This study presents the development and characterization of a targeted lipid nanoparticle (LNP) formulation for mRNA-based gene delivery based on the elimination of cholesterol and the subsequent introduction of targeting moieties. Because the high cholesterol content of conventional LNPs and its interaction with apolipoprotein E (ApoE) result in predominant hepatic accumulation in vivo, the introduction of targeting moieties to achieve extrahepatic delivery remains challenging. Thus, an ethanol and cholesterol-free formulation strategy, called "blue" lipid nanoparticle (BLNP), was applied to (i) investigate the impact of cholesterol on expression profiles and (ii) to enable the incorporation of targeting ligands. In vivo expression distribution analyses revealed no sign of toxicity for both formulations and an altered expression profile of BLNPs compared to classical cholesterol-containing LNPs with predominant expression in the liver. The lipid 1,2-dioctadecanoyl-sn-glycero-3-phosphoethanolamine (DSPE) was further conjugated to the hepatocyte-targeting dye DY635 and, following purification and characterization, incorporated into BLNPs at concentrations up to 1.1%. Uptake and transfection studies demonstrated successful nanoparticle uptake after 30 min and gene expression in various cell lines, with almost doubled mRNA expression in human HuH7 hepatocytes at optimized conditions. Incorporation of DSPE-DY635 enabled fluorescent tracking by flow cytometry and confocal laser scanning microscopy (CLSM). Cellular uptake studies revealed efficient and traceable uptake into HuH7 cells within 60 min. Overall, the successful integration of a targeting lipid into a cholesterol-free LNP platform provides a flexible strategy for the modular introduction of alternative targeting ligands and the development of controlled, tissue-specific mRNA delivery systems.
