1. siRNA Encapsulation into Exosomes by Electroporation
<ol>
	<li>Pre-chill the electroporation cuvette (see Table of Materials) on ice for 30 min before electroporation.</li>
	<li>Mix 7.0 &#181;g of exosomes (32 &#181;L from 7 x 1012 p/mL stock in PBS) with 0.33 &#181;g of siRNA (12 &#181;L from 2 &#181;M stock in RNase-free water) in the microcentrifuge tube. Make up the volume to 150 &#181;L with citric acid buffer (see Table of Materials). The exosome to siRNA molar ratio is 1:60 in this case.</li>
	<li>Transfer the mixture to electroporation cuvette. Cap the cuvette and place it in the cuvette holder of the electroporator (see Table of Materials). Rotate the turning wheel 180&#176; clockwise. 
	NOTE: The wheel must be turned completely to the locked position, in order for the cuvette to contact the electrodes.</li>
	<li>Select the desired electroporation program (e.g., X-01, X-05, A-20, T-20, T-30, etc.) and start electroporation by pressing the start button. 
	NOTE: A successful pulse is indicated by showing "OK"&#160;on the display.</li>
	<li>Once electroporated, remove the cuvette after turning back the wheel 180&#176; counterclockwise. Withdraw the sample from the cuvette with the plastic pipette for further processing.</li>
</ol>

<table><tbody><tr><td>Atto655-siRNA</td><td>Eurogentec</td><td>SQ-SIRNA</td><td>(Labelled-S) UGC-GCU-ACGAUC-GAC-GAU-G55; (UnlabelledAS) CAU-CGU-CGA-UCG-UAGCGC-A55.</td></tr><tr><td>Glass pipettes</td><td>Fisher Scientific</td><td>1156-6963</td><td></td></tr><tr><td>Amaxa Nucleofector I</td><td>Lonza</td><td>Nucleofector I</td><td>With Amaxa&amp;rsquo;s Nucleofector kits</td></tr><tr><td>Citric acid buffer with EDTA</td><td></td><td></td><td>Mix 0.1954 g citric acid and 0.2087 g disodium phosphate in 50 mL of deionised water. Add EDTA to 0.1 mM. Adjust pH to 4.4.</td></tr><tr><td>Microfuge tubes</td><td>Starlab</td><td>S1615-5500</td><td>1.5 ml</td></tr></tbody></table>

sirna encapsulation into exosomes using electroporation: an electric pulse based technique to load sirna into exosomes

siRNA Encapsulation into Exosomes Using Electroporation: An Electric Pulse Based Technique to Load siRNA into Exosomes

Source: Faruqu, F. N. et al. <a href='https://www.jove.com/video/58814' target='_blank'>Preparation of Exosomes for siRNA Delivery to Cancer Cells.</a> J. Vis. Exp. (2018)
This video describes an electroporation-based method for encapsulating siRNA into exosomes. The siRNA-loaded exosomes can then be used for therapeutic applications.

Source: Faruqu, F. N. et al. Preparation of Exosomes for siRNA Delivery to Cancer Cells. J. Vis. Exp. (2018)
This video describes an electroporation-based ...

Exosomes - nano-sized, cell-derived membrane vesicles - exhibit potential as therapeutic delivery vehicles for double-stranded small interfering RNA or siRNA.
Following release into the target cell's cytoplasm, siRNA binds to the RNA-induced silencing complex. The activated complex then recognizes and binds to specific mRNA, negatively regulating its expression and mediating gene silencing.&#160;&#160;
To encapsulate siRNA into exosomes, first, chill an electroporation cuvette on ice. Next, mix exosomes and specific siRNA in a tube in the desired ratio using a suitable electroporation buffer. Now, transfer this mixture to the pre-chilled cuvette.
Close the cuvette and place it in the cuvette holder of the electroporator. Rotate the turning wheel to ensure that the cuvette makes contact with the electroporation electrodes. Now, run the selected electroporation program.
During electroporation, the electric pulses disturb the phospholipid bilayer of the exosomes suspended in the conductive buffer. This results in the formation of transient pores in the exosome membrane, promoting the diffusion of siRNA into the vesicle. The buffer helps prevent siRNA aggregation.
Once the electroporation is complete, the pores reseal, entrapping the siRNA within. Finally, remove the cuvette from the electroporator and transfer the mixture into a fresh tube. Store the siRNA-encapsulated exosomes at low temperatures until further use.

Before starting the electroporation, pre-chill the electroporation cuvette on ice for 30 minutes. Mix 7 micrograms of exosomes with 0.33 micrograms of siRNA in the microcentrifuge tube. Add a citric acid buffer to achieve the volume of 150 microliters. Add the exosome-siRNA mixture to the electroporation cuvette using a plastic pipette and cap the cuvette.
Place the cuvette in the right orientation in the cuvette holder of the electroporator and rotate the turning wheel of the electroporator 180 degrees clockwise. Select the desired program and press the Start button to start electroporation. The display will indicate a successful pulse.
Then, turn back the wheel 180 degrees counterclockwise and remove the cuvette. Use a plastic pipette to remove the sample from the cuvette into a new microcentrifuge tube. Keep the tube on ice or in a fridge before further processing if not used immediately.

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4.3K Views. Exosomes - nano-sized, cell-derived membrane vesicles - exhibit potential as therapeutic delivery vehicles for double-stranded small interfering RNA or siRNA. Following release into the target cell's cytoplasm, siRNA binds to the RNA-induced silencing complex. The activated complex then recognizes and binds to specific mRNA, negatively regulating its expression and mediating gene silencing.   To encapsulate siRNA into exosomes, first, chill an electroporation cuvette o...

Abstract

Protocol

Disclosures

<table><tbody><tr><td>Atto655-siRNA</td><td>Eurogentec</td><td>SQ-SIRNA</td><td>(Labelled-S) UGC-GCU-ACGAUC-GAC-GAU-G55; (UnlabelledAS) CAU-CGU-CGA-UCG-UAGCGC-A55.</td></tr><tr><td>Glass pipettes</td><td>Fisher Scientific</td><td>1156-6963</td><td></td></tr><tr><td>Amaxa Nucleofector I</td><td>Lonza</td><td>Nucleofector I</td><td>With Amaxa&rsquo;s Nucleofector kits</td></tr><tr><td>Citric acid buffer with EDTA</td><td></td><td></td><td>Mix 0.1954 g citric acid and 0.2087 g disodium phosphate in 50 mL of deionised water. Add EDTA to 0.1 mM. Adjust pH to 4.4.</td></tr><tr><td>Microfuge tubes</td><td>Starlab</td><td>S1615-5500</td><td>1.5 ml</td></tr></tbody></table>

siRNA Encapsulation into Exosomes Using Electroporation: An Electric Pulse Based Technique to Load siRNA into Exosomes

In This Article

Abstract

Protocol

Disclosures

Materials

Tags

Name	Company	Catalog Number	Comments
Atto655-siRNA	Eurogentec	SQ-SIRNA	(Labelled-S) UGC-GCU-ACGAUC-GAC-GAU-G55; (UnlabelledAS) CAU-CGU-CGA-UCG-UAGCGC-A55.
Glass pipettes	Fisher Scientific	1156-6963
Amaxa Nucleofector I	Lonza	Nucleofector I	With Amaxa’s Nucleofector kits
Citric acid buffer with EDTA			Mix 0.1954 g citric acid and 0.2087 g disodium phosphate in 50 mL of deionised water. Add EDTA to 0.1 mM. Adjust pH to 4.4.
Microfuge tubes	Starlab	S1615-5500	1.5 ml