A novel ionizable lipid with comprehensive improvements in transfection potency, immune profile and safety of lipid nanoparticle.

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.