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

An Author Correction to this article was published on 15 June 2021

This article has been updated


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 (Open Science Framework) and can also be requested from the corresponding authors.

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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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Authors and Affiliations



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).

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