Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases. Santa Clara University researchers engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. The researchers developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. They characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. They demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line. Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα.
Exosome preparation and characterization
(A) A centrifuge-ultrafiltration-precipitation protocol used for exosome preparation. (B) Nanoparticle tracking analysis reveals exosomes in solution (left panel) along with particle size and distribution (right panel). (C) A dot-blot immuno-analysis was carried out using premade dot-blots to detect either exosomal makers, including CD63, CD81, ALIX, FLOT1, ICAM1, EpCam, ANXA5, and TSG10, or cytosolic protein GM130 for evaluating cellular contamination if nay. (D) Confocal images of isolated control and decoy exosomes.