Abstract

Lipid nanoparticles (LNPs) have emerged as a promising platform for mRNA delivery with widespread applications across various fields, including gene editing, disease treatment, and vaccine development. Improving mRNA expression and enhancing immune responses are essential for expanding their application in mRNA vaccines. In this study, we synthesized and screened a library of guanidine-based lipids (GLs) as the fifth component to enhance LNP-mediated mRNA transfection. After screening, GL9, featuring symmetrical and long alkyl chains, was found to be the optimal fifth component for incorporation into LNPs at a 5% molar ratio to enhance mRNA expression. GL9 addition broadly improved transfection across diverse cell types and LNP formulations, without significantly altering LNP physicochemical properties. Incorporating GL9 into LNP also increased spleen-targeted mRNA expression by 12-fold via intraperitoneal injection and enhanced expression by 2.7-fold after intramuscular administration in vivo. Furthermore, GL9 promoted dendritic cells maturation, highlighting its potential as an adjuvant for mRNA vaccines. Thus, GLs, which enhance both transfection and immune activation, were incorporated into five-component LNPs to establish a promising platform for mRNA vaccines and therapeutics, offering an innovative strategy to optimize LNP-mediated mRNA delivery.