Model polymer system for investigating the generation of hydrogen peroxide and its biological responses during the crosslinking of mussel adhesive moiety

Abstract

Mussel adhesive moiety, catechol, has been utilized to design a wide variety of biomaterials. However, the biocompatibility and biological responses associated with the byproducts generated during the curing process of catechol has never been characterized. An in situ curable polymer model system, 4-armed polyethylene glycol polymer end-capped with dopamine (PEG-D4), was used to characterize the production of hydrogen peroxide (H₂O₂) during the oxidative crosslinking of catechol. Although PEG-D4 cured rapidly (under 30s), catechol continues to polymerize over several hours to form a more densely crosslinked network over time. PEG-D4 hydrogels were examined at two different time points; 5min and 16h after initiation of crosslinking. Catechol in the 5min-cured PEG-D4 retained the ability to continue to crosslink and generated an order of magnitude higher H₂O₂ (40μM) over 6h when compared to 16h-cured samples that ceased to crosslink. H₂O₂ generated during catechol crosslinking exhibited localized cytotoxicity in culture and upregulated the expression of an antioxidant enzyme, peroxiredoxin 2, in primary dermal and tendon fibroblasts. Subcutaneous implantation study indicated that H₂O₂ released during oxidative crosslinking of PEG-D4 hydrogel promoted superoxide generation, macrophage recruitment, and M2 macrophage polarization in tissues surrounding the implant. Given the multitude of biological responses associated with H₂O₂, it is important to monitor and tailor the production of H₂O₂ generated from catechol-containing biomaterials for a given application.

Statement of significance: Remarkable underwater adhesion strategy employed by mussels has been utilized to design a wide variety of biomaterials ranging from tissue adhesives to drug carrier and tissue engineering scaffolds. Catechol is the main adhesive moiety that is widely incorporated to create an injectable biomaterials and bioadhesives. However, the biocompatibility and biological responses associated with the byproducts generated during the curing process of catechol has never been characterized. In this manuscript, we design a model system to systemically characterize the release of hydrogen peroxide (H₂O₂) during the crosslinking of catechol. Given the multitude of biological responses associated with H₂O₂ (i.e., wound healing, antimicrobial, chronic inflammation), its release from catechol-containing biomaterials need to be carefully monitored and controlled for a desired application.

Keywords: Cytotoxicity; Hydrogen peroxide; Macrophage polarization; Mussel adhesive protein; Oxidative crosslinking.