Lipid nanoparticles (LNPs) are essential nucleic acid delivery carriers, with cholesterol being crucial for their structural integrity and intracellular transport. Although C24-alkyl phytosterols (cholesterol analogs) promote gene transfection in HeLa cells, their broader impact on mRNA-LNP performance across organs and organ-derived cell lines remains unknown. We investigated the effects of C24-alkyl phytosterols on mRNA-LNP morphology, stability, transfection efficiency, and in vivo biodistribution before and after lyophilization. Freshly prepared β-sitosterol–containing mRNA-LNPs exhibited the highest transfection efficiency in most cell lines. Stigmasterol-containing mRNA-LNPs demonstrated superior perfo... More
Lipid nanoparticles (LNPs) are essential nucleic acid delivery carriers, with cholesterol being crucial for their structural integrity and intracellular transport. Although C24-alkyl phytosterols (cholesterol analogs) promote gene transfection in HeLa cells, their broader impact on mRNA-LNP performance across organs and organ-derived cell lines remains unknown. We investigated the effects of C24-alkyl phytosterols on mRNA-LNP morphology, stability, transfection efficiency, and in vivo biodistribution before and after lyophilization. Freshly prepared β-sitosterol–containing mRNA-LNPs exhibited the highest transfection efficiency in most cell lines. Stigmasterol-containing mRNA-LNPs demonstrated superior performance after lyophilization. In vivo, compared with cholesterol-based controls, β-sitosterol–formulated ALC-0315-based mRNA-LNPs and fucosterol–formulated MC3-based LNPs yielded increased bioluminescence in target organs. β-sitosterol–mRNA-LNPs delivering (MERTK)mRNA robustly restored visual function in a retinal degeneration rat model. These findings highlight how subtle structural modifications of cholesterol analogs can influence mRNA-LNP therapeutic efficacy, providing ideas for the rational design of next-generation gene delivery systems. We demonstrated the therapeutic effects of mRNA-LNPs in an animal model of eye disease (RCS rats), paving the way for application in ocular gene therapy.
