Colloidal Light Scattering Distorts Fluorometric Quantification of mRNA in Lipid Nanoparticles.

Accurate measurement of messenger RNA (mRNA) concentration and encapsulation efficiency remains a challenging task during mRNA-lipid nanoparticle (LNP) formulation development and quality control. RiboGreen-based fluorescence assays are often used for this purpose. LNP formulations are colloidal systems and may exhibit turbidity, which can interfere with fluorescence detection. The impact of LNP-related optical properties on assay performance remains insufficiently characterized. This study aimed to evaluate whether and how physicochemical factors associated with LNP-based drug-delivery systems influence fluorometric mRNA quantification. Empty LNPs, with the same lipid composition as mRNA-loaded LNPs, were used as a model matrix causing turbidity. Their effect on the RiboGreen fluorescence signal was assessed following dilution and then in different scenarios in the presence of free and encapsulated mRNA. The UV-Vis spectrum of the placebo dispersion was recorded to examine possible optical interference in the measurement range. We quantitatively characterized the suppression of fluorescence signals in the presence of LNPs in a concentration-dependent manner. These findings indicate that turbidity and LNP-associated matrix effects can bias RiboGreen-based mRNA quantification. To ensure reliable determination of mRNA concentration and accurate calculation of encapsulation efficiency in LNP formulations, method-specific dilution factors should be applied.