An Integrating Sphere Based Method for Quantifying RNA Encapsulation Efficiency in Lipid Nanoparticles without Lysis

Encapsulation efficiency (EE) is a critical quality attribute of lipid nanoparticle (LNP)-RNA formulations, determining the fraction of active RNA payload available for delivery and influencing therapeutic potency, dose accuracy, and product consistency. Conventional fluorescence-based EE assays, such as RiboGreen (RG), require detergent-mediated LNP lysis, which can introduce systematic bias arising from incomplete particle disruption and additional sample handling steps. Here, we report a lysis-free method for direct EE determination using Scatter-Free Absorption Spectroscopy (SFAS) within an integrating sphere. In this approach, RG fluorescence appears as a quantitative negative absorbance feature in the SFAS spectrum, enabling direct quantification of free RNA from intact LNP suspensions in a single measurement. Applied to SM-102 LNPs loaded with polyA or mRNA in PBS or Tris/sucrose buffers, the method yielded EE in close agreement with conventional plate-reader measurements. This approach provides a practical, formulation-independent, and potentially more robust strategy for standardized routine EE analysis and LNP quality-control workflows.