Together is Better: mRNA Co‐Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co‐Delivery and Protein Expression on the Single Cell Level

Liposomes can efficiently deliver messenger RNA (mRNA) into cells. When mRNA cocktails encoding different proteins are needed, a considerable challenge is to efficiently deliver all mRNAs into the cytosol of each individual cell. In this work, two methods are explored to co‐deliver varying ratiometric doses of mRNA encoding red (R) or green (G) fluorescent proteins and it is found that packaging mRNAs into the same lipoplexes (mingle‐lipoplexes) is crucial to efficiently deliver multiple mRNA types into the cytosol of individual cells according to the pre‐defined ratio. A mixture of lipoplexes containing only one mRNA type (single‐lipoplexes), however, seem to follow the “first come – first serve” principle, resulting in a large variation of R/G uptake and expression levels for individual cells leading to ratiometric dosing only on the population level, but rarely on the single‐cell level. These experimental observations are quantitatively explained by a theoretical framework based on the stochasticity of mRNA uptake in cells and endosomal escape of mingle‐ and single‐lipoplexes, respectively. Furthermore, the findings are confirmed in 3D retinal organoids and zebrafish embryos, where mingle‐lipoplexes outperformed single‐lipoplexes to reliably bring both mRNA types into single cells. This benefits applications that require a strict control of protein expression in individual cells.