Inside the Cell: Approaches to Evaluating mRNA Internalization and Trafficking

With the growing prominence of mRNA-based therapeutics and vaccines, accurately assessing the cellular uptake of mRNA complexes is a critical first step in evaluating both the efficiency of delivery systems and their downstream therapeutic potential. This is especially important when working with novel mRNA constructs, comparing different delivery vectors, or targeting diverse cell types. In this study, we present a suite of methods to quantify and visualize mRNA internalization following transfection of three types of human primary cells: esenchymal stromal cells, fibroblasts, and osteoblasts. We highlight the utility of fluorescent probes for both qualitative and quantitative assessment of mRNA uptake and intracellular trafficking. To dissect the pathways involved in uptake, we employed three distinct endocytic inhibitors— chlorpromazine, wortmannin, and genistein—each targeting specific endocytic mechanisms. Additionally, we provide protocols for the lipid-based transfection agents Lipofectamine 3000 and 3DFect, which can be adapted for use with similar vectors. Key methodologies such as flow cytometry and correlative light and electron microscopy, known as CLEM, are escribed in detail for their effectiveness in analyzing mRNA internalization. A deeper understanding of the internalization and intracellular fate of mRNA is ssential for the advancement of more efficient and safer mRNA-based delivery platforms.