Modulating Lipid Nanoparticles with Histidinamide‐Conjugated Cholesterol for Improved Intracellular Delivery of mRNA

Abstract Recently, mRNA‐based therapeutics, including vaccines, have gained significant attention in the field of gene therapy for treating various diseases. Among the various mRNA delivery vehicles, lipid nanoparticles (LNPs) have emerged as promising vehicles for packaging and delivering mRNA with low immunogenicity. However, while mRNA delivery has several advantages, the delivery efficiency and stability of LNPs remain challenging for mRNA therapy. In this study, an ionizable helper cholesterol analog, 3β[L‐histidinamide‐carbamoyl] cholesterol (Hchol) lipid is developed and incorporated into LNPs instead of cholesterol to enhance the LNP potency. The p K a values of the Hchol‐LNPs are ≈6.03 and 6.61 in MC3‐ and SM102‐based lipid formulations. Notably, the Hchol‐LNPs significantly improve the delivery efficiency by enhancing the endosomal escape of mRNA. Additionally, the Hchol‐LNPs are more effective in a red blood cell hemolysis at pH 5.5, indicating a synergistic effect of the protonated imidazole groups of Hchol and cholesterol on endosomal membrane destabilization. Furthermore, mRNA delivery is substantially enhanced in mice treated with Hchol‐LNPs. Importantly, LNP‐encapsulated SARS‐CoV‐2 spike mRNA vaccinations induce potent antigen‐specific antibodies against SARS‐CoV‐2. Overall, incorporating Hchol into LNP formulations enables efficient endosomal escape and stability, leading to an mRNA delivery vehicle with a higher delivery efficiency.