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Tissue nanotransfection

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Tissue nanotransfection (TNT) is an electroporation-based technique capable of gene and drug cargo delivery or transfection at the nanoscale. Furthermore, TNT is a scaffold-less tissue engineering (TE) technique that can be considered cell-only or tissue inducing depending on cellular or tissue level applications. The transfection method makes use of nanochannels to deliver cargo to tissues topically.  

History

Cargo delivery methods rely on carriers, for example nanoparticles, viral vectors, or physical approaches such as gene guns, microinjection, or electroporation[1][2][3][4][5][6][7][8][9] The various methods can be limited by size constraints or their ability to efficiently deliver cargo without damaging tissue. Electroporation is a physical method which harnesses an electric field to open pores in the normally semi-permeable cell membrane through which cargo can enter. In this process, the charges can be used to drive cargo in a specific direction.

Bulk electroporation (BEP) is the most conventional electroporation method. Benefits come in the form of high throughput and minimal set-up times.[7] The downside of BEP is that the cell membrane experiences an uneven distribution of the electric field and many membranes receive irreversible damage from which they can no longer close, thus leading to low cell viability.

Attempts have been made to miniaturize electroporation such as microelectroporation (MEP)[10] and nanochannel electroporation (NEP)[11] which uses electroporation approached to deliver cargo through micro/nanochannels respectively. These techniques have shown to have higher efficiency of delivery, increased uniform transfection, and increased cell viability compared to BEP.[12]

Technique

Tissue nanotransfection uses custom fabricated nanochannel arrays for nanoscale delivery of genetic cargo directly onto the surface of the skin. The postage stamp-sized chip is placed directly on the skin and an electric current is induced lasting for milliseconds to deliver the gene cargo with precise control. This approach delivers ample amounts of reprogramming factors to single-cells, creating potential for a powerful gene transfection and reprogramming method.[11][12] The delivered cargo then transforms the affected cells into a desired cell type without first transforming them to stem cells. TNT is a novel technique and has been used on mice models to successfully transfect fibroblasts into neuron-like cells along with rescue of ischemia in mice models with induced vasculature and perfusion [13]. Current methods require the fabricated TNT chip to be placed on the skin and the loading reservoir filled with a gene solution. An electrode (cathode) is placed into the well with a counter electrode (anode) placed under the chip intradermally (into the skin). The electric field generated delivers the genes.[13]

Initial TNT experiments showed that genes could be delivered to the skin of mice.[13] Once this was confirmed, a cocktail of gene factors (ABM) used by Vierbuchen[14] and collaborators to reprogram fibroblast into neurons was used.[12][13] Delivery of these factors demonstrated successful reprogramming in-vivo  and signals propagated from the epidermis to the dermis skin layers. This phenomenon is believed to be mediated by extracellular vesicles[15] and potentially other factors [18]. Successful reprogramming was determined by performing histology and electrophysiological tests to confirm the tissue behaved as functional neurons.[13]

Beyond inducing neurons, Gallego-Perez et al also set out to induce endothelial cells in an ischemic mouse limb that, without proper blood flow, becomes necrotic and decays. Using a patented cocktail of plasmids (Etv2, Fli1, Foxc2, or EFF), these factors were delivered to the tissue above the surgery site. Using various methods, including histology and laser speckle imaging, perfusion and the establishment of new vasculature was verified as early as 7 days post-treatment.[13]

The technique was developed to combat the limitations of current approaches, such as a shortage in donors to supply cell sources and the need to induce pluripotency.[14][15][16][17][18][19] Reprogramming cells in vivo takes advantage of readily available cells, bypassing the need for pre-processing.[20][21] Most reprogramming methods have a heavy reliance on viral transfection [22][23] TNT allows for implementation of a non-viral approach which is able to overcome issues of capsid size, increase safety, and increase deterministic reprogramming.[13]

Development

The tissue nanotransfection technique was developed as a method to efficiently and benignly deliver cargo to living tissues. This technique builds on the high-throughput nanoelectroporation methods developed for cell reprogramming applications by Dr. Lee and Dr. Gallego-Perez of Ohio State’s Chemical and Biomolecular Engineering department.. Development was a joint effort between OSU’s College of Engineering and College of Medicine led by Dr. Gallego-Perez (Ph.D), Dr. Lee (Ph.D), and Dr. Sen (Ph.D)

This technology was fabricated using cleanroom techniques and photolithography and deep reactive ion etching (DRIE) of silicon wafers to create nanochannels with backside etching of a reservoir for loading desired factors as described in Gallego-Perez et al 2017.[13] This chip is then connected to an electrical source capable of delivering an electrical field to drive the factors from the reservoir into the nanochannels, and onto the contacted tissue.  

References

  1. ^ Chen Z, Zhang A, Wang X, Zhu J, Fan Y, Yu H, Yang Z (2017). "The Advances of Carbon Nanotubes in Cancer Diagnostics and Therapeutics". Journal of Nanomaterials. 2017: 1–13. doi:10.1155/2017/3418932.
  2. ^ Kang C, Sun Y, Zhu J, Li W, Zhang A, Kuang T, Xie J, Yang Z (2016-09-30). "Delivery of Nanoparticles for Treatment of Brain Tumor". Current Drug Metabolism. 17 (8): 745–754. doi:10.2174/1389200217666160728152939. PMID 27469219.
  3. ^ Xie J, Yang Z, Zhou C, Zhu J, Lee RJ, Teng L (July 2016). "Nanotechnology for the delivery of phytochemicals in cancer therapy". Biotechnology Advances. 34 (4): 343–353. doi:10.1016/j.biotechadv.2016.04.002. PMID 27071534.
  4. ^ Chen Z, Chen Z, Zhang A, Hu J, Wang X, Yang Z (June 2016). "Electrospun nanofibers for cancer diagnosis and therapy". Biomaterials Science. 4 (6): 922–32. doi:10.1039/C6BM00070C. PMID 27048889.
  5. ^ Sha L, Chen Z, Chen Z, Zhang A, Yang Z (2016). "Polylactic Acid Based Nanocomposites: Promising Safe and Biodegradable Materials in Biomedical Field". International Journal of Polymer Science. 2016: 1–11. doi:10.1155/2016/6869154.
  6. ^ Xie J, Teng L, Yang Z, Zhou C, Liu Y, Yung BC, Lee RJ (2013). "A polyethylenimine-linoleic acid conjugate for antisense oligonucleotide delivery". BioMed Research International. 2013: 710502. doi:10.1155/2013/710502. PMC 3683435. PMID 23862153.
  7. ^ a b Shi J, Ma Y, Zhu J, Chen Y, Sun Y, Yao Y, Yang Z, Xie J (November 2018). "A Review on Electroporation-Based Intracellular Delivery". Molecules. 23 (11): 3044. doi:10.3390/molecules23113044. PMC 6278265. PMID 30469344.
  8. ^ Sun J, Wang X, Wu J, Jiang C, Shen J, Cooper MA, Zheng X, Liu Y, Yang Z, Wu D (April 2018). "Biomimetic Moth-eye Nanofabrication: Enhanced Antireflection with Superior Self-cleaning Characteristic". Scientific Reports. 8 (1): 5438. Bibcode:2018NatSR...8.5438S. doi:10.1038/s41598-018-23771-y. PMC 5883013. PMID 29615712.
  9. ^ Sun J, Kormakov S, Liu Y, Huang Y, Wu D, Yang Z (July 2018). "Recent Progress in Metal-Based Nanoparticles Mediated Photodynamic Therapy". Molecules. 23 (7): 1704. doi:10.3390/molecules23071704. PMC 6099795. PMID 30002333.
  10. ^ Kurosawa O, Oana H, Matsuoka S, Noma A, Kotera H, Washizu M (2006-12-01). "Electroporation through a micro-fabricated orifice and its application to the measurement of cell response to external stimuli". Measurement Science and Technology. 17 (12): 3127–3133. doi:10.1088/0957-0233/17/12/S02. S2CID 121182614.
  11. ^ a b Boukany PE, Morss A, Liao WC, Henslee B, Jung H, Zhang X, Yu B, Wang X, Wu Y, Li L, Gao K, Hu X, Zhao X, Hemminger O, Lu W, Lafyatis GP, Lee LJ (October 2011). "Nanochannel electroporation delivers precise amounts of biomolecules into living cells". Nature Nanotechnology. 6 (11): 747–54. Bibcode:2011NatNa...6..747B. doi:10.1038/nnano.2011.164. PMID 22002097.
  12. ^ a b c Gallego-Perez D, Otero JJ, Czeisler C, Ma J, Ortiz C, Gygli P, et al. (February 2016). "Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers". Nanomedicine. 12 (2): 399–409. doi:10.1016/j.nano.2015.11.015. PMC 5161095. PMID 26711960.
  13. ^ a b c d e f g h Gallego-Perez D, Pal D, Ghatak S, Malkoc V, Higuita-Castro N, Gnyawali S, Chang L, Liao WC, Shi J, Sinha M, Singh K, Steen E, Sunyecz A, Stewart R, Moore J, Ziebro T, Northcutt RG, Homsy M, Bertani P, Lu W, Roy S, Khanna S, Rink C, Sundaresan VB, Otero JJ, Lee LJ, Sen CK (October 2017). "Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue". Nature Nanotechnology. 12 (10): 974–979. Bibcode:2017NatNa..12..974G. doi:10.1038/nnano.2017.134. PMC 5814120. PMID 28785092.
  14. ^ a b Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Südhof TC, Wernig M (February 2010). "Direct conversion of fibroblasts to functional neurons by defined factors". Nature. 463 (7284): 1035–41. Bibcode:2010Natur.463.1035V. doi:10.1038/nature08797. PMC 2829121. PMID 20107439.
  15. ^ a b Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO (June 2007). "Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells". Nature Cell Biology. 9 (6): 654–9. doi:10.1038/ncb1596. PMID 17486113. S2CID 8599814.
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  • Veetil AT, Chakraborty K, Xiao K, Minter MR, Sisodia SS, Krishnan Y (December 2017). "Cell-targetable DNA nanocapsules for spatiotemporal release of caged bioactive small molecules". Nature Nanotechnology. 12 (12): 1183–1189. Bibcode:2017NatNa..12.1183V. doi:10.1038/nnano.2017.159. PMID 28825714.
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