Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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 68:2667–2688
Colombo M, Raposo G, Thery C (2014) Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30:255–289
Gould SJ, Raposo G (2013) As we wait: coping with an imperfect nomenclature for extracellular vesicles. J Extracell Vesicles 2:eCollection 2013
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 9:654–659
Ludwig AK, Giebel B (2012) Exosomes: small vesicles participating in intercellular communication. Int J Biochem Cell Biol 44:11–15
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 29:341–345
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 21:185–191
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 35:2383–2390
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 21:45–54
Taylor DD, Zacharias W, Gercel-Taylor C (2011) Exosome isolation for proteomic analyses and RNA profiling. Methods Mol Biol 728:235–246
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 784:181–195
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 7:2112–2126
Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1:841–845
Sugar IP, Neumann E (1984) Stochastic model for electric field-induced membrane pores electroporation. Biophys Chem 19:211–225
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 172:229–238
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 18:1606–1614
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 56:293–304
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Lamichhane, T.N., Jay, S.M. (2018). Production of Extracellular Vesicles Loaded with Therapeutic Cargo. In: Sirianni, R., Behkam, B. (eds) Targeted Drug Delivery. Methods in Molecular Biology, vol 1831. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8661-3_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8661-3_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8659-0
Online ISBN: 978-1-4939-8661-3
eBook Packages: Springer Protocols