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Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells

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Abstract

Using siRNAs to genetically manipulate immune cells is important to both basic immunological studies and therapeutic applications. However, siRNA delivery is challenging because primary immune cells are often sensitive to the delivery materials and generate immune responses. We have recently developed an amphiphilic dendrimer that is able to deliver siRNA to a variety of cells, including primary immune cells. We provide here a protocol for the synthesis of this dendrimer, as well as siRNA delivery to immune cells such as primary T and B cells, natural killer cells, macrophages, and primary microglia. The dendrimer synthesis entails straightforward click coupling followed by an amidation reaction, and the siRNA delivery protocol requires simple mixing of the siRNA and dendrimer in buffer, with subsequent application to the primary immune cells to achieve effective and functional siRNA delivery. This dendrimer-mediated siRNA delivery largely outperforms the standard electroporation technique, opening a new avenue for functional and therapeutic studies of the immune system. The whole protocol encompasses the dendrimer synthesis, which takes 10 days; the primary immune cell preparation, which takes 3–10 d, depending on the tissue source and cell type; the dendrimer-mediated siRNA delivery; and subsequent functional assays, which take an additional 3–6 d.

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Fig. 1: Schematic presentation of siRNA delivery mediated by the amphiphilic dendrimer AD.
Fig. 2: Chemical synthesis of the amphiphilic dendrimer AD.
Fig. 3: Experimental outline.
Fig. 4: Functional siRNA delivery mediated by AD in various primary immune cells.
Fig. 5: Experimental outline.

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Acknowledgements

This work was supported by La Ligue Nationale Contre le Cancer (EL2016.LNCC/LPP to L.P.); the NIH (grants R01AI29329, R01AI42552, and R01HL07470 to J.J.R., as well as grant P30CA033572 for City of Hope Core Facility support); National Science Centre Poland (no. 2017/25/B/NZ3/02483 to A.E.-M.); the French National Research Agency and the Italian Ministry of Health under the frame of the Era-Net EURONANOMED European Research projects ‘NANOGLIO’ (L.P., A.S., C.L., P.N.M.), ‘TARBRAINFECT’ (L.P.) and ‘NAN-4-TUM’ (L.P.); the China Scholarship Council (J.C., J.T.) and Bourse Eiffel du Campus France (J.C.); the Italian Association for Cancer Research (AIRC) (IG 2015 and IG 2019 to C.L; 22329 2018 to S.G.); and the Italian Research Foundation for ALS (AriSLA) (Pilot NKINALS 2019 to S.G.). This project has received funding from the European Union’s Horizon 2020 research and innovation program H2020 NMBP ‘SAFE-N-MEDTECH’ (L.P.; under grant agreement no. 814607) and ‘NEWDEAL’ (A.D., F.C., P.N.M.; under grant agreement no. 720905). This publication reflects only the authors’ views, and the Commission is not responsible for any use that may be made of the information it contains. This article is based upon work from COST Action CA 17140 ‘Cancer Nanomedicine from the Bench to the Bedside’, supported by COST (European Cooperation in Science and Technology). We thank A. Tintaru from Aix-Marseille University in France for NMR recording, G. Bernardini from the University of Rome La Sapienza in Italy for FACS analysis, and E. Sulpice and X. Gidrol from CEA/IRIG in Grenoble France for generously providing mouse JAK1 siRNA.

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Authors and Affiliations

  1. Aix-Marseille Université, Center Interdisciplinaire de Nanoscience de Marseille, UMR 7325, Equipe Labellisée Ligue Contre le Cancer, CNRS, Marseille, France

    Jiaxuan Chen, Jingjie Tang, Yifan Jiang & Ling Peng

  2. State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Drug Discovery, Center of Advanced Pharmaceutics and Biomaterials, China Pharmaceutical University, Nanjing, P. R. China

    Jiaxuan Chen & Xiaoxuan Liu

  3. Laboratory of Molecular Neurobiology, Neurobiology Center, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, Warsaw, Poland

    Aleksandra Ellert-Miklaszewska & Bozena Kaminska

  4. Department of Physiology and Pharmacology, Sapienza University of Rome, Rome, Italy

    Stefano Garofalo & Cristina Limatola

  5. Institute for Advanced Biosciences, University Grenoble-Alpes, Inserm U1209, CNRS 5309, La Tronche, France

    Arindam K. Dey, Flora Clément & Patrice N. Marche

  6. IRCCS Neuromed, Pozzilli, Italy

    Angela Santoni & Cristina Limatola

  7. Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope Medical Center, Monrovia, CA, USA

    John J. Rossi & Jiehua Zhou

Authors
  1. Jiaxuan Chen
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  2. Aleksandra Ellert-Miklaszewska
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  3. Stefano Garofalo
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  9. Xiaoxuan Liu
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  10. Bozena Kaminska
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  11. Angela Santoni
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  13. John J. Rossi
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  14. Jiehua Zhou
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  15. Ling Peng
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Contributions

Conceptualization, J.Z., L.P.; writing and editing, J.Z., L.P., J.C., Y.J., J.T., A.E.-M., B.K., C.L., S.G., A.S., A.K.D., F.C., P.N.M.; funding acquisition, L.P., J.J.R., B.K., A.E.-M., C.L., P.N.M.; all authors read and approved the final manuscript.

Corresponding authors

Correspondence to Jiehua Zhou or Ling Peng.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Jørn B. Christensen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Liu, X. et al. Angew. Chem. Int. Ed. 53, 11822–11827 (2014): https://doi.org/10.1002/anie.201406764

Ellert-Miklaszewska, A. et al. Nanomedicine (Lond.) 14, 2441–2458 (2019): https://doi.org/10.2217/nnm-2019-0176

Garofalo, S. et al. Nat. Commun. 11, 1773 (2020): https://doi.org/10.1038/s41467-020-15644-8

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Supplementary Information

Supplementary Figs. 1–8, Supplementary Results, Supplementary Methods and Supplementary References.

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Source Data Fig. 4

Statistical source data for Fig. 4a,b,c, d, f, g.

Source Data Fig. 4

Unprocessed western blots (gels) for Figs. 4e and 4h.

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Chen, J., Ellert-Miklaszewska, A., Garofalo, S. et al. Synthesis and use of an amphiphilic dendrimer for siRNA delivery into primary immune cells. Nat Protoc 16, 327–351 (2021). https://doi.org/10.1038/s41596-020-00418-9

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