Tissue nanotransfection (TNT) is an electromotive gene transfer technology that was developed to achieve tissue reprogramming in vivo. This protocol describes how to fabricate the required hardware, commonly referred to as a TNT chip, and use it for in vivo TNT. Silicon hollow-needle arrays for TNT applications are fabricated in a standardized and reproducible way. In <1 s, these silicon hollow-needle arrays can be used to deliver plasmids to a predetermined specific depth in murine skin in response to pulsed nanoporation. Tissue nanotransfection eliminates the need to use viral vectors, minimizing the risk of genomic integration or cell transformation. The TNT chip fabrication process typically takes 5–6 d, and in vivo TNT takes 30 min. This protocol does not require specific expertise beyond a clean room equipped for basic nanofabrication processes.
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The individual images used to compile the figures shown and additional examples of the types of results obtained are available from Yi Xuan upon reasonable request. The design lithography patterns we used for needle fabrication are available at https://doi.org/10.6084/m9.figshare.16528311. Source data are provided with this paper.
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This work made use of the Pritzker Nanofabrication Facility, which receives partial support from the SHyNE Resource, a node of the National Science Foundation’s National Nanotechnology Coordinated Infrastructure (NSF ECCS-2025633). We thank Parker Evans for his help in measuring the force applied during the TNT process. This work was supported in part by NIH grant DK128845; Department of Defense grants W81XWH-21-1-0097, W81XWH-21-1-0033 and W81XWH-20-1-251 to C.K.S.; Department of Defense grant W81XWH-21-1-0047 to S.K.; and NIH grant GM143572 to Y.X.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Tarun Bhattacharyya, Kui Cheng, Yuanyu Huang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key reference using this protocol
Gallego-Perez, D. et al. Nat. Nanotechnol. 12, 974–979 (2017): https://doi.org/10.1038/nnano.2017.134
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Xuan, Y., Ghatak, S., Clark, A. et al. Fabrication and use of silicon hollow-needle arrays to achieve tissue nanotransfection in mouse tissue in vivo. Nat Protoc 16, 5707–5738 (2021). https://doi.org/10.1038/s41596-021-00631-0
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