Enhancing RNA encapsulation quantification in lipid nanoparticles: Sustainable alternatives to Triton X-100 in the RiboGreen assay

To quantify concentration and encapsulation efficiency (EE) of mRNA in lipid nanoparticles (LNPs) the RiboGreen assay is extensively used. As part of this assay, a surfactant is used to release mRNA from LNPs for detection with the RiboGreen dye. So far, the surfactant of choice has been Triton X-100, which is harmful to human health and the environment. Alternatives to Triton X-100 are therefore needed, but surprisingly no such effort has yet been described in the literature. Here we show how three, less harmful, surfactants (Brij 93, Zwittergent 3–14 and Tween 20) compare to Triton X-100 for releasing mRNA from LNPs for detection with the RiboGreen assay. We found that Zwittergent 3–14 and Tween 20 at high concentrations (0.5 %) are at the minimum as effective as Triton X-100 at high concentration (0.5 %) across three different mRNA-LNP formulations. Interestingly, Tween 20 was the most effective at releasing mRNA from LNPs, across all concentration ranges explored (0.0025 %, 0.01 %, 0.1 % and to 0.5 % (v/v)) highlighting its potency at solubilizing the three different LNP formulations. Our results show that Tween 20 can be used as an alternative to Triton X-100 in the RiboGreen assay, resulting in more accurate quantification of the total mRNA concentration and EE%, as well as making the assay more environmentally friendly. Such improvement could potentially increase the likelihood of identifying therapeutically attractive hard-to-solubilize LNP-mRNA formulations that would be discharged when using Triton X-100 due to their apparent low EE values, as well as ensure more accurate mRNA dosing in both in vitro and in vivo studies.