Engineering and Monitoring the Sustained Release of Extracellular Vesicles from Hydrogels for In Vivo Therapeutic Applications.

Extracellular vesicles (EVs) are gaining interest in regenerative medicine and biomaterials have been shown to extend EV bioavailability following delivery. Here, we report the labeling of both hydrogels and EVs to better understand hydrogel design for sustained EV release into tissues. Shear-thinning hydrogels were engineered using guest-host (i.e., adamantane-cyclodextrin) modifications to hyaluronic acid (GH), as well as GH hydrogels with the addition of gelatin crosslinked via transglutaminase (GH+Gel) to temporally control hydrogel properties. When labeled with a near-IR dye and injected into rat myocardial tissue, the GH+Gel hydrogel was retained (>14 days) longer than the GH hydrogel alone (~7 days), likely due to the added gelatin network. To overcome challenges associated with common EV labeling methods, we utilized a highly versatile metabolic labeling methodology via the incorporation of Ac4ManNAz during EV synthesis to introduce azide groups that could then be reacted with DBCO-dyes. When injected in saline, EVs were cleared within 24 hours in hearts; however, hydrogels enhanced EV retention, with levels based on hydrogel degradation behavior, namely >14 days for GH+Gel hydrogel and ~7 days for GH hydrogel alone. These findings support the use of hydrogels in EV therapies to help retain their presence at desired tissue sites.