功能性配位基合成及應用在奈米氧化銅去毒性之研究

A functional ligand synthesis and applied in the detoxicity of CuO nanoparticles

陳冠宜(Kuan-Yi Chen)

指導教授 ： 黃鈺軫

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2010年)

摘要

當微粒縮小至奈米等級時，由於其特殊的反應性，使得環境與人體暴露並受其危害的機會也大幅增加。一般常用的奈米金屬氧化物中，奈米氧化銅具特別高的毒性。有研究顯示，當細胞胞飲吸入氧化銅奈米微粒後，氧化銅微粒會抑制細胞內抗氧化酵素活性，此外，除了微粒表面所誘發產生的活性氧化物質(ROS)外，另有來自於微粒表面其他未知的機制導致細胞死亡。這些ROS會氧化細胞內DNA等重要生體分子，且氧化銅微粒表面所解離出的銅離子也會傷害DNA分子。
本研究是全合成(2E,4E)-5-(4-hydroxyphenyl)penta-2,4-dienoic acid (HPPDA)分子，其具備可吸收ROS的共軛酚類結構，與可化學吸附在氧化銅微粒表面形成修飾型氧化銅的羧酸官能基，希望能以吸收氧化銅誘發產生的細胞內ROS、降低微粒與細胞接觸的活性表面積與減少銅離子從微粒表面解離的方式，減少氧化銅的奈米毒性，以增加奈米材料的環境友善性。
暴露HPPDA與常見的抗氧化劑，L-抗壞血酸(維生素C主結構)於A549細胞，由細胞致死率(MTT)與預發炎物質(IL-8)測試細胞毒性，結果發現HPPDA比抗壞血酸更具細胞耐受性。且在測試對H2O2 (其中一種ROS物質)吸收能力比較上，HPPDA比抗壞血酸吸收能力更強，因此以HPPDA修飾氧化銅奈米微粒的方式降低其細胞毒性是具可行性。
HPPDA在一般中性或微酸溶液中可部分解離羧酸根，使HPPDA能以羧酸官能基在氧化銅奈米微粒表面形成自組裝分子層(SAM)，此為功能性氧化銅微粒(fCuO)。fCuO在細胞致死率、刺激IL-8分泌、細胞內ROS的產生、細胞老化與氧化性DNA傷害上，都比奈米氧化銅毒性大幅下降許多。因此，HPPDA作為修飾奈米微粒配位基，在奈米材料的後處理上，可降低對人體健康的衝擊與增加環境友善性。而在比較都具備羧酸官能基但抗氧化能力不同的配位基:油酸、L-抗壞血酸、HPPDA，所修飾的氧化銅微粒的細胞致死率可發現，配位基降低奈米毒性的能力除了來自微粒與細胞接觸的活性表面積的減少外，配位基本身的抗氧化能力佔有很重要的因素。
此外，HPPDA由於具備優異的ROS吸收能力、特別的低細胞毒性與可直接修飾奈米微粒或可接上其他特定官能基的羧酸，因此未來也可做為抗氧化分子骨架，搭載無毒或磁性奈米生醫材料，可用以消除生體內氧化壓力。

關鍵字

奈米毒性 ； 抗氧化劑 ； 配位基

並列摘要

While particles reach nanoscale, specific properties of nanoparticles (NPs) may cause more hazard potential to health and environment. Among these common metal oxides NPs, the toxicity of CuO NP is highest. Some studies had reported that after pinocytosing CuO NPs, cells showed specific lethality because of reactive oxygen species (ROS) induced by NPs and some still unknown mechanisms from NPs surface. Furthermore, oxidative stress inducing from ROS and Cu2+ released from CuO NPs would cause DNA oxidative damage.
In this study, (2E, 4E)-5-(4-hydroxyphenyl) penta-2,4-dienoic acid (HPPDA), with conjugated phenols for ROS scavenging and carboxyl group for chemisorption on suface of CuO particles, was designed and synthesized. For scavenging intracellular ROS induced by CuO NPs, decreasing active surface of NPs contact with organisms, and lowering Cu2+ released from surface of particles, we modified CuO particles by coating HPPDA antioxidants to reduce nanotoxicity of CuO and increase environmentally friendly.HPPDA and L-ascorbic acid (a general antioxidant) exposed respectively to human lung carcinoma epithelial cell lines (A549) were study to compare the cytotoxicity through cell lethality and pre-inflammation response. Experimentally, HPPDA shows much more cell tolerance. In addition, the H2O2 scavenging capability of HPPDA is better than ascorbic acid. Thus, it is feasible to reduce cytotoxicity of CuO NPs by modification of HPPDA. HPPDA can be partial ionized its carboxyl group to form self-assembled monolayer (SAM) on the surface of CuO NPs under neutral or weak acidic situation, which was called functional CuO particles (fCuO). The results of cell lethality, stimulation of interleukin-8 secretion, production of intracellular ROS, cell aging and oxidative DNA damage, indicate that the cytotoxicity of fCuO is much lower than of naked CuO. Thus, in the after-treatment of NPs, HPPDA can be the suitable ligands for NPs modification to reduce their nanotoxicity. In addition, comparison of CuO NPs modified by different ligands: oleic acid, L-ascorbic acid and HPPDA (all of them are with carboxyl group but different in antioxidative capability) shows different toxicity in cell lethality. It also indicates that the capability of ligand for reducing nanotoxicity is based on not only decreasing active surface of NPs to contact directly with organisms by modification but the antioxidative capability of ligand itself.
Furthermore, due to outstanding antioxidative capability, very low cytotoxicity and possession of carboxyl group which can modify NPs directly or link other specific groups, HPPDA may be the framework combined with non-toxic or magnetic biomaterials applied for anti-aging in the future.

並列關鍵字

nanotoxicity ； antioxidant ； ligand

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