Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles
Abstract
Extracellular vesicles (EVs) are specialised endogenous carriers of proteins and nucleic acids and are involved in intercellular communication. EVs are therefore proposed as candidate drug delivery systems for the delivery of nucleic acids and other macromolecules. However, the preparation of EV-based drug delivery systems is hampered by the lack of techniques to load the vesicles with nucleic acids. In this work we have now characterised in detail the use of an electroporation method for this purpose. When EVs were electroporated with fluorescently labelled siRNA, siRNA retention was comparable with previously published results (20-25% based on fluorescence spectroscopy and fluorescence fluctuation spectroscopy), and electroporation with unlabelled siRNA resulted in significant siRNA retention in the EV pellet as measured by RT-PCR. Remarkably, when siRNA was electroporated in the absence of EVs, a similar or even greater siRNA retention was measured. Nanoparticle tracking analysis and confocal microscopy showed extensive formation of insoluble siRNA aggregates after electroporation, which could be dramatically reduced by addition of EDTA. Other strategies to reduce aggregate formation, including the use of cuvettes with conductive polymer electrodes and the use of an acidic citrate electroporation buffer, resulted in a more efficient reduction of siRNA precipitation than EDTA. However, under these conditions, siRNA retention was below 0.05% and no significant differences in…
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Current methods of loading therapeutic molecules into EVs are limited by low efficiency, toxicity and lack of scalability.
“Current methods of loading therapeutic molecules into EVs such as electroporation, genetic engineering of host cells and chemical conjugation, are limited by low efficiency, toxicity and lack of scalability20,21.”
From GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain (2021) · cites this paper as [20]
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- Authors
- Kooijmans, S. A. A., Stremersch, S., Braeckmans, K., de Smedt, S. C., Hendrix, A., Wood, M. J. A., Schiffelers, R. M., Raemdonck, K., & Vader, P.
- Affiliations
- University Medical Center Utrecht (NL)
- Ghent University (BE)
- Ghent University Hospital (BE)
- University of Oxford (GB)
- Corresponding author
- Raemdonck, Koen
- Vader, Pieter
- ORCID
- Venue
- Journal of Controlled Release
- Publisher
- Elsevier BV
- Published
- 2013-11
- Volume
- 172
- Issue
- 1
- Pages
- 229-238
- ISSN
- 0168-3659 (ISSN-L)
- OpenAlex
- W2072970273
- Cited by
- 663
- References
- 2
- Citations by year
- 2026: 77 · 2025: 58 · 2024: 74 · 2023: 74 · 2022: 68 · 2021: 72 · 2020: 55 · 2019: 43 · 2018: 33 · 2017: 35 · 2016: 38 · 2015: 17 · 2014: 18 · 2013: 1
- FWCI
- 11.04
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Controlled Release (publishedVersion)
- PubMed (publishedVersion)
- Ghent University Academic Bibliography (Ghent University) (submittedVersion)
- Funders
- European Union (260627)
- Research Foundation — Flanders (FWO)
- Ghent University Special Research Fund
- Research Foundation — Flanders
- Netherlands Organization for Scientific Research (NWO), The Hague, The Netherlands
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Microbial Inactivation Methods
- Keywords
- extracellular vesicles, electroporation, siRNA loading, nucleic acid delivery, EV-based drug delivery, EDTA, citrate buffer, conducting polymer electrodes
- MeSH terms
- Drug Carriers, Edetic Acid, Humans, Chemical Precipitation, Solubility, Electroporation, RNA, Small Interfering, Exosomes, HEK293 Cells
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