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

In order to record single-cell protein expression time courses in a highly parallel manner we employed an LISCA approach as published earlier (18) . HuH7, liver carcinoma, cells were seeded onto a microstructured cell culture channel slide (see Figure 1A ). In order to capture the very early events of protein expression the single-cell array was connected to a tailored perfusion system and scanning time-lapse data acquisition was started prior to transfection. The perfusion system allowed for fluid exchange and enabled the addition of transfection agents on the stage during the time-lapse measurement. mRNA complexes were added one hour after the beginning of the measurement and incubated for one hour. After the incubation period the transfection solution was exchanged for cell growth medium (indicated by the arrows and the gray bar in Figure 1B . Note that without the perfusion system, it is not possible to observe changes in the fluorescence intensity during and shortly after the time period of the transfection due to sample handling off the microscope. Secondly, with the use of a perfusion system, we prevent particle adsorption events after the incubation time by flushing out unbound nanocarriers. This is illustrated in Supplementary Figure 1A using fluorescently labeled lipoplexes before and after flushing. To investigate whether nanocarrier are taken up, we prepared lipoplexes containing mRNA with a Cy5 labeled fraction to visualize the lipoplexes during the time-lapse measurement. The fluorescence kinetics of the Cy5 labeled mRNAs shows the adsorption onto the cell surfaces (see Supplementary Figure 1B ). As a reference, we observed on areas with no cells that the fluorescence time courses of nanocarriers increases during the incubation period and abruptly decreases when the chamber is flushed with fresh carrier-free medium indicating that some partially adherent nanocarriers are rinsed away. Interestingly, in contrast, the fluorescence of nanocarriers adsorbed to cells not only does not decrease but increases within the next 1 hour after flushing. We interpret this time course such that firstly, nanocarriers are strongly adsorb and are not rinsed away and secondly, the fluorescence of the Cy5 labeled mRNAs is likely to increase during the time of uptake due to unpacking of the lipid-based carriers and thus dequenching the Cy5 fluorescence.