molecule-derived ionizable lipids to improve the efficacy and safety of mRNA delivery, offering key evidence and material candidates for developing new mRNA delivery systems with low immunogenicity.