Using ionizable guanidine to develop cholesterol-free three-component lipid nanoparticles for spleen-targeted mRNA delivery

Lipid nanoparticle (LNP) represents a key breakthrough in the realm of RNA therapeutics, particularly for the development of mRNA therapeutics and vaccines. Most LNPs developed to date rely on tertiary-amine based ionizable lipids and are mainly limited to liver delivery. Current strategies to alter the targeting performances of LNPs require four or more components, with cholesterol being a crucial element in the formulation. In this study, we introduce a novel approach utilizing an ionizable guanidine analogue, guanidino-carbonyl-pyrrole (GCP), to construct ionizable lipids. These GCP-based lipids can form multilayered, onion-like LNPs. These guanidine-based LNPs are highly sensitive to pH changes in the environment and demonstrate superior mRNA delivery capabilities both in vitro and in vivo. Unlike the commonly used amine-based lipids, GCP-lipids enable the formation of three-component, cholesterol-free LNPs that efficiently deliver functional mRNA to the spleen. Furthermore, we show that the new LNP system could transport mRNA to splenic immune cells with high efficacy, thus providing an exciting opportunity for future applications. Our findings underscore the advantages of ionizable guanidine-based lipids and could pave the way for the development of LNPs capable of targeting tissues beyond the liver.