Scatter-Free UV-Visible Spectroscopy for Accurate and Precise RNA Quantification in Complex RNA Nanoparticle Formulations.

Ribonucleic acid (RNA)-based drugs showed the potential for treating wide range of diseases. Their successful clinical use depends on developing complex nanoparticle (NP) formulations made from diverse biomaterials. Accurately quantifying total RNA concentration in complex formulations is challenging, often requiring expensive, low-throughput methods or fluorescence-based assays like RiboGreen that rely on effective NP disruption. This study evaluates scatter-free absorption spectroscopy (SFAS), a UV/Visible method that removes light scattering from NP components and enables accurate total RNA quantification in intact NPs. To validate SFAS, we employed diverse RNA formulations, including lipid NPs, polymer and dendrimer hybrid lipid NPs, and cyclodextrin nanocomplexes, which exhibit physicochemical characteristics that can interfere with RNA quantification. Data obtained with SFAS were compared to fluorescent-based assays utilizing RiboGreen and SYTO 9 dyes, which bind to RNA in free or encapsulated forms, respectively. SFAS demonstrated superior accuracy, precision, and reproducibility than fluorescence-based methods across all formulations, particularly those showing resistance to disruption. RNA quantification by SFAS was less influenced by NP composition and measurement conditions, unlike the RiboGreen and SYTO 9. These findings demonstrate SFAS as a versatile and reliable alternative to fluorescence-based assays for accurate quantification of total RNA concentration in complex RNA NP formulations.