Melanins are complex biological macromolecules that possess an array of physical and chemical properties of interest for biomaterials development. For instance, melanin’s broadband optical absorbance in the UV-visible range, extremely low radiative quantum yield (<0.05%) and photoconductivity make it an effective photoprotectant, while its antioxidant activity and strong affinity for metal cations/chelation ability provides excellent chemoprotection. These properties have spurred interest in using melanin for optoelectronics, functional biomaterials, tissue regeneration, drug release, bio-batteries, and other implantable applications where biocompatibility is important.
In addition to its essential physiological functions, abnormalities in melanin synthesis and structure have also been implicated in the development and progression of melanoma. Therefore, improved understanding of the structure, composition and function of natural and synthetic melanin will be useful for preventive as well as interventional healthcare.
Although the primary components of the principle mammalian forms of melanin (pheomelanin and eumelanin) are known, the secondary structure of melanin has not been unambiguously established, but is thought to be based on π-stacked oligomeric units of the ultimate monomeric precursors from the Raper-Mason pathways: 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The catechol/quinone moieties in these monomers are of tremendous interest for functional biomaterials because they provide a convenient motif for fundamental “click”-based surface functionalization that has broad utility for immobilizing ligands for selective protein binding and cell attachment. The carboxylic acid moiety in DHICA also provides a motif for carbodiimide chemistry and confers additional control over pH and zeta potential, which are both important determinants of the cellular and extracellular microenvironments. Therefore, synthetic methods to produce melanin with different ratios of these two monomers would be tremendously beneficial for selectively tuning the cell culture microenvironment and improving the biomimetic fidelity of functional biomaterial interfaces.
The synthesis of DHI films is well documented; however, there are no reports of pure DHICA films. Furthermore, we are unaware of any attempts to electrochemically synthesize melanin films using both precursors simultaneously. Here, we report chemical synthesis of DHICA, and electrochemical synthesis of melanin films using different ratios of DHI and DHICA in the synthesis solution, an extensive characterization of the resulting films, and evaluation of their biocompatibility.
The morphologies and surface properties of films synthesized from pure DHI were disntinct from those synthesized from DHICA. Specifically, films synthesized from DHICA displayed large stacked sheets of platelet-like aggregates, while films synthiseized from DHI were characterized by periodic undulations/wrinkles. Hybrid films displayed characteristics of both monomer-specific films, with small, platelet-like aggregations clustered near undulations. Fourier-transform infrared spectroscpy (FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed the presence of carboxylic acid moeities in films synthesized from monomer solutions containing DHICA, and water-contact angle measurements confimed that films containing DHICA were more hydrophilic than films synthesized from solutions without DHICA.
Primary progenitor cells were obtained from the hippocampi of E17.5 mouse embryos for cell culture, and cell phenotypes were determined by immunocytochemistry using nestin to identify highly undifferentiated cells, ß-III-tubulin to identify neurons, and glial fibrillary acidic protein (GFAP) (ab7260, Abcam) to detect astroglial cells. Progenitor cells adhered preferentially on films synthesized from monomer solutions containing both DHI and DHICA, potentially due to the unique morphology of these films, which contained both macro- and mirco-scale features.