Simple heat-assisted method for mRNA loading into empty lipid nanoparticles enables robust in vitro and in vivo delivery

mRNA-lipid nanoparticles (LNP) have emerged as a versatile modality, enabling diverse applications by changing encoded mRNA sequences. However, the conventional mRNA-LNP preparation process poses significant barriers to non-specialists because it requires specialized equipment and technical expertise. In this study, we aimed to report a simple post-encapsulation approach where mRNA is mixed with preformed empty LNP in solution to form mRNA-LNP for effective in vitro and in vivo delivery. Process optimization revealed that heating mRNA and empty LNP at 75°C and pH 4.0 achieved approximately 90% mRNA encapsulation while maintaining an LNP size of approximately 100 nm. The resulting post-encapsulated LNP were comprehensively compared with conventional LNP prepared via microfluidic mixing. Both LNP formulations exhibited over 90% cellular uptake and comparable concentration-dependent expression in vitro. The intravenous administration of post-encapsulated LNP in mice resulted in robust luciferase expression in the liver and spleen at 0.01 mg/kg, with no notable changes in serum chemistry at 1 and 3 mg/kg. However, the activity of the post-encapsulated LNP was slightly reduced compared with that of the conventional LNP. Sucrose density gradient centrifugation indicated that post-encapsulated mRNA-LNP formed heterogeneous populations with varying mRNA/lipid ratios, in contrast to conventional mRNA-LNP. These findings support a working hypothesis that this heterogeneity could contribute to the reduced in vivo activity. Our simple post-encapsulation method potentially could lower entry barriers for non-specialists in this field and accelerate RNA-LNP research through its compatibility with 96-well-format preparation.