Synthesis of melanin nanoparticles and their photosensitizing properties

Abstract

Melanins are multifunctional bio-macromolecules found throughout nature, yet they remain as poorly understood class of biomacromolecules compared with other biomolecules such as proteins. Researchers have extensively characterized the molecular building blocks of melanins but their overall architecture has not yet been described. Melanins are categorized chemically into two classes

eumelanin (black) and pheomelanin (red). Although these two types of melanins have common biosynthetic origins, specific molecular reactions occur in the early stage of the synthetic procedure, which differentiate the types of melanin. The function of melanins is defined by their intriguing physicochemical properties including broadband monotonic absorbance of UV-visible light, extremely low radiative relaxation of photo-excited electronic states, and powerful anti-oxidant and free radical scavenging abilities. Because of their strong broadband absorption of UV visible light with non-radiative relaxation, melanins have been thought to serve an important photoprotective role and numerous protection-related researches on melanins have been carried out. In direct contradiction with the photoprotective properties, melanins also show photosensitizing properties upon UV light irradiation. There are some implications of melanins in disease-related events such as photochemistry with the production of DNA strand breaks, suggesting a role of the pigments in carcinogenesis, and neurodegenerative disorders like Parkinsons disease. In this regard, research and establishment of the relationship between structure and physicochemically related biological functions is a starting point in understanding the ambivalent biological behaviors of melanins. However, the melanin models suggested so far have some limitations such as the lack of solubility in aqueous solutions which is valuable for studying the basic physicochemical properties of melanins and strong binding to proteins in biological environments. In particular, the lack of synthetic method to generate melanin models with their nano-sized particle structure is an intrinsic limitation because the particle characteristic is one of the most prominent features of melanins in nature and a starting point to represent their unique physicochemical properties. In this thesis, we demonstrated a simple method to synthesize melanin models with nanoparticulate characteristic which have physicochemical properties similar to those of enzyme-synthesized ones. Furthermore, we examined the photosensitizing function and photo biological function related to chemical components by comparing different types of melanin models. In addition, we examined the oxidation dependent transition of the photo-physical properties of melanins based on the chemical analysis methods of melanins. Chapter 1 briefly describes the research background of melanins with their structural feature, types and physicochemical properties of natural melanins related to their biological functions and analytical methods by oxidation of melanins. In chapter 2, we demonstrated a simple method to synthesize pheomelanin nanoparticles (PMNPs) through the polymerization of 3, 4-dihydroxyphenylalanine (DOPA) in the presence of cysteine by KMnO4. The synthesized pheomelanin nanoparticles had a diameter of approximately 100 nm, exhibited high dispersion stability in neutral water and various culture media and possessed morphology similar to that of naturally occurring pheomelanins. The efficiency of photoinduced generation of hydroxyl radicals from PMNPs was determined and related in vitro cell experiments were carried out, with data being compared with those from eumelanin-type nanoparticles (EMNPs) and natural sepia melanin nanoparticles. Endocytosed PMNPs showed the highest phototoxicity (~50% viability) to UV-irradiated HeLa cells, confirming the direct relationship between phototoxic efficiency and the generation of hydroxyl radicals through the complex processes of O2 sensitization. In Chapter 3, we assessed the oxidation-dependent photophysical properties of melanins through oxidation of water-dispersible Melanin-like Nanoparticles (MelNps) similar to natural melanins. Oxidation caused an absorption decrement in visible light, increment in the UV region and changes in morphology, especially the oxidation of MelNps with an alkaline hydrogen peroxide environment. The photooxidation of 1, 5-dihydroxynaphthalene (DHN) with singlet oxygen generated by oxidized melanins and direct detection of the singlet oxygen indicated the significant photoreactivity of oxidized melanins. The oxidation of melanins also affected their photoreactivity as well as their efficiency of radical quenching, which was proven through radical quenching experiments.