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Together is Better: mRNA Co-Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co-Delivery and Protein Expression on the Single Cell Level
来源: | 作者:Heyang Zhang 1, Jeroen Bussmann 2, Florian H Huhnke 3, Joke Devoldere 1, An-Katrien Minnaert 1, Wim Jiskoot 2, Friedhelm Serwane 3 4 5 6, Joachim Spatz 3 7, Magnus Röding 8 9, Stefaan C De Smedt 1 10, Kevin Braeckmans 1 11, Katrien Remaut 1 10 | 发布时间: 2022-01-06 | 331 次浏览 | 分享到:

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.

Keywords: cellular uptake; mingle/single-mRNA lipoplex; protein expression; single cell; theoretical modeling.