Cholesterol Analogs and Vitamin-Conjugated Ionizable Lipids Enhance Lipid Nanoparticle Delivery for In Vitro and In Vivo Applications

LNP-mediated mRNA delivery encounters challenges with insufficient efficacy, poor safety, and unstable performance. Moreover, the distinct requirements of in vitro and in vivo applications call for specific LNP strategies. To address this, we developed two complementary LNP strategies tailored to different application scenarios: one optimized for in vitro transfection efficiency and the other enhanced for in vivo delivery performance. For the in vitro-oriented strategy, β-sitosterol was used to replace cholesterol in LNPs. The resulting β-sitosterol-based LNPs displayed distinct internal fingerprint structures visualized by Cryo-TEM and markedly enhanced in vitro protein expression by two orders of magnitude, making it a high-efficiency tool for in vitro mRNA delivery. However, this modification did not enhance in vivo transfection efficiency and led to a significant reduction in LNP stability, confirming its suitability for in vitro use only. For the in vivo-oriented strategy, eight vitamin-conjugated ionizable lipids were synthesized. Six of these lipids were selected for animal screening, and two exhibited higher in vivo protein expression levels compared with the commercial LNP control. Accelerated stability studies demonstrated that LNPs containing these vitamin-conjugated ionizable lipids had good stability. Furthermore, the optimized in vivo-oriented LNP was used to encapsulate varicella-zoster virus (VZV) mRNA and respiratory syncytial virus (RSV) mRNA, and animal immunization experiments showed that it induced superior immunogenicity at a lower dose than the commercial delivery system. In vivo safety studies further confirmed that the in vivo-oriented LNPs had a better safety profile than commercial LNPs, with no obvious cytotoxicity or adverse reactions. Herein, we developed two complementary LNP strategies based on cholesterol analogs and vitamin-conjugated ionizable lipids, which address the limitations of traditional LNP systems, providing targeted solutions for both in vitro and in vivo translational applications.