Controlling matrix-bound extracellular vesicle release to guide wound repair using alginate biomaterials

Introduction: : Extracellular vesicles (EVs) are lipid bilayer-bound nanoparticles that encapsulate cellular signaling molecules such as RNAs and proteins and play a role in biological processes such as tissue homeostasis, repair, and regeneration. Interestingly, a new population of EVs were recently discovered to natively reside within the extracellular matrix of non-mineralized tissues and in vitro-cultured cell sheets (termed “matrix-bound nanovesicles”, MBVs) with implications in immune modulation and tissue repair. There is significant interest in the use of EVs toward therapeutic applications, yet the controlled release and targeted delivery of EVs is challenging due to their small size (50-200 nm) and rapid systemic clearance in the body. Cellular signaling factors are critical to the development of tissue-mimetic biomaterials for wound repair and MBVs have yet to be incorporated into biomaterial platforms for controlled delivery. Here, we present a tunable alginate hydrogel platform to assess the impact of material and mechanical properties on MBV release and sequestration. We hypothesize that controlled spatiotemporal MBV release from hydrogels can be achieved by modulating biomaterial stiffness, viscoelasticity, and ligand presentation.

Materials and

Methods: : Primary human dermal fibroblasts were cultured in the presence of L-ascorbic acid to induce extracellular matrix deposition and cell sheet formation in vitro. MBVs were isolated from fibroblast cell sheets by enzymatic extracellular matrix digestion, differential centrifugation, and ultracentrifugation. MBV enrichment was verified using nanoparticle tracking analysis (size, quantity), transmission electron microscopy (morphology), and Western blotting (surface marker expression). Alginate hydrogel scaffolds of different molecular weights ( < 75kDa, >200kDa) were fabricated using varying concentrations of calcium crosslinker (CaSO4). We incorporated these MBV into alginate hydrogels of different stiffness (2, 8, 20kPa) and viscoelasticity (slow- and fast-relaxing) and measured their release into surrounding culture medium over a period of 7 days using nanoparticle tracking analysis. At day 7, alginate hydrogels were chelated using EDTA to release remaining sequestered MBVs and quantify particle retention.

Results, Conclusions, and Discussions:: Successful MBV isolation from human dermal fibroblast cell sheets was confirmed with an average particle size of 162 nm, vesicle-like morphology, and expression of EV-associated surface markers (Alix, CD63, ITGB1). As originally hypothesized, we found MBV release to decrease with an increase in stiffness across both slow- and fast-relaxing hydrogels, likely due to differences in matrix flexibility. On the other hand, fast-relaxing hydrogels release more MBV than slow-relaxing hydrogels at the same stiffness. This stress relaxation-dependent difference in release could be attributed to an increase in alginate chain length and increased entanglement that obstructs MBV diffusion. After 7 days of incubation in culture medium, soft, fast-relaxing hydrogels released ~33.13% of all recovered MBVs compared to ~0.38% released by stiff, slow-relaxing hydrogels. Here, we show that MBVs can be effectively incorporated into alginate hydrogels and MBV release from these hydrogels can be controlled by modulating biomaterial stiffness and viscoelasticity. Our results suggest a promising biomaterial design strategy to more precisely control MBV release and sequestration to address challenges associated with their limited therapeutic timeline.

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