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