Molecular Dynamics‐Guided Sterol Engineering of mRNA‐Lipid Nanoparticles Reprograms Biodistribution and Enhances Spleen‐Selective Immunity

ABSTRACT Multicomponent membrane organization of mRNA‐lipid nanoparticles (LNPs) critically determines their formulation behavior, organ selectivity, and immunological outcomes. However, compared to ionizable lipids, sterols remain a relatively underexplored design axis. This study discusses the engineering of a library of nine bile acid‐derived sterols with different hydroxylation patterns and alkyl tail lengths, and systematically maps how sterol structure governs formulation‐level properties and organ‐level expression profiles. After integrating physicochemical characterization with all‐atom molecular dynamics (MD) simulations, the experimentally observed formulation behaviors correlate with MD‐derived membrane structural descriptors. These descriptors provide a quantitative evaluation framework for prioritizing sterol chemotypes based on their predicted encapsulation performance and membrane organization, supporting the notion that sterol‐dependent membrane organization provides a structural basis for formulation properties, including mRNA encapsulation. Moreover, substituting cholesterol with bile acid‐derived sterols consistently attenuated hepatic expression and shifted organ‐level expression toward spleen‐dominant profiles, which is central to immune priming and adaptive immune activation. Among bile acid‐derived sterols, CA‐20 LNPs functionally enhance antigen‐specific humoral immunity and elicit antigen‐specific cellular immune responses, including improved memory‐associated immune features, while maintaining an acute safety profile. Collectively, these results establish sterol engineering as a powerful design strategy for modulating LNP formulation properties, in vivo fate, and immunological function.