應用與比較靜電式氣液介面暴露系統與沉浸式暴露法於奈米銀毒性測試結果

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林子皓(Zi-Hao Lin) 查詢紙本館藏

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應用與比較靜電式氣液介面暴露系統與沉浸式暴露法於奈米銀毒性測試結果
(Evaluation and comparison of ESP-ALI exposure system and submerged method for AgNPs toxicity test)

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摘要(中)

奈米科技是近年來最炙手可熱的產業之一，越來越多的奈米產品被應用在工業與日常生活中。然而，隨著奈米科技的蓬勃發展，奈米材料的潛在毒性也逐漸受到人們的重視。許多研究人員投身其中，致力於了解奈米物質的傳輸與毒性機制，期望能提供政府機構未來制定相關法規的參考資料。過去奈米物質毒性實驗中大多採用沉浸式in vitro的方式，此方法雖然操作較為簡單且成本較低，但近年來越來越多研究人員對此方法提出質疑，認為應用此一方法所進行的生物毒性實驗無法完全代表暴露物質原本的毒性強弱。氣液介面式暴露技術能克服傳統沉浸式暴露的缺點並可模擬真實情況中微粒與人類呼吸道的暴露行為，故許多學者開始採用此一方式進行生物毒性實驗，但氣液介面暴露技術仍然存在部分尚未釐清的疑慮，本研究目標即為觀察氣相奈米銀微粒沉浸於液相之後其物化特性的變化程度，並實際建立一套靜電式氣液介面(ESP-ALI)暴露系統進行生物毒性實驗，再與沉浸式實驗結果相比及討論兩種實驗方法的差異性。
氣相奈米銀微粒沉浸於兩種不同溶液(DI水與DMEM-H medium)後，其粒徑大小皆有明顯上升的趨勢，顯示氣相奈米銀微粒沉浸於液體之後會發生劇烈的聚集行為。界達電位方面，沉浸於DI水的奈米銀微粒維持在-20至-30 mV，DMEM-H medium的奈米銀微粒則維持在-5至-15 mV，細胞介達電位多為負值，奈米銀微粒與生物接觸機率降低。銀離子釋出實驗方面，暴露四小時後，DI水中銀離子濃度可達0.8至1.5 ppb，DMEM-H medium中銀離子濃度為3.5至5.1 ppb，此一範圍的銀離子濃度足以造成部分生物死亡，此外，DMEM-H medium中的無機鹽類會與銀銀子結合，故DMEM-H medium的銀離子濃度會隨存放時間增長而下降。
實際應用ESP-ALI暴露系統於生物實驗方面，本研究所建立的暴露系統於暴露時間三小時以內可使細胞活性維持在80%以上。實驗結果顯示本研究所觀測的幾項生物指標(死亡率、細胞自我吞噬與細胞凋亡)，ESP-ALI暴露系統皆僅需低於沉浸式實驗的劑量即可誘發相近程度的毒性反應，表示沉浸式實驗確實存在劑量高估的疑慮。細胞壞死方面，應用沉浸式暴露法僅需更低劑量便可誘發細胞產生細胞壞死，顯示不同暴露方式會導致受測細胞死亡機制不同。
本研究實際觀測了氣相奈米銀微粒沉浸於液體之後的物化特性變化，並比較兩種不同暴露方式(ESP-ALI與沉浸式)實驗結果的劑量差異，未來將持續改進ESP-ALI暴露系統效能與穩定性，期望提供更為準確的生物毒性實驗方法。

摘要(英)

Traditional submerged exposure method has some drawbacks and limits which may influence test results. An air-liquid-interface (ALI) exposure method can conquer those disadvantages of submerged exposure method, so more and more researchers apply this method to do nanomaterials toxicity test. However, the physical and chemical properties of test materials which may change in the ‘exposure processes’ is rarely be evaluated by ALI exposure method. In addition, the broad-spectrum antimicrobial properties of silver nanoparticles (AgNPs) make its use in numerous household products, water and air puriﬁcation. Researchers also have been investigating the potential toxicity of AgNPs. This research investigated the physical and chemical properties of air phase silver nanoparticles (AgNPs) when they immersed into different liquid, and established an ESP-ALI exposure system to do silver nanoparticles biological toxicity test, and then compared these results with traditional submerged exposure method.
After immersed into DI water, particle size of liquid phase AgNPs would become larger than air phase AgNPs. It presented AgNPs would aggregate dramatically when air phase AgNPs immersed into liquids. Zeta potential of liquid phase AgNPs would approach to -38 mV, and then increase with storage time. In brief, particle size becoming larger and zeta potential showed a negative value implied that its toxicity would decrease when air phase AgNPs immersed into DI water. In terms of water quality, pH and dissolved oxygen in AgNPs suspension solution maintained a constant value with storage time increasing. Thus it implied that air phase AgNPs immersed into DI water would not affect pH and dissolved oxygen of DI water. Conductivity increased with increasing storage time perhaps due to the Ag+ ion release. Furthermore, the released Ag+ concentrations increased almost linearly within 4 hours. After exposure at 4-hour point, Ag+ concentrations of AgNPs suspension would approach to 0.8~1.5 ppb, this concentration range may make some organisms dead.
Furthermore, after immersed into DMEM-H medium, liquid phase AgNPs would aggregate significantly, and particle size can be even larger than one immersed into DI water. This is because under high ionic strength condition, the attractive force between particles became dominant over the repulsive force. Zeta potential of liquid phase AgNPs would approach to -5 mV initially, and then decrease with storage time increasing. In terms of water quality, pH and dissolved oxygen would not change with storage time increasing, and however, conductivity had a trend of rise first and then fall. It may be because Ag+ can bind with Cl- and decrease the ionic strength of sample. Released Ag+ concentrations also had a similar trend with conductivity of AgNPs suspension solution.
Our ESP-ALI exposure system can make cell viability above 80% when exposure time shorter than 3 hours. It presented our ESP-ALI exposure system can use in short exposure experiments. Compared with submerged exposure method, ESP-ALI exposure system only needed lower AgNPs dose to make biomarkers easily detected (cell viability, cell autophagy and apoptosis), and it implied submerged exposure method had a disadvantage of overestimated dose. However, compared with ESP-ALI exposure system, submerged exposure method needed lower dose to induce necrosis, and it implied different stresses on the test cell via different exposure methods might cause different cell death mechanisms.

關鍵字(中)

★ 奈米銀微粒
★ 奈米毒理學
★ ESP-ALI暴露系統

關鍵字(英)

論文目次

摘要……… I
Abstract….. III
致謝……… V
目錄……… VI
圖目錄…… .VIII
表目錄........ …...X
符號說明… XI
一、前言 1
1-1 研究緣起 1
1-2 文獻回顧 5
1-2-1 奈米銀對於細胞的毒性機制 5
1-3 研究目的 16
二、研究方法 17
2-1 奈米銀物化特性變化實驗流程 17
2-2 ESP-ALI系統應用於生物暴露實驗流程 23
2-2-1 ESP-ALI設計與操作條件 23
2-2-2 氣膠生成與暴露系統 24
2-2-3 細胞培養與生物毒性暴露實驗 26
2-2-4 沉浸式in vitro生物暴露實驗 32
三、結果與討論 33
3-1 氣相奈米銀微粒沉浸於液相中物化特性的改變 33
3-1-1 奈米銀氣膠合成 33
3-1-2 溶液中奈米銀微粒物化特性變化(以DI水做為收集溶液) 42
3-1-3 溶液中奈米銀微粒物化特性變化(以DMEM-H medium做為收集溶液)…… 52
3-2 ESP-ALI生物暴露實驗結果 60
3-2-1 ESP-ALI模組測試結果 60
3-2-2 實際應用ESP-ALI於生物暴露實驗結果 65
四、結論 85
參考文獻…. 90
附錄……... 99
A. 90 plus particle size analyzer 測試 99
B. DI水靜置4小時後，水質條件隨時間的變化 100
C. DMEM-H medium靜置4小時後，水質條件隨時間的變化 101
D. 細胞自我吞噬流式細胞儀結果(ESP-ALI，18小時) 102
E. 細胞凋亡與壞死流式細胞儀結果(ESP-ALI，18小時) 104
F. 細胞氧化壓力生成流式細胞儀結果(ESP-ALI) 107
G. 細胞自我吞噬流式細胞儀結果(傳統沉浸式，18小時) 108
H. 細胞凋亡與壞死流式細胞儀結果(傳統沉浸式，18小時) 111
委員意見回覆 114

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指導教授

蕭大智(Ta-Chih Hsiao)

審核日期

2015-7-7

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