Transfection Potency of Lipid Nanoparticles Containing mRNA Depends on Relative Loading Levels

When formulating mRNA into lipid nanoparticles (LNP), various copy numbers of mRNA are encapsulated, leading to a distribution of mRNA loading levels within the LNPs. It is unclear whether the mRNA loading level affects the functional delivery of the message. Here we show that depending on the mRNA loading level, LNPs exhibit distinct mass densities and can be fractionated via ultracentrifugation. Upon fractionation, we investigated if mRNA loading levels influence LNP sizing, lipid composition, and morphology. We further conducted in vitro and in vivo functional delivery of mRNA and found that the LNP fraction with the highest mRNA loading levels was the least transfection competent.