Ionizable cationic lipid is critical for construction of lipid nanoparticles (LNPs) for mRNA delivery. Here, we reported the rational design and evaluation of FS01, a novel ionizable cationic lipid incorporating an ortho-butylphenyl-modified hydrophobic tail into a squaramide-based lipid headgroup architecture. Molecular dynamics simulations revealed that FS01 enhances mRNA stability through π-π stacking interactions between its aromatic tail and nucleobases aromatic rings, alongside hydrogen bonding via the squaramide headgroup. FS01-LNPs demonstrated smaller particle sizes (∼ 70 nm), high encapsulation efficiency (> 90 %), and superior mRNA delivery performance across intramuscular, subcutaneous, and intravenous routes in mice compared to FDA-approved lipids (Dlin-MC3-DMA, SM-102, ALC-0315). In prophylactic vaccine models (Varicella-zoster virus and Hepatitis B virus), FS01-LNP formulations elicited robust antigen-specific antibodies, memory B cells, and Th1-biased T cell responses, outperforming benchmark LNPs. Further, transcriptomic profiling and safety assessments demonstrated that FS01-LNP induced a well-balanced innate immune activation with minimal inflammation and liver toxicity, contrasting with the pronounced reactogenicity of Dlin-MC3-DMA and ALC-0315 LNPs. These findings highlighted FS01 as a promising ionizable lipid candidate for mRNA therapeutics, offering enhanced delivery efficiency, immunogenicity, and safety, with potential applications extending beyond vaccines to gene editing and protein replacement therapies.
