- 學位論文
牛血清白蛋白(Bovine Serum Albumin, BSA)修飾之奈米金粒子的毒性評估測試
Toxicity Assessment of BSA-Coated Gold Nanoparticles
摘要
奈米金粒子(Gold nanoparticles，AuNPs)的應用與研究逐年成長，基於其具有低反應性和良好生物相容性的特性，適用於微米級(micro)與奈米級(nano)粒子的合成與製造，目前已知運用在多種領域，例如藥物運輸、基因傳遞以和生物影像......等。由於粒子表面可以附著塗層材料，在AuNPs外層形成一層包膜，使得AuNPs能夠獲得包覆物質的特性、功能。在本篇研究選用的塗層材料是胎牛血清白蛋白(Bovine serum albumin，BSA)，過去研究已證實BSA具有高度生物相容之特性，利用化學反應將金奈米粒子進行BSA包覆性處理，合成BSA-coated AuNPs (以下以AuNP,BSA表示)。為了評估AuNP,BSA在生物體內是否引起不良反應，利用斑馬魚(Danio rerio)的胚胎作為動物模式進行毒性試驗。因為胚胎對毒性物質的高敏感性、斑馬魚發育時間短以及斑馬魚的半透明體便於觀察生理變化，使用此動物模式是目前毒性評估的標準實驗方法之一。除了胚胎存活率和生理發育觀察作為AuNP,BSA毒性測試，另外亦使用Q-PCR和Cytokine assay進行細胞激素IL-6和TNF-α的檢測，評估暴露於奈米金顆粒等成分中是否引起免疫發炎反應。研究數據顯示，胚胎經過72小時的浸泡於各溶液中，純奈米金粒子AuNPs對斑馬魚胚胎僅具有無至低毒性反應；AuNP,BSA則對斑馬魚胚胎產生較高毒性作用。Q-PCR結果發現，暴露於AuNPs的斑馬魚胚胎，IL-6 mRNA 表現略有減少，而TNF-α mRNA表現與對照組無顯著差異；暴露於AuNPs,BSA的組別，IL-6 mRNA表現略有下降、TNF-α mRNA表現則顯著上升。 此外，利用MTT assay進行細胞實驗，測試AuNPs和AuNPs,BSA對老鼠單核球細胞(RAW264.7)和正常人類纖維細胞(Wi-38)是否有毒殺效果。實驗結果顯示，使用高濃度100μM的AuNPs-BSA培養24小時之後，兩種細胞株仍有80.91%和78.52%的存活率。使用細胞激素IL-6和TNF-α檢測RAW264.7 cells的狀態細胞培養液(condition medium)，AuNPs和AuNPs,BSA兩組的TNF-α levels均有顯著增加，尤其是AuNPs,BSA組別。然而細胞暴露在AuNPs和AuNPs-BSA中無偵測到IL-6 levels。除了以上實驗之外，使用合成AuNPs,BSA時所使用的相同BSA劑量，進行6小時細胞實驗，以便確認BSA本身是否會影響細胞激素的表現。從IL-6 levels和TNF-α levels結果可見，RAW264.7 cells暴露於BSA時所產生的IL-6量遠高於AuNPs-BSA的組別所產生的。本篇研究顯示胎牛血清白蛋白包覆金奈米粒子(AuNPs,BSA)對斑馬魚胚胎會引起免疫反應，但是對老鼠免疫細胞或人類正常纖維細胞的毒殺能力低，未來可針對包覆性材料與奈米金顆粒大小進行進一步探討。
並列摘要
Gold nanoparticles (AuNP) have become a tool of great interest due to its low reactivity and biocompatibility, making it a suitable material for constructing micro and nanoparticles. AuNPs can be used for various applications such as drug delivery, gene delivery, and bioimaging. This is possible because AuNPs can be functionalized with a coat, which allows the particle to take on the properties of the coat. The coating material used in this study is Bovine Serum Albumin (BSA), which has been previously shown to be biocompatible. In order to determine whether BSA-coated AuNPs (AuNP,BSA) can induce an adverse response, toxicity assessments of the AuNP,BSA were performed using zebrafish (Danio rerio) embryos. Zebrafish embryos were chosen as a model for toxicity assessments because of their sensitivity to toxic substances and their rapid development. Furthermore, the Zebrafish’s semi-transparent body allows for ease of observation of physiological changes. In addition to the toxicity assessment, Q-PCR and Cytokine assay were performed to assess any changes in IL-6 and TNF-α levels in order to determine if exposure to the AuNPs causes an inflammatory response.In this investigation, it was found that uncoated AuNPs exhibited little to no toxic effect on zebrafish embryos after 72 hours of exposure, while AuNP,BSA exerted some toxic effect. Q-PCR results of the zebrafish IL-6 and TNF-α showed that exposure to AuNPs slightly reduced IL-6 mRNA levels, while TNF-α levels did not differ from control. Exposure to AuNP,BSA also slightly reduced IL-6 mRNA levels. However, exposure to AuNP,BSA increased TNF-α mRNA levels. In addition, MTT assay was performed on mouse monocyte cell line RAW264.7 and human fibroblast cell line Wi-38 to determine if the AuNPs have any toxic effects in vitro. Results indicated that cells were viable after 24-hour exposure to up to 100 μM AuNP or AuNP,BSA. Furthermore, the cytokine assay for IL-6 and TNF-α showed that exposure to 100 μMAuNP or AuNP,BSA increased TNF-α secreted by RAW264.7, although cells exposed to AuNP,BSA secreted higher levels of TNF-α. However, cells exposed to either nanoparticle did not produce any IL-6. Additionally, a cytokine assay was performed after 6 hour exposure to the equivalent amount of BSA used to make a 100 μM AuNP,BSA solution to determine if BSA alone affected cytokine levels. Results showed that cells exposed to BSA alone produced greater levels of IL-6 and TNF-α compared to the control.