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Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation

Nature Biomedical Engineering volume 4, pages 69–83 (2020)Cite this article

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Abstract

Exosomes are attractive as nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and their ability to penetrate physiological barriers that are impermeable to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs, into cell-secreted exosomes leads to low yields. Here we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared with bulk electroporation and other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and a more than 103-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic phosphatase and tensin homologue (PTEN)-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced inhibition of tumour growth and increased survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

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Fig. 1: CNP generates large quantities of EVs loaded with transcribed mRNAs.
Fig. 2: Exosomes, rather than MVs, contain functionally transcribed mRNAs after CNP transfection.
Fig. 3: CNP-induced MVB formation.
Fig. 4: CNP-induced exosome secretion is associated with Ca2+ ion influx after CNP.
Fig. 5: CNP increases exosome release through HSP–p53–TASP6 signalling pathway.
Fig. 6: In vitro study of CNP-generated exosomes for gene therapy and immunogenicity evaluation in mice.
Fig. 7: In vivo therapeutic efficacy of CNP-generated exosomes in a U87 orthotopic glioma model.
Fig. 8: In vivo therapeutic efficacy of CNP-generated exosomes in a GL261 orthotopic glioma model.

Data availability

The datasets generated and analysed during the study are publicly available at http://osf.io/byahe (Open Science Framework) and can also be requested from the corresponding authors.

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References

  1. Fraietta, J. A. et al. Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells. Nature 558, 307–312 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Borducchi, E. N. et al. Ad26/MVA therapeutic vaccination with TLR7 stimulation in SIV-infected rhesus monkeys. Nature 540, 284–287 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Dickson, D. UK scientists test liposome gene therapy technique. Nature 365, 4 (1993).

    CAS  PubMed  Google Scholar 

  4. Lam, F. C. et al. Enhanced efficacy of combined temozolomide and bromodomain inhibitor therapy for gliomas using targeted nanoparticles. Nat. Commun. 9, 1991 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Sun, T. et al. Closed-loop control of targeted ultrasound drug delivery across the blood–brain/tumor barriers in a rat glioma model. Proc. Natl Acad. Sci. USA 114, E10281–E10290 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Singh, A. et al. Multifunctional photonics nanoparticles for crossing the blood-brain barrier and effecting optically trackable brain theranostics. Adv. Funct. Mater. 26, 7057–7066 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Erel-Akbaba, G. et al. Radiation-induced targeted nanoparticle-based gene delivery for brain tumor therapy. ACS Nano. 13, 4028–4040 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Alvarez-Erviti, L. et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29, 341–345 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Kamerkar, S. et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature 546, 498–503 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Andaloussi, S. E., Mager, I., Breakefield, X. O. & Wood, M. J. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat. Rev. Drug Discov. 12, 347–357 (2013).

    Article  CAS  Google Scholar 

  11. Squadrito, M. L., Cianciaruso, C., Hansen, S. K. & De Palma, M. EVIR: chimeric receptors that enhance dendritic cell cross-dressing with tumor antigens. Nat. Methods 15, 183–186 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yeo, R. W. et al. Mesenchymal stem cell: an efficient mass producer of exosomes for drug delivery. Adv. Drug Deliv. Rev. 65, 336–341 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Stewart, M. P. et al. In vitro and ex vivo strategies for intracellular delivery. Nature 538, 183–192 (2016).

    Article  CAS  PubMed  Google Scholar 

  14. Usman, W. M. et al. Efficient RNA drug delivery using red blood cell extracellular vesicles. Nat. Commun. 9, 2359 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Kojima, R. et al. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nat. Commun. 9, 1305 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Wang, Q. et al. ARMMs as a versatile platform for intracellular delivery of macromolecules. Nat. Commun. 9, 960 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Boukany, P. E. et al. Nanochannel electroporation delivers precise amounts of biomolecules into living cells. Nat. Nanotechnol. 6, 747–754 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Gallego-Perez, D. et al. Topical tissue nano-transfection mediates non-viral stroma reprogramming and rescue. Nat. Nanotechnol. 12, 974–979 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654–659 (2007).

    Article  CAS  PubMed  Google Scholar 

  20. Tricarico, C., Clancy, J. & D'Souza-Schorey, C. Biology and biogenesis of shed microvesicles. Small GTPases 8, 220–232 (2017).

    Article  CAS  PubMed  Google Scholar 

  21. Khushman, M. et al. Exosomal markers (CD63 and CD9) expression pattern using immunohistochemistry in resected malignant and nonmalignant pancreatic specimens. Pancreas 46, 782–788 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yang, Z. et al. Functional exosome-mimic for delivery of siRNA to cancer: in vitro and in vivo evaluation. J. Control. Release 243, 160–171 (2016).

    Article  CAS  PubMed  Google Scholar 

  23. Sako, Y., Minoghchi, S. & Yanagida, T. Single-molecule imaging of EGFR signalling on the surface of living cells. Nat. Cell Biol. 2, 168–172 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. Lee, L. J. et al. Extracellular mRNA detected by tethered lipoplex nanoparticle biochip for lung adenocarcinoma detection. Am. J. Respir. Crit. Care Med. 193, 1431–1433 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Savina, A., Furlan, M., Vidal, M. & Colombo, M. I. Exosome release is regulated by a calcium-dependent mechanism in K562 cells. J. Biol. Chem. 278, 20083–20090 (2003).

    Article  CAS  PubMed  Google Scholar 

  26. Messenger, S. W., Woo, S. S., Sun, Z. & Martin, T. F. J. A Ca2+-stimulated exosome release pathway in cancer cells is regulated by Munc13-4. J. Cell Biol. 217, 2877–2890 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Muller, L., Schaupp, A., Walerych, D., Wegele, H. & Buchner, J. Hsp90 regulates the activity of wild type p53 under physiological and elevated temperatures. J. Biol. Chem. 279, 48846–48854 (2004).

    Article  PubMed  CAS  Google Scholar 

  28. Walerych, D. et al. Hsp70 molecular chaperones are required to support p53 tumor suppressor activity under stress conditions. Oncogene 28, 4284–4294 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Yu, X., Harris, S. L. & Levine, A. J. The regulation of exosome secretion: a novel function of the p53 protein. Cancer Res. 66, 4795–4801 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Lespagnol, A. et al. Exosome secretion, including the DNA damage-induced p53-dependent secretory pathway, is severely compromised in TSAP6/Steap3-null mice. Cell Death Differ. 15, 1723–1733 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Ricklefs, F. L. et al. Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles. Sci. Adv. 4, eaar2766 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Zhang, Y. et al. Hypothalamic stem cells control ageing speed partly through exosomal miRNAs. Nature 548, 52–57 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhang, L. et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature 527, 100–104 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hergenreider, E. et al. Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. Nat. Cell Biol. 14, 249–256 (2012).

    Article  CAS  PubMed  Google Scholar 

  35. Wei, Z. et al. Coding and noncoding landscape of extracellular RNA released by human glioma stem cells. Nat. Commun. 8, 1145 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Lobb, R. J. et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J. Extracell. Vesicles 4, 27031 (2015).

    Article  PubMed  Google Scholar 

  37. Lee, D. et al. Protective effect of α-mangostin against iodixanol-induced apoptotic damage in LLC-PK1 cells. Bioorg. Medicinal Chem. Lett. 26, 3806–3809 (2016).

    Article  CAS  Google Scholar 

  38. Mendt, M. et al. Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight 3, 99263 (2018).

    Article  PubMed  Google Scholar 

  39. Kao, C.-Y. & Papoutsakis, E. T. Extracellular vesicles: exosomes, microparticles, their parts, and their targets to enable their biomanufacturing and clinical applications. Curr. Opin. Biotechnol. 60, 89–98 (2019).

    Article  CAS  PubMed  Google Scholar 

  40. Gallego-Perez, D. et al. Deterministic transfection drives efficient nonviral reprogramming and uncovers reprogramming barriers. Nanomedicine 12, 399–409 (2016).

    Article  CAS  PubMed  Google Scholar 

  41. Wei, X. et al. A d-peptide ligand of nicotine acetylcholine receptors for brain-targeted drug delivery. Angew. Chem. 54, 3023–3027 (2015).

    Article  CAS  Google Scholar 

  42. Chung, E. J. et al. Fibrin-binding, peptide amphiphile micelles for targeting glioblastoma. Biomaterials 35, 1249–1256 (2014).

    Article  CAS  PubMed  Google Scholar 

  43. Wang, W., li, J., Wu, K., Azhati, B. & Rexiati, M. Culture and identification of mouse bone marrow-derived dendritic cells and their capability to induce T lymphocyte proliferation. Med. Sci. Monit. 22, 244–250 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Lutz, M. B. et al. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223, 77–92 (1999).

    Article  CAS  PubMed  Google Scholar 

  45. Thery, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluid. Curr. Protoc. Cell Biol. 30, 3.22.1–3.22.29 (2006).

    Article  Google Scholar 

  46. Kowal, J. et al. Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. Proc. Natl Acad. Sci. USA 113, E968–E977 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Xu, Y. et al. Microscopic structure of the polymer-induced liquid precursor for calcium carbonate. Nat. Commun. 9, 2582 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Ross, D., Gaitan, M. & Locascio, L. E. Temperature measurement in microfluidic systems using a temperature-dependent fluorescent dye. Anal. Chem. 73, 4117–4123 (2001).

    Article  CAS  PubMed  Google Scholar 

  49. Mane, D. R., Kale, A. D. & Belaldavar, C. Validation of immunoexpression of tenascin-C in oral precancerous and cancerous tissues using ImageJ analysis with novel immunohistochemistry profiler plugin: an immunohistochemical quantitative analysis. J. Oral. Maxillofac. Pathol. 21, 211–217 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was partially supported by the National Science Foundation of the USA (EEC-0914790, L.J.L.), the National Natural Science Foundation of China (no. 81502999, L.T. and no. 81773758, T.L.), the National Heart, Lung, and Blood Institute (R01HL132355, J.O.), the National Institute of Neurological Disorders and Stroke Grant (R01 NS104315, B.Y.S.K.), the Cancer Prevention and Research Institute of Texas (RR180017, W.J.), the American Brain Tumor Association (DG1900021) and the National Cancer Institute (K08 CA241070, W.J.). We acknowledge J. Perrino (Stanford University) for TEM imaging, which was supported in part by the National Center for Research Resources (1S10RR026780-01). This work’s contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. The authors thank D. Hollingshead at Nanotech West Lab, the Ohio State University for assisting with CNP device fabrication, X. Huang (Department of Biophysics, Peking University) for providing critical help on cryo-EM imaging, F. Meng (School of Life Sciences, Jilin University) for exosome preparation and confocal microscopy, and A. L. Chun of Science Storylab and J. Feinberg of UT Southwestern Medical Center for their editorial services.

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Author notes

  1. These authors contributed equally: Zhaogang Yang, Junfeng Shi.

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA

    Zhaogang Yang, Junfeng Shi, Jingyao Sun, Xinmei Wang, Yifan Ma, Veysi Malkoc, Kwang J. Kwak, Yamin Fan & L. James Lee

  2. Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Zhaogang Yang, Yifan Wang, Weiye Deng & Wen Jiang

  3. School of Life Sciences, Jilin University, Changchun, China

    Jing Xie, Yarong Zhao, Xiuting Zhao & Lesheng Teng

  4. College of Pharmacy, Jinan University, Guangzhou, China

    Tongzheng Liu

  5. College of Medicine, The Ohio State University, Columbus, OH, USA

    Chiling Chiang

  6. Department of Neurological Surgery, Mayo Clinic, Jacksonville, FL, USA

    Yuanxin Chen, Yuan Fu & Betty Y. S. Kim

  7. Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    Chen Kang

  8. Department of Biophysics, Health Science Center, Peking University, Beijing, China

    Changcheng Yin

  9. Institute for Stem Cell Biology, Stanford University, Palo Alto, CA, USA

    June Rhee & Andrew S. Lee

  10. Department of Electrical and Computer Engineering, The Ohio State University, Columbus, OH, USA

    Paul Bertani & Wu Lu

  11. Department of Neuroscience, The Ohio State University, Columbus, OH, USA

    Jose Otero

  12. Peak Center for Pituitary and Brain Tumor, Department of Neurosurgery, Houston Methodist Research institute, Weill-Cornell Medical College, Houston, TX, USA

    Kyuson Yun

  13. Institute of Cancer Research, Shenzhen Bay Laboratory, Shenzhen, China

    Andrew S. Lee

  14. Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Betty Y. S. Kim

Authors
  1. Zhaogang Yang
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Contributions

L.J.L. conceived the work; L.J.L., L.T., W.J. and B.Y.S.K. supervised the research; L.J.L., J.Shi and Z.Y. developed the technology; L.J.L., Z.Y., W.J. L.T. and B.Y.S.K. designed the experiments; L.J.L., Z.Y., W.J., B.Y.S.K., K.Y. and J.O. provided intellectual input; L.J.L., Z.Y., W.J., J.Shi and B.Y.S.K. wrote the manuscript with input from all authors; Z.Y., W.J., B.Y.S.K., J.X. and Y.C. prepared figures; J.Shi and J.Sun prepared videos. J.Shi and P.B. designed and fabricated CNP chips with input from W.L.; Z.Y., J.Sun, X.W., V.M., K.J.K., J.X., Y.M., J.Sun, Y.Z., X.Z., C.K. and C.C. conducted CNP experiments and collected, and sorted EVs; T.L., Z.Y. and Y.Fan designed and constructed plasmids; J.X., Y.C., Y.Z., Y.Fu and X.Z. injected mice orthotopically with tumour cells; Z.Y., X.W., Y.W., W.D., K.J.K., Y.Z. and X.Z. extracted RNA and quantified RNA loading in EVs by TLN and RT–qPCR analyses; J.X., Y.Z., X.Z. and L.T. stained cells and tissues for PTEN. C.Y. performed the cryo-EM experiment and J.Shi, J.R. and A.S.L. prepared and analysed TEM images.

Corresponding authors

Correspondence to Lesheng Teng, Betty Y. S. Kim or L. James Lee.

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Supplementary information

Supplementary Information

Supplementary Figures

Reporting Summary

Supplementary Video 1

Massive formation of multivesicular bodies 4 hours after cellular nanoelectroporation delivering CD63-GFP plasmid.

Supplementary Video 2

Mouse embryonic fibroblasts transfected by bulk electroporation only exhibit weak green fluorescence.

Supplementary Video 3

CD63-GFP release after cellular nanoelectroporation (3x slow motion).

Supplementary Video 4

Fluorescent signal of the diffusion of propidium iodide dye via anchored cell membrane pores in mouse embryonic fibroblasts in cellular nanoelectroporation.

Supplementary Video 5

Diffusion of propidium iodide in bulk electroporation.

Supplementary Video 6

Diffusion of propidium iodide through ‘bottom’ nanochannels.

Supplementary Video 7

Temperature rise by joule heating during cellular nanoporation, as measured with the temperature-sensitive fluorescent dye Rhodamine B.

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Yang, Z., Shi, J., Xie, J. et al. Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation. Nat Biomed Eng 4, 69–83 (2020). https://doi.org/10.1038/s41551-019-0485-1

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