(M1130-02-09) Alternative Detergents to Triton X-100 for Disrupting Lipid Nanoparticles and Enhancing RNA Encapsulation Quantification in RiboGreen Assay

Purpose: The accurate measurement of RNA encapsulation efficiency (EE) and total RNA concentration is crucial for the development of lipid nanoparticle (LNP)-based RNA delivery systems. Triton X-100 has long been a standard surfactant in the RiboGreen assay for disrupting LNPs, but its negative impact on human health and environmental sustainability necessitates the search for alternatives.^1-2 This study investigates alternative surfactants that offer effective LNP disruption while minimizing health and environmental risks. We aim to improve the accuracy of RNA quantification in RiboGreen assays, with a focus on enhancing RNA EE measurements. The outcomes of this research have broad implications for both academic research and the pharmaceutical industry, promoting more sustainable, accurate, and environmentally friendly practices in RNA delivery system development and quality control.

Methods: dsRNA-LNPs were prepared using an ANP microfluidic system. The lipid blend consisted of ALC-0315, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5. The particle size, polydispersity index (PDI), and particle concentration of the LNPs were characterized using Stunner, while the zeta potential was measured using a Zetasizer. The total RNA content and EE were determined using the RiboGreen assay, employing various non-ionic surfactants, including Virodex TXR-1, Virodex TXR-2, and Tween 20 in different purities (Tween 20 pharma, super-refined polysorbate 20, and Tween 20 HP LSA). Calibration curves for each surfactant were prepared through serial dilutions (0.0025%, 0.005%, 0.01%, 0.1%, 0.25%, and 0.5% w/v) in combination with serial concentrations of RNA (0, 3.125, 6.25, 12.5, 25, 50, and 100 ng/mL). The dsRNA-LNPs were either diluted 1:1 in TE buffer or in varying concentrations of the surfactants to conduct the RiboGreen assay. The total RNA released by the surfactants was quantified and compared to the expected total RNA amount, along with the EE, to assess the LNP disruption capabilities of each surfactant.

Results: The LNPs were successfully prepared with an average size of 114.5 nm, PDI < 0.1, a slightly negative zeta potential (-5.1 mV), and a particle concentration of 2.6E+10 particles/mL. As shown in Figure 1A, the calibration curves for each surfactant demonstrated a clear correlation between the dsRNA concentration and fluorescence intensity across the different concentrations of surfactants, which are similar to the curve without surfactants. Although, the linear calibration curves for all surfactants are lower compared to the no surfactant control (Figure 1B), we concluded based on the linearity that all surfactants are compatible with the RiboGreen assay. These calibration curves were used to determine total RNA and EE afterwards. Notably, alternative surfactants such as Virodex TXR-1, Virodex TXR-2, especially Tween 20 (including its variants) were able to release the comparable amount of total RNA at a low concentration of 0.0025% as Triton X-100 at a much higher concentration of 0.5% (Figure 2A). This observation indicates that these alternative surfactants are highly efficient at disrupting lipid nanoparticles even at significantly lower concentrations, making them more environmentally friendly and less toxic compared to Triton X-100, which requires higher concentrations for similar performance. In terms of EE, all surfactants tested maintained high RNA encapsulation values, generally exceeding 90% (Figure 2B). These results suggest that Tween 20 is a more potent surfactant for disrupting LNPs than Triton X-100, which is consistent with findings from David et al., who attributed this to the significantly lower CMC range of Tween 20 compared to Triton X-100.³

Conclusion: This study demonstrates that alternative surfactants, including Virodex TXR-1, Virodex TXR-2, and polysorbate 20 variants, can effectively release total dsRNA and achieve similar RNA quantification results as Triton X-100, even at much lower concentrations, making them safer and more environmentally friendly alternatives. Furthermore, these surfactants do not compromise RNA encapsulation efficiency, maintaining high EE values. Moving forward, we plan to extend this investigation by testing a broader range of LNP formulations with varying levels of rigidity to assess the generalizability of these surfactants across different LNP structures. Additionally, we will explore more potential surfactant candidates to further optimize RNA release and quantification, aiming for a more comprehensive, sustainable, and accurate approach to LNP disruption and RNA analysis in future applications.

References: 1. National Library of Medicine (US), National Center for Biotechnology Information; 2004-. PubChem Compound Summary for CID 5590, Triton X-100; [cited 2025 Jan. 21]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Triton-X-100.
2. Schober, Gretchen B., Sandra Story, and Dev P. Arya. "A careful look at lipid nanoparticle characterization: analysis of benchmark formulations for encapsulation of RNA cargo size gradient." Scientific Reports 14.1 (2024): 2403.
3. Schultz, David, et al. "Enhancing RNA encapsulation quantification in lipid nanoparticles: Sustainable alternatives to Triton X-100 in the RiboGreen assay." European Journal of Pharmaceutics and Biopharmaceutics 205 (2024): 114571.

Acknowledgements: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported.

Table 1. Lipid nanoparticle composition, size as hydrodynamic diameter, polydispersity index (PDI), zeta potential, and particle concentration (n = 3).

Figure 1. Calibration curve of Triton X-100, Virodex TXR-1, Virodex TXR-1, Tween 20 pharma, SR polysorbate 20, and Tween 20 HP LSA at serial concentration with 0, 3.125, 6.25, 12.5, 25, 50, or 100 ng/mL mRNA measured with the RiboGreen assay (A); Calibration curve of each surfactant at concentration of 0.5% with 0, 3.125, 6.25, 12.5, 25, 50, or 100 ng/mL mRNA (B).

Figure 2. Difference in measured total mRNA after surfactant-based LNP disruption (A); Measured encapsulation efficiency (EE) using various surfactants (B). Triton X-100 values are compared to the other surfactants at each concentration.