Abstract
Lipid nanoparticles (LNPs) have enabled the effective delivery of RNA therapeutics and mRNA vaccines. However, their broader applications are limited by the suboptimal stability and endosomal escape efficiency. Here, we present an easy-to-adopt post-assembly crosslinking approach to enhance the structural and functional stability of mRNA LNPs. By leveraging a series of cholesterol derivatives and crosslinking methods, we induce crosslinks of the lipid components following mRNA LNP assembly to form the crosslinked LNPs (cLNPs). We systematically evaluated crosslinking parameters and identified optimal conditions that enhance both the physical stability and transfection efficiency of cLNPs. Our findings demonstrate that cLNPs exhibit improved structural integrity under storage and lyophilization conditions, as well as increased extracellular stability and endosomal escape efficiency, resulting in improved performance of mRNA LNPs both in vitro and in vivo . This crosslinking strategy represents a critical advance in LNP engineering, enabling more resilient LNPs and broadening the applicability of LNP-based therapies for gene therapy and vaccine delivery. Our work lays the foundation for developing next-generation LNPs with superior stability and delivery efficiency, broadening the impact of RNA therapeutics and vaccines.