A comparative study of enzyme initiators for crosslinking phenol-functionalized hydrogels for cell encapsulation

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Abstract
Background: Dityrosine crosslinking in proteins is a bioinspired method of forming hydrogels. This study compares oxidative enzyme initiators for their relative crosslinking efficiency and cytocompatibility using the same phenol group and the same material platform. Four common enzyme and enzyme-like oxidative initiators were probed for resulting material properties and cell viability post-encapsulation. Results: All four initiators can be used to form phenol-crosslinked hydrogels, however gelation rates are dependent on enzyme type, concentration, and the oxidant. Horseradish peroxidase (HRP) or hematin with hydrogen peroxide led to a more rapid poly (vinyl alcohol)-tyramine (PVA-Tyr) polymerization (10–60 min) because a high oxidant concentration was dissolved within the macromer solution at the onset of crosslinking, whereas laccase and tyrosinase require oxygen diffusion to crosslink phenol residues and therefore took longer to gel (2.5+ hours). The use of hydrogen peroxide as an oxidant reduced cell viability immediately post-encapsulation. Laccase- and tyrosinase-mediated encapsulation of cells resulted in higher cell viability immediately post-encapsulation and significantly higher cell proliferation after one week of culture. Conclusions: Overall this study demonstrates that HRP/H 2 O 2 , hematin/H 2 O 2 , laccase, and tyrosinase can create injectable, in situ phenol-crosslinked hydrogels, however oxidant type and concentration are critical parameters to assess when phenol crosslinking hydrogels for cell-based applications.
Funding Information
  • University of New South Wales (PS35129)

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