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

The delivery time distributions after mRNA transfection, are measured here for the first time at the single cell level, reveal that the delivery process occurs over a time period of only a few hours (see Figure 2C and Figure 4A ). The FWHM of the distributions fitted by Gamma distribution functions increase with increasing FBS concentration (see Supplementary Figure 3A ). This finding is in agreement with the literature that there is only a small window of opportunity for successful carrier release before the nucleic acid nanocarriers are trapped in late endosomes or lysosomes (12) . Since we apply a pulsed incubation in our experiments, with a period restricted to only an hour, the observed onset time distribution corresponds to the delivery time distribution. Note that the onset time distribution in the case of continuing lipoplex incubation exhibits a much broader and prolonged distribution (see Supplementary Figure 7 ) Hence, absorption and uptake is a continuous process, while if mRNA loaded particles are trapped in endosomes or lysosomes they are subject to degradation and an intact release becomes less likely with time. This scenario explains why we do not observe late expression onsets in pulsed incubation. This mechanism, however, would suggest that cells with short delivery times show higher expression efficiency. However, we observed no correlation between delivery time and expression rate at the single-cell level (see Figure 4B ). This means that there is no intrinsic mechanism linking the delivery time with expression efficiency and that the expression rates' variance is independent of the delivery times' variance. However, effects induced by external changes such as the level of blood serum proteins (FBS) act on both delivery time and expression rate in a systematic manner. We found that the transfection efficiency decreases, and delivery time increases for Lipofectamine mediated transfection, while the opposite occurs for LNPs. In case of Lipofectamine unspecific protein adsorption, which leads to the formation of a protein corona on the lipoplex surface, explains this effect (27, 28) . The protein corona changes the structure and surface charge of lipoplexes, which influences the interaction between nanocarriers and cells for example during adsorption or endosomal uptake (29) . Interestingly, the protein corona has different effects on delivery time and expression rate for LNPs, where delivery shifts to faster times and higher expression rates. It is known that different nanocarrier formulations lead to different targeting of endocytotic pathways (30) to different quantities of proteins forming the corona (31). In particular LNP formulations containing ionizable lipids and dissociable PEG lipid, the serum effect has been explained by apolipoprotein E (apoE) adsorption, (32) , which has been shown to lead to increased uptake in hepatocytes via apoE receptors (33, 34) . The reason why LNPs without FBS are still able to transfect might be because HuH7 cells secret apoE resulting in a lower concentration of apoE in the medium than with FBS present (35) . We showed that protein adsorption on LNPs does not only play a role in faster delivery times but also influence the expression rates. However, similar to the Lipofectamine lipoplexes we did not observe a correlation of delivery time with expression rate for LNPs at the single-cell level.