RiboGreen-Based Encapsulation Efficiency Measurement for Lipid Nanoparticles: Evaluation of Total RNA Assay and Surrogate-Matrix Correction of Free RNA Fluorescence Bias

Abstract The RiboGreen fluorescence assay is widely utilized to determine mRNA encapsulation efficiency (EE %) in lipid nanoparticle (LNP) formulations by separately quantifying free and total mRNA, but both measurements often exhibit high variability and inconsistent performance, compromising accurate EE % measurement. We evaluated each assay using both LNP and targeted lipid nanoparticles (tLNP) formulations to identify root causes of poor performance. The total RNA assay performed acceptably, with %CV below 13% and inter-run recovery of 92−113% as assessed using nanoparticles with known mRNA concentration and different ionizable lipids, confirming complete RNA−lipid dissociation under Triton X-100 lysis. Progressive fluorescent signal decay was observed in the free RNA assay during prolonged incubation, highlighting the importance of an optimal time window for plate reading. More strikingly, the free assay yielded a systematic underestimation of over 30% when calibrated in conventional Trizma-EDTA (TE) buffer, inflating apparent EE % irrespective of dilution factor. Parallelism testing revealed that the calibration curve in TE buffer failed to match the responses in the sample matrix, attributable to nanoparticle-mediated fluorescence attenuation. Substituting the TE calibration buffer with empty nanoparticles (LNPs without mRNA) as a surrogate matrix restored inter-day spike-in recovery to 84−102% across 1−10% spike levels with %CV ≤ 18% at the lower limit of quantitation (LOQ), enabling accurate EE % reporting up to 99%. Matrix-matched calibration is therefore essential for reliable EE % determination and represents a broadly applicable solution for method qualification.