Overview

Headache-free loading of siRNAs and miRNAs into extracellular vesicles

Whether you’re engineering extracellular vesicles (EVs) as part of therapeutics development¹ or using them as a tool for functional studies of EV-mediated nucleic acid delivery², getting the desired miRNA or siRNA cargo into EVs is a critical first step. SBI helps you efficiently and effortlessly get this step done with the Exo-Fect™ siRNA/miRNA Transfection Kit, a first-in-class reagent specifically designed for loading small RNAs into EVs.

Unlike electroporation, a common method for introducing small RNAs into EVs³, the Exo-Fect workflow does not require access to specialized equipment or the same time-consuming optimization of voltage and timing. Instead, it leverages our proprietary Cell-Penetrating Peptide (CPP) technology to transfer siRNAs or miRNAs to the inside of your EVs.

This next-generation EV transfection reagent offers the following benefits:

• Efficient delivery—siRNA/miRNA-loaded EVs deliver cargo to as many as 95% of target cells*
• Validated efficacy—siRNA-loaded EVs lead to significant knockdown (>80%) of target gene expression in transfected cells**
• No toxicity—unlike commonly-used lipid-based transfection reagents, the Exo-Fect siRNA/miRNA Transfection Reagent causes no toxicity in tested cells
• Fast, easy workflow—go from prepared small RNA to ready-to-use, RNA-loaded EVs in less than 1.5-hours and with only 15-minutes of hands-on time
• Minimal background—a clean-up step ensures complete removal of free small RNA and excess transfection reagent for clean, ready-to-use, RNA-loaded EVs

*As assessed by exposing non-fluorescent target cells to EVs loaded with Cy3-labeled control siRNA using Exo-Fect siRNA/miRNA transfection reagent, and then measuring the percentage of fluorescent cells (HeLa, HUVEC, and HEK293 cells were all tested).

** As assessed using qPCR analysis of HPRT gene expression in cells treated with either HPRT-targeting siRNA or control siRNA.

References

1. Chen Z, et al. Therapeutic Potential of Mesenchymal Cell-Derived miRNA-150-5p-Expressing Exosomes in Rheumatoid Arthritis Mediated by the Modulation of MMP14 and VEGF. J Immunol. 2018 Oct 15;201(8):2472-2482. PMCID: PMC6176104.
2. Pegtel DM, et al. Functional delivery of viral miRNAs via exosomes. Proc Natl Acad Sci U S A. 2010;107(14):6328-33. PMCID: PMC2851954.
3. Alvarez-Erviti, L et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011 Apr; 29:341–345. PMID: 21423189.

How It Works

Quick and easy loading of siRNA or miRNA into EVs

The Exo-Fect siRNA/miRNA Transfection Kit workflow is quick and easy—complete in 1.5 hours and with only 15-minutes of hands-on time.

The workflow consists of two steps:

1. Load your siRNA or miRNA into already-isolated EVs.
   1. Incubate siRNA or miRNA with Exo-Fect siRNA/miRNA Transfection reagent for 15-minutes at room temperature.
   2. Add isolated EVs to the mixture and incubate at 37°C for 1-hour.
2. Clean-up the reaction by removing free siRNA/miRNA, transfection reagent, and free siRNA/miRNA-Exo-Fect complexes
   1. Transfer reaction to the included, pre-washed spin-column and incubate with gentle rotation for 10-minutes at room temperature.
   2. Collect EVs by centrifuging the spin-column for 30-seconds at 1,000 x g. Loaded EVs are ready for use in downstream applications.

Supporting Data

See the outstanding efficiency of the Exo-Fect siRNA/miRNA Transfection Kit

Figure 1. Exo-Fect siRNA/miRNA Transfection Reagent efficiently loads siRNA into EVs. (A) Cy3-labeled control siRNA were loaded into EVs, delivered to primary HUVECs, and cells imaged 36-48 hours post transfection. Shown are the bright-field images (left panels), fluorescence images (middle panels), and merged images (right panels). The top row of images are cells minus EVs. The bottom row shows cells treated with EVs that were loaded with Cy3-labeled siRNA. EV treatment results in the transfer of Cy3-labeled siRNA to most of the imaged cells (bottom row). The negligible fluorescence seen in the minus EV treated cells (top row) is due to the minute traces of labeled siRNA that escaped the clean-up step. (B) Quantitation of the EV cargo delivery efficiency shown in (A). (C) EV transfection is highly robust with loaded EVs efficiently transferring cargo to a range of different cell types.

Figure 2. Exo-Fect siRNA/miRNA reagent is non-toxic to cells exposed to treated EVs. (A) HeLa cells exposed to mock treatments, control siRNA-loaded EVs, and HPRT siRNA-loaded EVs show high viability regardless of treatment conditions. (B) Measurement of cytokines also shows the low toxicity of the Exo-Fect siRNA/miRNA reagent. Conditioned medium from cells exposed to untransfected EVs, mock EVs, EVs loaded with control siRNA, or EVs loaded with GAPDH-targeting siRNA all show low levels of cytokines, indicating that all are healthy, non-stressed cells.

Figure 3. EVs loaded with siRNA using Exo-Fect siRNA/miRNA reagent efficiently deliver functional cargo into recipient cells. (A) Only cells treated with EVs containing HPRT-targeting siRNA show a knock-down of HPRT expression via qPCR. Mock-treated cells and cells exposed to EVs loaded with control siRNA all show normal HPRT expression. (B) The knock-down in HPRT expression is further confirmed at the protein level, as demonstrated via Western blot.

Figure 4. EVs loaded with anti-miRNA using Exo-Fect siRNA/miRNA reagent are functional. EVs loaded with an miR-16 inhibitor using Exo-Fect siRNA/miRNA reagent strongly reduce miR-16 expression in HeLa cells. miR-16 expression was assessed by qPCR 24-hours post-transfection using U6 RNA as a reference.

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