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 des