The dynamic process of mRNA delivery by lipid nanoparticles in vivo

Lipid nanoparticles (LNPs) have achieved clinical success in mRNA delivery. Nowadays, many studies have reported on the development of new LNP delivery systems or the exploration of new applications using clinically approved LNPs. However, the correlation among LNP biodistribution, mRNA release, and mRNA expression in vivo remains largely unknown. Therefore, we herein report a systemic study of the dynamic process of LNP mRNA delivery in vivo. To accomplish this, we used LNPs labeled with the lipophilic dye DiR to encapsulate Cy5-labeled luciferase mRNA. This enabled us to observe the biodistribution of LNPs and mRNA, together with mRNA expression in real-time through fluorescence and bioluminescence imaging. By utilizing the fluorescence resonance energy transfer (FRET) characteristic of Cy5 and DiR, the dissociation of mRNA from LNP could be monitored by detecting recovery of the Cy5 fluorescence signal from FRET. Based on this convenient live imaging method, via intramuscular and intravenous administration, respectively, we investigated in vivo mRNA delivery by LNPs based on the two most widely used ionizable lipids: MC3 and SM-102, but with different degradability properties. The metabolism of ionizable lipids was also determined by liquid chromatography-mass spectrometry. The dynamic process of mRNA dissociation from LNP and mRNA expression in vivo, which depends on the type of ionizable lipids and the route of administration, was revealed. Overall, this study leads to a better understanding of mRNA-LNP delivery dynamics, developing new LNP systems, and selecting suitable LNPs for specific applications.