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.