Microencapsulation of small extracellular vesicles with nanoporous biomaterials results in a sustained-release preparation to enhance targeting and persistence after intravascular delivery

Background \u0026 Aim Mesenchymal stromal cells (MSCs) have shown promise as therapy for treatment of cardiovascular disease. The therapeutic effect of MSCs is predominately paracrine, involving the release of small ( Methods, Results \u0026 Conclusion MSC-derived small EVs were collected during 24h of serum-free culture and purified using sequential ultracentrifugation or tangential flow filtration. ZetaView nanoparticle tracking revealed a population of small EVs having a median size of 108±53 nm, positive for the tetraspanin CD63 by Western blot analysis. Pkh26 membrane labelling of small EVs had negligible impact on EV size (median size 102±42 nm). Agarose (2%) or agarose-gelatin (1%-1%) microgels were formed using vortex-emulsion or microfluidic encapsulation, resulting microgels of differing size (34±15 mm and 59±3 mm, respectively). Free EVs, EV-loaded microgels, or empty microgels were plated with human umbilical vein endothelial cells (HUVEC) and time-lapse imaging over 48h demonstrated transfer of EVs from microgels to HUVEC. While by 48h free EVs were readily taken up by HUVECs, encapsulated EVs demonstrated a slower uptake during this period (64% vs 26% cells/FOV), consistent with sustained release kinetics. Biodegradability of microgels was assessed in vivo using the rat monocrotaline (MCT) model of pulmonary arterial hypertension. Three days post MCT empty microgels were delivered by intrajugular injection. Right ventricular systolic pressure was assessed at day 1, 3 and 7. IV delivery of 500,000 agarose-gelatin or agarose microgels had no impact on right ventricular systolic pressure compared to vehicle (saline). Gelatin-agarose microgels demonstrated \u003e2-fold more rapid degradation and clearance from the pulmonary vascular bed from day 1 through to day 7 post compared with agarose microgels. Microencapsulation of small EVs in nanoporous biomaterials produces microgels which are efficiently trapped in the microcirculation with tunable release of EVs depending on biomaterial composition and gel degradation rate.