Protocol | Published:

Exosome-mediated delivery of siRNA in vitro and in vivo

Nature Protocols volume 7, pages 21122126 (2012) | Download Citation



The use of small interfering RNAs (siRNAs) to induce gene silencing has opened a new avenue in drug discovery. However, their therapeutic potential is hampered by inadequate tissue-specific delivery. Exosomes are promising tools for drug delivery across different biological barriers. Here we show how exosomes derived from cultured cells can be harnessed for delivery of siRNA in vitro and in vivo. This protocol first describes the generation of targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. Next, we explain how to purify and characterize exosomes from transfected cell supernatant. Next, we detail crucial steps for loading siRNA into exosomes. Finally, we outline how to use exosomes to efficiently deliver siRNA in vitro and in vivo in mouse brain. Examples of anticipated results in which exosome-mediated siRNA delivery is evaluated by functional assays and imaging are also provided. The entire protocol takes 3 weeks.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


NCBI Reference Sequence


  1. 1.

    , , & Exosomes: proteomic insights and diagnostic potential. Expert Rev. Proteomics 6, 267–283 (2009).

  2. 2.

    et al. Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells. Blood 104, 3257–3266 (2004).

  3. 3.

    et al. Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes. Cancer Immunol. Immunother. 55, 808–818 (2006).

  4. 4.

    et al. Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia 20, 847–856 (2006).

  5. 5.

    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).

  6. 6.

    et al. Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells. Nat. Commun. 2, 282 (2011).

  7. 7.

    et al. Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes. Blood 119, 756–766 (2012).

  8. 8.

    , , & Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity. Nat. Cell Biol. 11, 1143–1149 (2009).

  9. 9.

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

  10. 10.

    & The immunogenicity of dendritic cell-derived exosomes. Blood Cells Mol. Dis. 35, 94–110 (2005).

  11. 11.

    et al. APP and BACE1 miRNA genetic variability has no major role in risk for Alzheimer disease. Hum. Mutat. 30, 1207–1213 (2009).

  12. 12.

    et al. Targeting of the Bmi-1 oncogene/stem cell renewal factor by microRNA-128 inhibits glioma proliferation and self-renewal. Cancer Res. 68, 9125–9130 (2008).

  13. 13.

    et al. microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma. Cancer Res. 68, 3566–3572 (2008).

  14. 14.

    , , & Exosome lipidomics unravels lipid sorting at the level of multivesicular bodies. Biochimie 89, 205–212 (2007).

  15. 15.

    et al. Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region to the brain. Mol. Ther. 19, 1769–1779 (2011).

  16. 16.

    Rabies virus binding to an acetylcholine receptor alpha-subunit peptide. J. Mol. Recognit. 3, 82–88 (1990).

  17. 17.

    , & Cell-penetrating peptides: mechanisms and applications. Curr. Pharm. Des. 11, 3597–3611 (2005).

  18. 18.

    , , & Peptide-based matrices as drug delivery vehicles. Curr. Pharm. Des. 16, 1167–1178 (2010).

  19. 19.

    & Targeting RNA to treat neuromuscular disease. Nat. Rev. Drug Discov. 10, 621–637 (2011).

  20. 20.

    et al. Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo. Nucleic Acids Res. 39, 3972–3987 (2011).

  21. 21.

    , & A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16010–16017 (1997).

  22. 22.

    , , & Designing highly active siRNAs for therapeutic applications. FEBS. J. 277, 4806–4813 (2010).

  23. 23.

    et al. Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis. Nanomedicine 7, 780–788 (2011).

Download references


S.E.-A. was supported by a postdoctoral research fellowship from the Swedish Society of Medical Research (SSMF). C.G. was supported by a Wellcome Trust Technology Development Grant GR087730. Y.L. is funded by the Agency of Science, Technology and Research (A*STAR), Singapore.

Author information

Author notes

    • Samir El-Andaloussi
    • , Yi Lee
    •  & Samira Lakhal-Littleton

    These authors contributed equally to this work.


  1. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.

    • Samir El-Andaloussi
    • , Yi Lee
    • , Samira Lakhal-Littleton
    • , Jinghuan Li
    •  & Matthew J A Wood
  2. Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden.

    • Samir El-Andaloussi
  3. Molecular Engineering Laboratory, Proteos, Agency for Science Technology and Research, Singapore, Singapore.

    • Yiqi Seow
  4. Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Oxford, UK.

    • Chris Gardiner
    •  & Ian L Sargent
  5. University Department of Clinical Neurosciences, Institute of Neurology, University College London, London, UK.

    • Lydia Alvarez-Erviti


  1. Search for Samir El-Andaloussi in:

  2. Search for Yi Lee in:

  3. Search for Samira Lakhal-Littleton in:

  4. Search for Jinghuan Li in:

  5. Search for Yiqi Seow in:

  6. Search for Chris Gardiner in:

  7. Search for Lydia Alvarez-Erviti in:

  8. Search for Ian L Sargent in:

  9. Search for Matthew J A Wood in:


M.J.A.W., L.A.-E. and Y.S. initially developed the protocol. S.E.-A., S.L.-L., Y.L. and M.J.A.W. drafted and wrote most of the protocol. All authors contributed with data: S.E.-A. performed in vitro knockdown studies with Tat exosomes, Y.L. provided the CellMask data, S.L.-L. ran flow cytometry and provided BACE-1 in vitro RNAi data, Y.S. performed the cloning and provided the constructs, J.L. wrote the western blotting protocol, L.A.-E. provided in vivo RNAi data and western blot photographs, and C.G. and I.L.S. performed the NTA analysis.

Competing interests

Patents filed are as follows: WO2010/119256, priority date April 2009; UK1121070.5 and UK1121069.7, filed December 2011.

Corresponding author

Correspondence to Matthew J A Wood.

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    Detection of exosomal markers by western blot. Flotillin-1 and Lamp-1 are both present in BMDC cells (C) and BMDC-derived exosomes (E) using 20μg of protein sample for loading on gels. These markers are normally membrane proteins that are present on the surface of exosomes and inside cells depending on the cell type. A list of exosomal markers found in exosomes derived from different cells and organisms can be found in the exosome database: ExoCarta (

  2. 2.

    Supplementary Figure 2

    Knockdown of BACE-1 in N2A cells following treatment with 10μg RVG-exosomes per well and increasing amounts of siRNA in a 24 well plate. Optimal ratio is 1:1 according to the observed RNAi responses. This ratio might vary between cells wherefore it is advised to screen the ratio in initial experiments. N=3 and error bars represents the SD.

About this article

Publication history



Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.