Separation and Enrichment of Extracellular Vesicles in the Presence of Virions

Begin with a purified culture supernatant from virus-infected cells. This supernatant contains extracellular vesicles, or EVs, including multiple exosome subpopulations and exosome-like vesicles, along with HIV virions and host protein complexes.
Take a tube containing a density gradient medium, with the highest-density layer at the bottom and progressively lower-density layers above.
Add the EV suspension to the top of the gradient.
Centrifuge to fractionate the sample according to particle density, enriching EVs in specific fractions while concentrating HIV virions in higher-density fractions.
Then, collect equal volumes from each layer into separate tubes.
Introduce hydrogel nanoparticles with porous shells and embedded core ligands into each tube.
Incubate under rotation to allow the EVs to enter the porous shells, bind to the ligands through their surface proteins, and concentrate within nanoparticle-EV complexes.
Centrifuge to pellet the nanoparticle-EV complexes, discard the supernatant, and wash with buffer.
The EV-enriched fractions are now ready for downstream analysis.
Prepare the iodixanol density gradient medium with 11 density fractions, ranging from 6 to 18% iodixanol, as described in the manuscript. Mix each tube by vortexing and layer the density fractions into a clean and dry swinging bucket ultracentrifuge tube.
Add the re-suspended EV pellet to the top of the layered gradient and ultracentrifuge the tube at 10,000 times g and 4 degrees Celsius for 90 minutes. Carefully transfer each fraction from the ultracentrifuge tube to a new microcentrifuge tube. Prepare a 30% slurry of nanoparticles for EV fraction enrichment by mixing equal volumes of NT80, NT82, and 1X PBS.
Vortex the nanoparticle mixture to ensure homogeneity. Add 30 microliters of the slurry to each density fraction and mix by either pipetting or inverting the tubes. The most critical step is the addition of the nanoparticles to concentrate EVs, following the density gradient separation.
Without this, EV recovery is poor. Rotate the nanoparticle-containing density fractions overnight at 4 degrees Celsius. Then, centrifuge the density fractions at 20,000 times g for 5 minutes at room temperature. Discard the liquid and wash the EV pellet twice with 1X PBS.