Production of Extracellular Vesicles Loaded with Therapeutic Cargo

  • Tek N. Lamichhane
  • Steven M. JayEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1831)


Extracellular vesicles (EVs) are biological nanoparticles comprising exosomes, microvesicles, and other heterogeneous nanoscopic vesicle populations that are produced by most cell types. In addition to their putative roles as critical mediators of intercellular communication, EVs have begun to be harnessed as drug delivery vehicles, with early evidence indicating they may have significant advantages over synthetic nanoparticle delivery systems for particular applications. Targeted delivery of EV-encapsulated cargo has already been realized and may have broad applicability; however, methods for producing and purifying EVs and loading them with therapeutic molecules have yet to be standardized. In this chapter, we outline steps for EV isolation and characterization and compare current methods for active and passive loading of EVs with payloads of short interfering RNA (siRNA) or small molecules, with the results revealing that active loading via electroporation increases loading efficiency of siRNA but not of Rhodamine B, a model for a small molecule drug, in HEK293T-derived EVs. The methods described here may inform future design of targeted delivery of nucleic acids or small molecules via EVs.

Key words

Extracellular vesicles (EVs) Exosomes siRNA Small molecules Electroporation Drug delivery Cancer therapeutics 



This work was supported by NIH R00 grant HL112905, by an ORAU Ralph E. Power Junior Faculty Enhancement Award, and by two University of Maryland Tier 1 seed grants (all to S.M.J.). The authors thank Rini Pek, Navein Arumugasaamy and Anjana Jeyaram for their helpful contributions.


  1. 1.
    Gyorgy B, Szabo TG, Pasztoi M, Pal Z, Misjak P, Aradi B, Laszlo V, Pallinger E, Pap E, Kittel A, Nagy G, Falus A, Buzas EI (2011) Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci 68:2667–2688CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Colombo M, Raposo G, Thery C (2014) Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30:255–289CrossRefGoogle Scholar
  3. 3.
    Gould SJ, Raposo G (2013) As we wait: coping with an imperfect nomenclature for extracellular vesicles. J Extracell Vesicles 2:eCollection 2013CrossRefGoogle Scholar
  4. 4.
    Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659CrossRefGoogle Scholar
  5. 5.
    Ludwig AK, Giebel B (2012) Exosomes: small vesicles participating in intercellular communication. Int J Biochem Cell Biol 44:11–15CrossRefPubMedGoogle Scholar
  6. 6.
    Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, Wood MJ (2011) Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol 29:341–345CrossRefGoogle Scholar
  7. 7.
    Ohno S, Takanashi M, Sudo K, Ueda S, Ishikawa A, Matsuyama N, Fujita K, Mizutani T, Ohgi T, Ochiya T, Gotoh N, Kuroda M (2013) Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol Ther 21:185–191CrossRefPubMedGoogle Scholar
  8. 8.
    Tian Y, Li S, Song J, Ji T, Zhu M, Anderson GJ, Wei J, Nie G (2014) A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials 35:2383–2390CrossRefPubMedGoogle Scholar
  9. 9.
    Lamichhane TN, Sokic S, Schardt JS, Raiker RS, Lin JW, Jay SM (2015) Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine. Tissue Eng Part B Rev 21:45–54CrossRefPubMedGoogle Scholar
  10. 10.
    Taylor DD, Zacharias W, Gercel-Taylor C (2011) Exosome isolation for proteomic analyses and RNA profiling. Methods Mol Biol 728:235–246CrossRefPubMedGoogle Scholar
  11. 11.
    Rani S, O'Brien K, Kelleher FC, Corcoran C, Germano S, Radomski MW, Crown J, O'Driscoll L (2011) Isolation of exosomes for subsequent mRNA, MicroRNA, and protein profiling. Methods Mol Biol 784:181–195CrossRefPubMedGoogle Scholar
  12. 12.
    El-Andaloussi S, Lee Y, Lakhal-Littleton S, Li J, Seow Y, Gardiner C, Alvarez-Erviti L, Sargent IL, Wood MJ (2012) Exosome-mediated delivery of siRNA in vitro and in vivo. Nat Protoc 7:2112–2126CrossRefPubMedGoogle Scholar
  13. 13.
    Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1:841–845PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Sugar IP, Neumann E (1984) Stochastic model for electric field-induced membrane pores electroporation. Biophys Chem 19:211–225CrossRefPubMedGoogle Scholar
  15. 15.
    Kooijmans SA, Stremersch S, Braeckmans K, de Smedt SC, Hendrix A, Wood MJ, Schiffelers RM, Raemdonck K, Vader P (2013) Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles. J Control Release 172:229–238CrossRefPubMedGoogle Scholar
  16. 16.
    Sun D, Zhuang X, Xiang X, Liu Y, Zhang S, Liu C, Barnes S, Grizzle W, Miller D, Zhang HG (2010) A novel nanoparticle drug delivery system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes. Mol Ther 18:1606–1614CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ (2012) Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods 56:293–304CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Fischell Department of BioengineeringUniversity of MarylandCollege ParkUSA
  2. 2.Program in Oncology, Marlene and Stewart Greenebaum Cancer CenterUniversity of MarylandCollege ParkUSA
  3. 3.Program in Molecular and Cell BiologyUniversity of MarylandCollege ParkUSA

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