This article details a method for loading dopamine into exosomes, which are lipid-based extracellular nanovesicles. The process involves using saponin to enhance membrane permeability, allowing dopamine to be encapsulated within the exosomes for potential neurotherapeutic applications.
Begin with a tube containing a suspension of exosomes, which are lipid-based extracellular nanovesicles.
Add phosphate-buffered saline or PBS to the tube to obtain uniform exosome dispersion.
Aspirate the exosome solution and filter it using a membrane filter to remove residual contaminants.
Transfer this filtered exosome suspension into a fresh tube.
Add a solution containing dopamine, a neurotherapeutic agent, followed by saponin, a surfactant, to the exosome suspension.
Incubate the mixture at physiological temperature to enhance exosomal membrane permeability.
Saponin selectively removes membrane-bound cholesterols from the exosome membrane, creating pores in the exosomal lipid bilayers.
Dopamine diffuses through these pores into the exosomes.
Over time, lipids in the exosomal membrane redistribute, and the pores close, encapsulating dopamine within exosomes.
Next, ultracentrifuge the mixture solution to pellet the dopamine-loaded exosomes.
Remove the supernatant containing free dopamine and saponin.
The pellet containing the dopamine-loaded exosomes is ready for further studies.
Add 3 milliliters of PBS to the exosome suspension collected earlier by ultracentrifugation, and sterilize the diluted suspension by filtration using a 0.22 micron filter.
Then transfer 500 microliters of the sterilized exosome suspension to another tube.
Next, sequentially add 500 microliters of 0.5 milligrams per milliliter dopamine solution and saponin into the sterilized suspension. After incubation, ultracentrifuge the suspension.
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