- 學位論文
黑色素的奈米化與其在生物醫學上之應用
NANONIZATION OF MELANIN AND ITS APPLICATIONS TO BIOMEDICINE
摘要
黑色素是人體組織中最普遍的異質生物聚合物之一，但其分子結構至今還無法完全被解析出來。黑色素具有很寬的吸收光譜，並且可生產永久自由基和光致自由基，皮膚表層的黑色素細胞中有許多與免疫系統相關聯的酶，有助於幫助人體對抗微生物，其中的黑色素可能就扮演重要的角色。此外研究指出黑色素細胞能保護皮膚不受紫外線的傷害，靠的不僅是經由吸收作用衰減紫外線強度，還有清除自由基和活性氧的功能，更多的研究指出黑色素具有抗輻射、抗氧化、抗癌、抗蛇毒、抗病毒和清除重金屬離子等功能。在這些潛在應用中，不論是天然黑色素或人工合成黑色素都無法在適合生物生存的溶劑中被溶解，大量的沉澱聚集形成更大的顆粒，造成其效用大大降低。於是我們著手開發利用飛秒脈衝雷射與機械粉碎的技術將黑色素奈米化，製造黑色素奈米顆粒使之可以分散於水溶液中，以便更可靠地研究黑色素的生物功能，並增強其作為藥物的療效。結果顯示被奈米化的烏賊和合成黑色素呈片狀，顆粒大小約為直徑42.5奈米與高度0.95奈米，可以均勻分散在水溶液中超過一週不會沉澱，理論上，此技術適用於任何種類的黑色素，而且用飛秒脈衝雷射進行奈米化的製程還可用來解析黑色素的結構，我們推斷烏賊黑色素是由許多奈米薄片聚集而成，其間是靠凡得瓦力和疏水作用力來維繫的，而合成黑色素是由許多奈米薄片堆疊而成的微米薄片，其間靠的是π–π相互作用力來維繫的，此作用力比前者大。在生物醫學應用方面，雖然過去研究指出黑色素在皮膚中具有抗微生物能力，但我們的實驗證明並非黑色素奈米顆粒直接殺死微生物，同時也證明黑色素奈米顆粒在有無照光的情況下都不具有細胞毒性，更加確認它做為藥物的可行性。此外，我們證明了奈米化後大大提高黑色素對抗急性氧化壓力和清除重金屬離子的效用，甚至不需要外加照光來增強其效果，對於日後做為注射劑應用到人體會更加方便。
並列摘要
Melanin is one of the most ubiquitous heterogeneous biological polymer widespread in our body tissue. However, its complete molecular hierarchical structure is still unknown. Melanin has a broadband absorption spectrum and it can generate permanent and light-induced free radicals. In the human epidermis, melanocytes have numerous enzymes with capabilities in antimicrobial defense and functional links to the immune system, in which melanin may play an activating role. Furthermore, it is believed that melanocytes in the human epidermis play a key role in protecting our skin from the damaging effects of UV radiation by scavenging free radicals and reactive oxygen species, besides simply attenuating the radiation. It is well established that melanin has multiple functions such as anti-radiation, antioxidant, antitumor, antivenin, anti-virus, and removing heavy metal ions. In all these potential applications, insolubility of both natural melanin and synthetic melanin in bio-compatible solvent leads to quick precipitation and formation of large aggregates, drastically reducing the efficacy in in vivo and in vitro experiments. We deliberately set out to develop techniques based on photo-fragmentation with femtosecond laser pulses and mechanical smashing respectively for nanonization and dispersibilization of melanin, in order to more reliably study the biological functions of melanin and to promote the efficacy of melanin as medicine. It was found that both Sepia melanin and synthetic melanin particles processed with either method represent flaky shape with the diameter of ~42.5 nm and height of ~0.95 nm. Therefore, they can disperse in water and avoid precipitation for more than a week. In principle, the techniques can be applied to any kind of melanin. Amount them, the nanonization process by femtosecond laser pulses also serves as a top-down approach for resolving melanin structure. We inferred that in Sepia melanin the aggregation of nano-flakes is mediated by van der Waals interaction and hydrophobic interaction, whereas in synthetic melanin the formation of micro-flakes from nano-flakes is mediated by π–π interaction, which is substantially stronger than the former. As for the biomedical applications, experiments on the antimicrobial efficacy concluded that even if melanin plays a role in antimicrobial capability of skin, as proposed previously by others, it does not result from direct killing of microbe by melanin nanoparticle. In addition, this shows that melanin nanoparticle whether illuminated or not is not cytotoxic, therefore promises its use as medicine. Moreover, we demonstrated that nanonization dramatically improves the efficacy of melanin against acute oxidative stress and heavy metal ions. The effect was even more prominent without simultaneous light irradiation, promising for effective in vivo intravenous application to the whole body.