Poster N14 - Sepia Melanin for the Local Deactivation of Matrix Metalloproteinases

Introduction: Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that degrade the extracellular matrix (ECM), and serve essential functions in wound healing and tissue remodeling. However, overexpression of MMPs is implicated in chronic wound environments and cancer metastasis. Systemic circulation of MMP inhibitors causes severe side effects, and therefore, local deactivation of MMPs is highly desirable. In this research, sepia melanin, a natural biopigment, is explored for deactivation of MMPs, through chelation of zinc ions. Melanins have high affinities toward metal ions through various chemical functional groups, which we demonstrate can be exploited for the deactivation of MMPs. Zinc binding to melanin and MMP deactivation in the presence of melanin are examined using zinc and MMP assays, and functional in vitro tissue studies. These results demonstrate the potential applications of melanin for local deactivation of MMPs.

Materials and

Methods: Ion Binding: The zinc-binding capacity of melanin was measured using a commercially available zinc quantification kit (Abcam). Melanin was incubated in 1 mM Zn(NO3)2, in the presence or absence of 5 mM Ca(NO3)2 and the remaining zinc in solution was quantified. Zinc binding to melanin was confirmed by XPS.
MMP Deactivation: The capacity for melanin to deactivate MMPs was tested using a commercially available MMP activity assay (Abcam). Active MMPs were incubated with melanin at a range of concentrations (1-50 mg/mL), and the MMP activity after 4 hours incubation was measured.
Functional ECM degradation study: A functional assay was developed measuring the ability of melanin to protect against the degradation of gelatin hydrogel in the presence of MMPs. Gelatin hydrogel was incubated with Collagenase I (a bacterial MMP analog) in the presence or absence of melanin (200 mg/mL) at 37 °C, and the remaining mass of hydrogel was massed after 48 hours incubation to determine protection against degradation of the model ECM.
Cell Study: Cell culture media containing collagenase I (1 µg/mL) was either treated with melanin (200 mg/mL) for 4 hours, or untreated, and added to human dermal fibroblasts (hDF) cultured on the surface of gelatin hydrogels. After 24 hours incubation, the cells were fixed in paraformaldehyde and stained with phalloidin and DAPI for fluorescence microscopy.

Results, Conclusions, and Discussions: Fig. 1a describes the mechanism of MMP deactivation by melanin. Local depletion of zinc ions by melanin leads to the loss of zinc ions from MMPs, resulting in the loss of MMP activity. Melanin selectively bound to zinc ions in solution, even in the presence of calcium ions (not shown), demonstrating the feasibility of this approach.
Incubation of active MMPs with melanin significantly reduces their activity (Fig. 1b). We found that MMP-9 activity decreased with increasing melanin concentration from around 40% deactivation at 1 mg/mL melanin to ~ 98% deactivation at 50 mg/mL melanin. A similar trend was observed with MMP-1 and MMP-2 (not shown).
Pretreatment of collagenase-containing media with melanin delays the degradation of gelatin hydrogels and preserves cellular growth on the gel surface. hDFs spread and proliferated well on the gelatin hydrogel surface (Fig. 1c). However, collagenase-containing media severely disrupts the gel surface, leading to cell shrinkage and detachment (Fig. 1d). When the collagenase-containing media was pretreated with melanin, the degradative effect of collagenase was significantly reduced, preserving cell growth and spreading on the hydrogel (Fig. 1e).
In summary, melanin was found to effectively bind to zinc ions in solution, and deactivate zinc-dependent MMPs as shown through zinc and MMP assays, and functional cell-based assays. These findings indicate that melanin has high potential to be used for biomedical applications that require local MMP deactivation.

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