Development of a novel modified hyaluronic acid hydrogel for the enhanced binding and controlled release of extracellular vesicles

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) demonstrate tremendous therapeutic potential in tissue regeneration applications, however, experience limited efficacy due to rapid clearance post-delivery in vivo. Here, a novel method for retaining EVs within hyaluronic acid (HA)-based hydrogel carriers was explored and parameters were optimized to develop a hydrogel with repeatably and significantly higher EV retentive capacity compared to the existing and widely employed methacrylated hyaluronic acid (Me-HA). EV characterization provided evidence of the pro-proliferative nature of the nanoparticles, including in the presence of hydrogel. Fluorescent as well as fluorescent/bioluminescent EVs were developed to enable tracking during hydrogel optimization for improved EV binding. Methods for quantifying EVs, including several commercially available alternatives, were evaluated in the presence of hydrogel to ensure that those in which hydrogels interfered were not used in tests concerning material development for EV retention. Ultimately, fluorescent EVs experienced very little interference from hydrogels and maintained their fluorescent intensity over the course of six weeks. Thus, they were selected for use in quantifying EV release from materials over time. Novel hydrogel formulations were developed and optimized for the crosslinker density, crosslinking timing, molecular weight, type and degree of modification, and material weight/volume (w/v) concentration that led to the highest EV retention over time and suitable hydrogel mechanical properties. The optimal formulation allowed for more EV retention both on thin layers of hydrogel and within hydrogel scaffolds compared to Me-HA and other novel formulations, as well as in the presence of PBS alone and HA degradation enzyme. Materials were shown to be cytocompatible and exhibited MSC binding ability, and fluorescent EVs encapsulated within hydrogels that were implanted subcutaneously in black mice could be seen through preliminary in vivo imaging analysis.

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University of Galway

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CC BY-NC-ND

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University of Galway Theses (PhD Theses)