This article discusses the use of exosomes as therapeutic delivery vehicles for siRNA. It details the process of encapsulating siRNA into exosomes through electroporation, highlighting the mechanism of gene silencing.
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 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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