Rapid and Accurate Quantification of RNA in Lipid Nanoparticles by Scatter-Free UV/Visible Spectroscopy

UV/visible spectroscopy is the method of choice for RNA quantification, thanks to its simplicity and accuracy. However, it cannot be used to quantify RNA in lipid nanoparticles (LNPs), such as those used for drug delivery in mRNA vaccines, because of light scattering by LNPs. Alternative methods such as the RiboGreen fluorescence assay require much more sample preparation and lack reproducibility and accuracy. Here we propose and demonstrate an alternative approach using an integrating sphere setup to measure scatter-free absorption spectra. RNA spectra of RNA-loaded LNPs can then be directly measured, and the total RNA concentration can be deduced. The method shows very good linearity and precision (∼1.5%), and the accuracy is estimated to be ∼5% when applied to various mRNA-LNP formulations. This work paves the way for the routine characterization of payload concentration in LNP formulation research.