Physiochemical Characterization of Firefly Luciferase mRNA Nanoparticles Using Dynamic Light Scattering

Abstract

Gene therapy using messenger RNA (mRNA) delivery has recently shown potential for applications in gene editing, cellular reprogramming, and cancer immunotherapy.  Although these modalities are promising, the large size of mRNA transcripts and their enzymatic susceptibility render its delivery challenging. For safety and efficacy considerations, size and heterogeneity (polydispersity) of mRNA nanoparticles need to be characterized. Dynamic Light Scattering (DLS) is an effective, non-invasive method for measuring nanocomplex size as well as the polydispersity index (PDI) or the particle distribution of the sample. However, parameters must be optimized to ensure valid and reproducible size measurements. In this study, Firefly Luciferase mRNA is prepared with a commercial transfection reagent, Lipofectamine MessengerMAX, and then diluted in two dispersants. Four experimental methods were created to evaluate the measurement variables. The stability of cationic, lipid-based nanoparticles was also assessed using size changes over time. After analyzing the data, the recommended parameters for DLS measurements are automatic runs and ten measurements. In addition, dilutions should be avoided to maintain high-quality reports and to minimize variability. The preliminary conclusion for Lipid-Nanoparticle stability is that size increases over time at 4 degrees C. The future implications of these results are a method for assessing stability and size constraints upon mRNA delivery.