Advertisement

Preparation and Isolation of siRNA-Loaded Extracellular Vesicles

  • Pieter Vader
  • Imre Mäger
  • Yi Lee
  • Joel Z. Nordin
  • Samir E. L. Andaloussi
  • Matthew J. A. Wood
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1545)

Abstract

RNA interference (RNAi) has tremendous potential for specific silencing of disease-causing genes. Its clinical usage however critically depends on the development of carrier systems that can transport the RNAi-mediating small interfering RNA (siRNA) molecules to the cytosol of target cells. Recent reports have suggested that extracellular vesicles (EVs) form a natural transport system through which biomolecules, including RNA, is exchanged between cells. Therefore, EVs are increasingly being considered as potential therapeutic siRNA delivery systems.

In this chapter we describe a method for preparing siRNA-loaded EVs, including a robust, scalable method to isolate them from cell culture supernatants.

Key words

Extracellular vesicles Exosomes Microvesicles siRNA Size-exclusion chromatography Drug delivery 

Notes

Acknowledgments

P.V. was supported by a Rubicon Fellowship from the Netherlands Organisation for Scientific Research (NWO). I.M. is supported by a Postdoctoral MOBILITAS Fellowship of the Estonian Science Foundation. Y.L. is funded by the Agency of Science, Technology and Research (A*STAR), Singapore. SELA is supported by the Swedish Research Council (VR-MED and EuroNanoMedII) as well as the Swedish Society of Medical Research (SSMF).

References

  1. 1.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811CrossRefPubMedGoogle Scholar
  2. 2.
    Kanasty R, Dorkin JR, Vegas A, Anderson D (2013) Delivery materials for siRNA therapeutics. Nat Mater 12(11):967–977CrossRefPubMedGoogle Scholar
  3. 3.
    El Andaloussi S, Mager I, Breakefield XO, Wood MJ (2013) Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 12(5):347–357CrossRefGoogle Scholar
  4. 4.
    Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200(4):373–383CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Valadi H, Ekstrom K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9(6):654–659CrossRefPubMedGoogle Scholar
  6. 6.
    Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MA, Hopmans ES, Lindenberg JL, de Gruijl TD, Würdinger T, Middeldorp JM (2010) Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A 107(14):6328–6333CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kosaka N, Iguchi H, Yoshioka Y, Takeshita F, Matsuki Y, Ochiya T (2010) Secretory mechanisms and intercellular transfer of microRNAs in living cells. J Biol Chem 285(23):17442–17452CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    van der Pol E, Boing AN, Harrison P, Sturk A, Nieuwland R (2012) Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 64(3):676–705CrossRefPubMedGoogle Scholar
  9. 9.
    Seow Y, Wood MJ (2009) Biological gene delivery vehicles: beyond viral vectors. Mol Ther 17(5):767–777CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    van Dommelen SM, Vader P, Lakhal S, Kooijmans SA, van Solinge WW, Wood MJ, Schiffelers RM (2012) Microvesicles and exosomes: opportunities for cell-derived membrane vesicles in drug delivery. J Control Release 161(2):635–644CrossRefPubMedGoogle Scholar
  11. 11.
    Marcus ME, Leonard JN (2013) FedExosomes: engineering therapeutic biological nanoparticles that truly deliver. Pharmaceuticals (Basel) 6(5):659–680CrossRefGoogle Scholar
  12. 12.
    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(4):341–345CrossRefPubMedGoogle Scholar
  13. 13.
    Wahlgren J, De LKT, Brisslert M, Vaziri Sani F, Telemo E, Sunnerhagen P, Valadi H (2012) Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes. Nucleic Acids Res 40(17):e130CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Kooijmans SA, Stremersch S, Braeckmans K, de Smedt SC, Hendrix A, Wood MJ (2013) Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles. J Control Release 172(1):229–238CrossRefPubMedGoogle Scholar
  15. 15.
    Zhang Y, Li L, Yu J, Zhu D, Zhang Y, Li X, Gu H, Zhang CY, Zen K (2014) Microvesicle-mediated delivery of transforming growth factor beta1 siRNA for the suppression of tumor growth in mice. Biomaterials 35(14):4390–4400CrossRefPubMedGoogle Scholar
  16. 16.
    Witwer KW, Buzas EI, Bemis LT, Bora A, Lässer C, Lötvall J, Nolte-'t Hoen EN, Piper MG, Sivaraman S, Skog J, Théry C, Wauben MH, Hochberg F (2013) Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles 2Google Scholar
  17. 17.
    Squadrito ML, Baer C, Burdet F, Maderna C, Gilfillan GD, Lyle R, Ibberson M, De Palma M (2014) Endogenous RNAs modulate microRNA sorting to exosomes and transfer to acceptor cells. Cell Rep 8(5):1432–1446CrossRefPubMedGoogle Scholar
  18. 18.
    Villarroya-Beltri C, Baixauli F, Gutierrez-Vazquez C, Sánchez-Madrid F, Mittelbrunn M (2014) Sorting it out: regulation of exosome loading. Semin Cancer Biol 28C:3–13CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Pieter Vader
    • 1
    • 2
  • Imre Mäger
    • 1
    • 3
  • Yi Lee
    • 1
  • Joel Z. Nordin
    • 4
  • Samir E. L. Andaloussi
    • 1
    • 4
  • Matthew J. A. Wood
    • 1
  1. 1.Department of Physiology, Anatomy and GeneticsUniversity of OxfordOxfordUK
  2. 2.Department of Clinical Chemistry and HaematologyUMC UtrechtUtrechtThe Netherlands
  3. 3.Institute of TechnologyUniversity of TartuTartuEstonia
  4. 4.Department of Laboratory MedicineKarolinska InstitutetHuddingeSweden

Personalised recommendations