Formulating a Dopamine-Loaded Exosome Carrier System

Samenvatting

Source: Yavuz, B., et al. Formulating and Characterizing an Exosome-based Dopamine Carrier System. J. Vis. Exp. (2022)

This video demonstrates the formulation of dopamine-loaded exosomes, focusing on saponin-induced membrane permeabilization to enhance dopamine encapsulation within exosomes.

Protocol

1. Dopamine loading into exosomes

NOTE: After the characterization of Wharton's Jelly mesenchymal stem cells (WJ-MSCs) exosomes is completed, dopamine-loaded exosomes were obtained as a drug delivery system. Drug loading into exosomes is performed using the incubation method.

1. Add 3 mL of phosphate-buffered saline (PBS) to the exosome suspension. Sterilize the diluted suspension by filtration using 0.22 µm filters. Transfer 500 µL of the sterilized exosome suspension to another tube.
2. Prepare dopamine solution (0.5 mg/mL) with distilled water. Add 500 µL of dopamine solution into the tubes containing the exosomes.
3. Add saponin to the suspension in step 1.2. Incubate the prepared exosome-dopamine suspension for 24 h at 37 °C. NOTE: Saponin concentration should not exceed 0.002% of the total solution.
4. Ultracentrifuge the suspension at 90,000 × g for 70 min to remove free dopamine and saponin from the medium. Store the isolated exosomes at -20 °C until use for further analysis. NOTE: Add 0.02% ascorbic acid for the stability of the prepared dopamine solution.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
0.22 µm membrane filter	Aisimo		Used for the sterilization process
15 mL Falcon tube	Nest		Used in cell culture step
50 mL Falcon tube	Nest		Used in cell culture step
Centrifugal	Hitachi	CF16RN	Used in the exosome isolation step
Dopamine hydrochloride H8502-10G	Sigma	H8502-10G	Used in exosome dopamine loading
Optima XPN-100 Ultracentrifuge	Beckman Coulter		Used in the exosome isolation step
PBS tablet	Biomatik	43602	In the preparation of the PBS solution
Saponin	Sigma	47036-50G-F	It was used by adding it to the total solution in the exosome dopamine loading process.
Wharton’s jelly mesenchymal stem cells	ATCC