Correlation of mRNA delivery timing and protein expression in lipid-based transfection

A. Reiser; D. Woschée; N. Mehrotra; R. Krzyszton; H. H. Strey; J. O. Rädler

Here we study the time-to-expression after mRNA delivery as well as the expression rate using high-throughput single-cell analysis of eGFP reporter fluorescence. We employ micropatterned single-cell arrays and automated live-cell time-lapse microscopy to collect individual fluorescence time courses. Fitting the time courses to a translation-maturation model, we obtain best estimates for the onset time and expression rate distributions. The single-cell data reveal large cell-to-cell variability and the insight that the delivery time and expression rate do not correlate at the single-cell level. Nevertheless, both parameters change in a concerted manner as a function of external parameters such as the amount of serum. In mRNA transfection experiments using Lipofectamine lipoplexes the efficiency decreases, and delivery timing increases as a function of the fraction of serum protein. In contrast, for lipid nanoparticles (LNPs) containing the ionizable cationic lipid DLin-MC3-DMA efficiency improves and timing shortens in the course of increasing serum.