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Optimized Protocol for Plasma-Derived Extracellular Vesicles Loading with Synthetic miRNA Mimic Using Electroporation

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Extracellular Vesicles in Diagnosis and Therapy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2504))

Abstract

Extracellular vesicles (EVs) are a population of particles naturally released by cells to transport biological messages, including nucleic acids. Thus, EVs represent an ideal vehicle to deliver therapeutic miRNAs. The current challenge is the development of efficient protocols to load EVs with exogenous miRNAs. Human plasma is an abundant source of EVs which can be manipulated for therapeutic applications. Despite numerous techniques are currently available to load EVs, all of them present issues which limit their clinical application. Among all, electroporation was shown to be superior to other protocols and to efficiently load plasma-derived EVs with miRNAs. However, also the electroporation procedure presents issues that can reduce the miRNA delivery. In this chapter, we describe a protocol to isolate EVs from human plasma, to load synthetic miRNA mimics using electroporation, to evaluate EV integrity and miRNA loading into EVs. Finally, the analysis of EV functionality allows to investigate the ability of engineered EVs to transfer the miRNAs to target cells and to exploit their biological effects.

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References

  1. Yáñez-Mó M, Siljander PR, Andreu Z et al (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4:27066. https://doi.org/10.3402/jev.v4.27066

    Article  PubMed  Google Scholar 

  2. Armstrong JP, Holme MN, Stevens MM (2017) Re-engineering extracellular vesicles as smart nanoscale therapeutics. ACS Nano 11(1):69–83. https://doi.org/10.1021/acsnano.6b07607

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Di Leva G, Garofalo M, Croce CM (2014) MicroRNAs in cancer. Annu Rev Pathol 9:287–314. https://doi.org/10.1146/annurev-pathol-012513-104715

    Article  CAS  PubMed  Google Scholar 

  4. Romero-Cordoba SL, Salido-Guadarrama I, Rodriguez-Dorantes M, Hidalgo-Miranda A (2014) miRNA biogenesis: biological impact in the development of cancer. Cancer Biol Ther 15(11):1444–1455. https://doi.org/10.4161/15384047.2014.955442

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Qian H, Tay CY, Setyawati MI, Chia SL, Lee DS, Leong DT (2017) Protecting microRNAs from RNase degradation with steric DNA nanostructures. Chem Sci 8(2):1062–1067. https://doi.org/10.1039/c6sc01829g

    Article  CAS  PubMed  Google Scholar 

  6. Didiot MC, Hall LM, Coles AH, Haraszti RA, Godinho BM, Chase K, Sapp E, Ly S, Alterman JF, Hassler MR, Echeverria D, Raj L, Morrissey DV, DiFiglia M, Aronin N, Khvorova A (2016) Exosome-mediated delivery of hydrophobically modified siRNA for Huntingtin mRNA silencing. Mol Ther 24(10):1836–1847. https://doi.org/10.1038/mt.2016.126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lamichhane TN, Jeyaram A, Patel DB, Parajuli B, Livingston NK, Arumugasaamy N, Schardt JS, Jay SM (2016) Oncogene knockdown via active loading of small RNAs into extracellular vesicles by sonication. Cell Mol Bioeng 9(3):315–324. https://doi.org/10.1007/s12195-016-0457-4

    Article  CAS  PubMed  Google Scholar 

  8. Luan X, Sansanaphongpricha K, Myers I, Chen H, Yuan H, Sun D (2017) Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacol Sin 38(6):754–763. https://doi.org/10.1038/aps.2017.12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhang D, Lee H, Zhu Z, Minhas JK, Jin Y (2017) Enrichment of selective miRNAs in exosomes and delivery of exosomal miRNAs in vitro and in vivo. Am J Physiol Lung Cell Mol Physiol 312(1):L110–L121. https://doi.org/10.1152/ajplung.00423.2016

    Article  PubMed  Google Scholar 

  10. Tapparo M, Pomatto MAC, Deregibus MC, Papadimitriou E, Cavallari C, D’Antico S, Collino F, Camussi G (2021) Serum derived extracellular vesicles mediated delivery of synthetic miRNAs in human endothelial cells. Front Mol Biosci 8:636587. https://doi.org/10.3389/fmolb.2021.636587

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. O’Loughlin AJ, Mäger I, de Jong OG, Varela MA, Schiffelers RM, El Andaloussi S, Wood MJA, Vader P (2017) Functional delivery of lipid-conjugated siRNA by extracellular vesicles. Mol Ther 25(7):1580–1587. https://doi.org/10.1016/j.ymthe.2017.03.021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fuhrmann G, Serio A, Mazo M, Nair R, Stevens MM (2015) Active loading into extracellular vesicles significantly improves the cellular uptake and photodynamic effect of porphyrins. J Control Release 205:35–44. https://doi.org/10.1016/j.jconrel.2014.11.029

    Article  CAS  PubMed  Google Scholar 

  13. Haney MJ, Klyachko NL, Zhao Y, Gupta R, Plotnikova EG, He Z, Patel T, Piroyan A, Sokolsky M, Kabanov AV, Batrakova EV (2015) Exosomes as drug delivery vehicles for Parkinson’s disease therapy. J Control Release 207:18–30. https://doi.org/10.1016/j.jconrel.2015.03.033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wahlgren J, De L Karlson T, 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):e130. https://doi.org/10.1093/nar/gks463

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kooijmans SAA, Stremersch S, Braeckmans K, de Smedt SC, Hendrix A, Wood MJA, Schiffelers RM, Raemdonck K, Vader P (2013) Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles. J Control Release 172(1):229–238. https://doi.org/10.1016/j.jconrel.2013.08.014

    Article  CAS  PubMed  Google Scholar 

  16. Johnsen KB, Gudbergsson JM, Skov MN, Christiansen G, Gurevich L, Moos T, Duroux M (2016) Evaluation of electroporation-induced adverse effects on adipose-derived stem cell exosomes. Cytotechnology 68(5):2125–2138. https://doi.org/10.1007/s10616-016-9952-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lamichhane TN, Raiker RS, Jay SM (2015) Exogenous DNA loading into extracellular vesicles via electroporation is size-dependent and enables limited gene delivery. Mol Pharm 12(10):3650–3657. https://doi.org/10.1021/acs.molpharmaceut.5b00364

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yang Z, Xie J, Zhu J, Kang C, Chiang C, Wang X, Wang X, Kuan T, Chen F, Chen Z et al (2016) Functional exosome-mimic for delivery of siRNA to cancer: in vitro and in vivo evaluation. J Control Release 243:160–171. https://doi.org/10.1016/j.jconrel.2016.10.008

    Article  CAS  PubMed  Google Scholar 

  19. Pomatto MAC, Bussolati B, D’Antico S, Ghiotto S, Tetta C, Brizzi MF, Camussi G (2019) Improved loading of plasma-derived extracellular vesicles to encapsulate antitumor miRNAs. Mol Ther Methods Clin Dev 13:133–144. https://doi.org/10.1016/j.omtm.2019.01.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

This work was supported by 2i3t Incubatore di Imprese e per il Trasferimento Tecnologico dell’Università degli Studi di Torino and Unicyte AG. GC was a component of Scientific Advisory Board of Unicyte AG, CG and MACP were named as inventors in a related patent (EP3833744A1).

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Authors and Affiliations

  1. Department of Medical Sciences, University of Turin, Turin, Italy

    Margherita A. C. Pomatto, Federica Negro & Giovanni Camussi

Authors
  1. Margherita A. C. Pomatto
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  2. Federica Negro
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  3. Giovanni Camussi
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Corresponding author

Correspondence to Margherita A. C. Pomatto .

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Editors and Affiliations

  1. National Center for Global Health, Istituto Superiore di Sanità, Rome, Italy

    Maurizio Federico

  2. National Center for Global Health, Istituto Superiore di Sanità, Rome, Italy

    Barbara Ridolfi

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Pomatto, M.A.C., Negro, F., Camussi, G. (2022). Optimized Protocol for Plasma-Derived Extracellular Vesicles Loading with Synthetic miRNA Mimic Using Electroporation. In: Federico, M., Ridolfi, B. (eds) Extracellular Vesicles in Diagnosis and Therapy. Methods in Molecular Biology, vol 2504. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2341-1_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2341-1_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2340-4

  • Online ISBN: 978-1-0716-2341-1

  • eBook Packages: Springer Protocols

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