Hydrogen peroxide generation and biocompatibility of hydrogel-bound mussel adhesive moiety

Abstract

To decouple the extracellular oxidative toxicity of catechol adhesive moiety from its intracellular non-oxidative toxicity, dopamine was chemically bound to a non-degradable polyacrylamide hydrogel through photo-initiated polymerization of dopamine methacrylamide (DMA) with acrylamide monomers. Network-bound dopamine released cytotoxic levels of H2O2 when its catechol side chain oxidized to quinone. Introduction of catalase at a concentration as low as 7.5 U/mL counteracted the cytotoxic effect of H2O2 and enhanced the viability and proliferation rate of fibroblasts. These results indicated that H2O2 generation is one of the main contributors to the cytotoxicity of dopamine in culture. Additionally, catalase is a potentially useful supplement to suppress the elevated oxidative stress found in typical culture conditions and can more accurately evaluate the biocompatibility of mussel-mimetic biomaterials. The release of H2O2 also induced a higher foreign body reaction to catechol-modified hydrogel when it was implanted subcutaneously in rat. Given that H2O2 has a multitude of biological effects, both beneficiary and deleterious, regulation of H2O2 production from catechol-containing biomaterials is necessary to optimize the performance of these materials for a desired application.

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

“…Anti-CD163 antibody (sc-58965) and goat anti-mouse IgG (sc-2781) were purchased from Santa Cruz Biotechnology (Dallas, Texas). Rat dermal fibroblasts and rat tendon fibroblasts were isolated from rat dermal tissue and tendon, respectively, and identified with anti-S100A4 antibody (ab27957) and goat anti-rabbit IgG H&L (Alexa Fluor 488) [17]. PEG-D4 was prepared following a previously published protocol [26] using 4-arm 10k Da N hydroxysuccinimide ester activated poly (ethylene glycol) purchased from JenKem U.S.A, Inc. (Allen, TX).…”

“…H 2 O 2 concentration was measured using the ferrous ion oxidation xylenol orange (FOX) assay by Quantitative Peroxide Assay Kit [17, 27]. 20 μL of the hydrogel extract was mixed with 200 μL of the FOX reagent and incubated at room temperature for 20 min and its absorbance at 590 nm was assessed using a plate reader (SynergyTM HT, BioTek).…”

“…Our lab recently identified the generation of hydrogen peroxide (H 2 O 2 ) during autoxidation of catechol as the source of its cytotoxicity in culture [17]. H 2 O 2 is one of the major reactive oxygen species (ROS) released during normal wound healing response and its biological functions is highly dependent on its concentration [18].…”

Recent approaches in designing bioadhesive materials inspired by mussel adhesive protein

“…During catechol oxidation, reactive oxygen species (ROS) such as super oxide anion ( ${\mathrm{O}}_2^{•}$¯) and hydrogen peroxide (H 2 O 2 ) are generated 93, 94. Catechol‐containing hydrogel released 10 2 −10 3 µM of H 2 O 2 during a period of 48 h in culture, which reduced cell viability 93.…”

“…Catechol‐containing hydrogel released 10 2 −10 3 µM of H 2 O 2 during a period of 48 h in culture, which reduced cell viability 93. The cytotoxic effect of ROS was prevented in the presence of catalase, indicating that the release of ROS is a source of cytotoxicity of catechol in culture 93, 95.…”