Together is Better: mRNA Co‐Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co‐Delivery and Protein Expression on the Single Cell Level
Abstract 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...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2022-02-01
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| Series: | Advanced Science |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/advs.202102072 |
| _version_ | 1819275665371824128 |
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| author | Heyang Zhang Jeroen Bussmann Florian H. Huhnke Joke Devoldere An‐Katrien Minnaert Wim Jiskoot Friedhelm Serwane Joachim Spatz Magnus Röding Stefaan C. De Smedt Kevin Braeckmans Katrien Remaut |
| author_facet | Heyang Zhang Jeroen Bussmann Florian H. Huhnke Joke Devoldere An‐Katrien Minnaert Wim Jiskoot Friedhelm Serwane Joachim Spatz Magnus Röding Stefaan C. De Smedt Kevin Braeckmans Katrien Remaut |
| author_sort | Heyang Zhang |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-12-23T23:27:56Z |
| format | Article |
| id | doaj.art-836eb56c915b40afa530d03660979760 |
| institution | Directory Open Access Journal |
| issn | 2198-3844 |
| language | English |
| last_indexed | 2024-12-23T23:27:56Z |
| publishDate | 2022-02-01 |
| publisher | Wiley |
| record_format | Article |
| series | Advanced Science |
| spelling | doaj.art-836eb56c915b40afa530d036609797602022-12-21T17:26:10ZengWileyAdvanced Science2198-38442022-02-0194n/an/a10.1002/advs.202102072Together is Better: mRNA Co‐Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co‐Delivery and Protein Expression on the Single Cell LevelHeyang Zhang0Jeroen Bussmann1Florian H. Huhnke2Joke Devoldere3An‐Katrien Minnaert4Wim Jiskoot5Friedhelm Serwane6Joachim Spatz7Magnus Röding8Stefaan C. De Smedt9Kevin Braeckmans10Katrien Remaut11Laboratory of General Biochemistry and Physical Pharmacy Faculty of Pharmaceutical Sciences Ghent University Ghent 9000 BelgiumDivision of BioTherapeutics Leiden Academic Center for Drug Research Leiden University Leiden 2333 CC The NetherlandsMax Planck Institute for Medical Research Department of Cellular Biophysics 70569 Stuttgart GermanyLaboratory of General Biochemistry and Physical Pharmacy Faculty of Pharmaceutical Sciences Ghent University Ghent 9000 BelgiumLaboratory of General Biochemistry and Physical Pharmacy Faculty of Pharmaceutical Sciences Ghent University Ghent 9000 BelgiumDivision of BioTherapeutics Leiden Academic Center for Drug Research Leiden University Leiden 2333 CC The NetherlandsMax Planck Institute for Medical Research Department of Cellular Biophysics 70569 Stuttgart GermanyMax Planck Institute for Medical Research Department of Cellular Biophysics 70569 Stuttgart GermanyRISE Research Institutes of Sweden Bioeconomy and Health, Agriculture and Food Göteborg 41276 SwedenLaboratory of General Biochemistry and Physical Pharmacy Faculty of Pharmaceutical Sciences Ghent University Ghent 9000 BelgiumLaboratory of General Biochemistry and Physical Pharmacy Faculty of Pharmaceutical Sciences Ghent University Ghent 9000 BelgiumLaboratory of General Biochemistry and Physical Pharmacy Faculty of Pharmaceutical Sciences Ghent University Ghent 9000 BelgiumAbstract 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.https://doi.org/10.1002/advs.202102072cellular uptakemingle/single‐mRNA lipoplexprotein expressionsingle celltheoretical modeling |
| spellingShingle | Heyang Zhang Jeroen Bussmann Florian H. Huhnke Joke Devoldere An‐Katrien Minnaert Wim Jiskoot Friedhelm Serwane Joachim Spatz Magnus Röding Stefaan C. De Smedt Kevin Braeckmans Katrien Remaut Together is Better: mRNA Co‐Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co‐Delivery and Protein Expression on the Single Cell Level Advanced Science cellular uptake mingle/single‐mRNA lipoplex protein expression single cell theoretical modeling |
| title | Together is Better: mRNA Co‐Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co‐Delivery and Protein Expression on the Single Cell Level |
| title_full | Together is Better: mRNA Co‐Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co‐Delivery and Protein Expression on the Single Cell Level |
| title_fullStr | Together is Better: mRNA Co‐Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co‐Delivery and Protein Expression on the Single Cell Level |
| title_full_unstemmed | Together is Better: mRNA Co‐Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co‐Delivery and Protein Expression on the Single Cell Level |
| title_short | Together is Better: mRNA Co‐Encapsulation in Lipoplexes is Required to Obtain Ratiometric Co‐Delivery and Protein Expression on the Single Cell Level |
| title_sort | together is better mrna co encapsulation in lipoplexes is required to obtain ratiometric co delivery and protein expression on the single cell level |
| topic | cellular uptake mingle/single‐mRNA lipoplex protein expression single cell theoretical modeling |
| url | https://doi.org/10.1002/advs.202102072 |
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