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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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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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.
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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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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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DOI: https://doi.org/10.1038/s41596-020-00418-9
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