Schultz David, Münter Rasmus D, Cantín Alex M, Kempen Paul J, Jahnke Nadin, Andresen Thomas L, Simonsen Jens B, Urquhart Andrew J
Department of Health Technology, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
Department of Health Technology, Technical University of Denmark, 2800 Kongens Lyngby, Denmark; National Centre for Nano Fabrication and Characterization, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
Eur J Pharm Biopharm. 2024 Dec;205:114571. doi: 10.1016/j.ejpb.2024.114571. Epub 2024 Oct 28.
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.
为了量化脂质纳米颗粒（LNP）中mRNA的浓度和包封效率（EE），广泛使用了RiboGreen测定法。作为该测定法的一部分，使用表面活性剂从LNP中释放mRNA，以便用RiboGreen染料进行检测。到目前为止，所选用的表面活性剂一直是Triton X-100，它对人类健康和环境有害。因此需要Triton X-100的替代品，但令人惊讶的是，文献中尚未描述此类研究。在这里，我们展示了三种危害较小的表面活性剂（Brij 93、两性离子去污剂3-14和吐温20）与Triton X-100相比，在通过RiboGreen测定法从LNP中释放mRNA方面的情况。我们发现，在三种不同的mRNA-LNP制剂中，高浓度（0.5%）的两性离子去污剂3-14和吐温20至少与高浓度（0.5%）的Triton X-100一样有效。有趣的是，在所有探索的浓度范围（0.0025%、0.01%、0.1%和0.5%（v/v））内，吐温20从LNP中释放mRNA的效果最为显著，突出了其溶解三种不同LNP制剂的能力。我们的结果表明，吐温20可在RiboGreen测定法中用作Triton X-100的替代品，从而更准确地定量总mRNA浓度和EE%，并使测定法更环保。这种改进可能会增加识别具有治疗吸引力但难以溶解的LNP-mRNA制剂的可能性，这些制剂在使用Triton X-100时由于其明显较低的EE值而被排除，同时也能确保在体外和体内研究中更准确地进行mRNA给药。
