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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48759

Title: 功能性奈米材料應用於偵測纖溶相關蛋白與循環腫瘤細胞及腫瘤細胞的光治療
Applications of Functional Nanomaterials for Detection of Fibrinogen-Related Protein and Circulating Tumor Cells, and Cancer Cell Phototherapy
Authors: Wei-Jane Chiu
邱緯真
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 奈米材料;纖溶酶;雷射脫附游離質譜;循環腫瘤細胞;腫瘤光治療
nanomaterials;plasmin;laser desorption/ionization mass spectrometry;circulating tumor cells;tumor phototherapy
Date: 2016
Issue Date: 2018-08-22T06:31:03Z
Abstract: 本研究中我們主要為開發功能性奈米材料應用於偵測纖溶相關蛋白(Fibrinolytic-Related Proteins)、循環腫瘤細胞(Circulating Tumor Cells; CTCs)及光治療(Phototherapy)腫瘤細胞(Tumor Cells)。於偵測纖溶相關蛋白及循環腫瘤細胞部分,結合雷射脫附游離質譜(Laser desorption/ionization Mass Spectrometry; LDI-MS)作為一分析工具進行檢測。纖溶系統為一藉由纖溶酶(plasmin)將纖維蛋白降解而回復正常血管功能的機制,於第一個研究工作中,在仿生理條件下將修飾纖維蛋白原的金奈米粒子(FibrinogenGold Nanoparticles; FibAu NPs)探針和纖溶酶反應後,降解金奈米粒子表面之纖維蛋白原,導致金奈米粒子聚集,再將其吸附上混合纖維素脂膜(Mixed Cellulose Ester Membrane; MCEM)。因纖溶酶降解FibAu NPs後使其與MCEM吸付能力降低,故在脈衝雷射(355 nm, 6 ns)照射MCEM下,碎裂Au NPs所形成金團簇訊號強度會下降,藉由金團簇訊號改變可定量溶液中纖溶酶。MCEM在LDI-MS中可降低背景雜訊干擾,提高偵測靈敏性亦提供良好的再現性(相對標準偏差小於5%)。利用此方法可成功的在人類血清環境中高度靈敏地偵測纖溶酶(偵測極限: ca. 0.1 nM) 並具有高度選擇性。此簡單、快速、靈敏、高通量的檢測法,於臨床醫學檢驗蛋白活性上相當具有發展性。於第二個研究中我們探討金奈米薄膜(Gold Nanofilms; Au NFs)於不同厚度(10100 nm)在脈衝雷射(355 nm, 6 ns)照射下形成Au NPs大小、密度以及金團簇訊號強度的變化,研究發現Au NFs厚度、脈衝雷射能量與金奈米粒子的形成有相當的關係,較薄Au NFs與高雷射能量易形成高密度小粒子的Au NPs。隨後,我們藉由金硫鍵結(Au-S bond)於Au NFs(厚度20 nm)表面修飾具與特定腫瘤細胞專一性結合MUC1的功能性核苷酸適合體(MUC1 Aptamer; AptMUC1),合成出具專一性之AptMUC1−Au NFs偵測平台,結合雷射脫附游離質譜可透過監測金團簇訊號強度變化靈敏地在人類全血環境中高選擇性的檢測乳癌腫瘤細胞(MCF-7),其偵測極限可達至10顆癌細胞。我們所開發出偵測循環腫瘤細胞的方法具有簡單、快速和易操作等優點,將來可應用於腫瘤轉移的研究。於第三部分研究中,我們致力於發展奈米材料應用於結合腫瘤細胞的光熱治療(Photothermal Therapy; PTT)及光動力治療(Photodynamic Therapy; PDT)的雙重療效(PTT/PDT)試劑並成功的在荷瘤老鼠實驗上獲得良好的治療效果。我們利用二價鐵離子在三羥甲基氨基甲烷硼酸鹽溶液反應下會與還原型氧化石墨烯(reduced Graphene Oxide; rGO)自組裝形成氫氧化鐵/氧化鐵石墨烯複合(FeOxH‐rGO)的方式簡易製備雙重光治療之複合型奈米材料。奈米石墨烯及其氧化衍生物具有較大的比表面積和優異的光熱效果等性質,已成為奈米醫學領域中備受關注的研究重點。由於氧化石墨烯特殊表面結構及官能基使其在紅外光區域有很強的吸收及光熱轉換效率,大幅增加了腫瘤細胞光熱治療的應用性。而我們所製備的FeOxH‐rGO表面沉積之不規則氫氧化鐵/氧化鐵在光照射下則會進行類似Fenton反應於水溶液中產生氧化自由基(Reactive Oxygen Speices; ROS)促進腫瘤細胞凋亡達到光動力治療之效果。我們使用808 nm雷射作為FeOxH–rGO進行腫瘤細胞光治療的光源,在體外(in vitro)及體內(in vivo)動物實驗部分均有相當顯著的治療效果。此一結合光熱及光動力治療(PTT/PDT)效果之奈米複合型材料,不僅製備簡易、成本較低,其以近紅外光源做為啟動治療開關達到雙重治療效果,大幅降低了在臨床腫瘤細胞治療上風險及後遺症,在臨床治療上相當具有發展潛力。
In this thesis, we describe a pulsed-laser desorption/ionization mass spectrometry (LDI-MS)-based approach for the detection of plasmin with subnanomolar sensitivity through the analysis of gold (Au) clusters desorbed from fibrinogen-modified gold nanoparticles (Fib−Au NPs) on a mixed cellulose ester membrane (MCEM) and for the detection of tumor cells through the analysis of gold cluster ions [Aun]+ from aptamer-modified gold nanofilms (Au NFs). In the first part, the sensing mechanism of Fib−Au NPs probe is based on the plasmin-mediated cleavage of the Fib−Au NPs and the reduced interaction between Fib−Au NPs and MCEM. The Fib−Au NPs were deposited onto the MCEM to form a highly efficient background-free surface assisted LDI substrate. Under pulsed laser irradiation (355 nm, 6 ns), the cleaved Fib−Au NPs decreased the adsorbed on MCEM. As a result, the intensities of the signals of the Au clusters decreased in the mass spectra. This approach provided a highly amplified target labeling indicator for the analysis of plasmin. Under optimized conditions, this probe was highly sensitive (limit of detection: ca. 0.1 nM) and selective (by at least 1000-fold over other enzymes and proteins) toward plasmin; it also improved the reproducibility (<5%) of ion production by presenting a more-homogeneous substrate surface relative to surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) analysis. Relative to conventional assays, this new probe for plasmin offers the advantages of high sensitivity and selectivity and high throughput, with great potential for practical studies of fibrinolytic-related proteins. In our second study, we observed not only the transformation of Au NFs to gold nanoparticles (Au NPs) but also the formation of gold cluster ions ([Aun]+; n = 1−5) under the irradiation of nanosecond pulsed-laser. The sizes and density of the formed Au NPs and the abundance of [Aun]+ were highly dependent on the thickness of Au NFs (10100 nm). The Au NFs form highly dense Au NPs on substrate and favor to desorption and ionization of gold cluster ions. The signal intensities of the [Aun]+ through the monitoring by mass spectrometry were decreased with increasing the thickness of Au NFs from 10 nm to 100 nm and after modified with thiolated DNA. We further demonstrated that the mucin1-binding aptamer modified Au NFs (AptMUC1−Au NFs) could selectively enrich of MCF-7 cells (human breast adenocarcinoma cell line) in blood samples and coupling with LDI-MS analysis could selectivly detection of MCF-7 cells as low as 10 cells in blood samples by monitoring the [Aun]+. This approach offers the advantages of high sensitivity, selectivity and high throughput for the detection of circulating tumor cells (CTCs), showing great potential as a powerful analysis platform for the application in clinical diagnosis of tumor metastasis. In the third study, we employed iron hydroxide/oxide immobilized-reduced graphene oxide (FeOxH–rGO) nanocomposites as a combination theory of photothermal therapy (PTT) and hotodynamic therapy (PDT) agent for cancer therapy. We investigated the PTT and PDT therapy abilities of FeOxH–rGO nanocomposites in cell line and in living mice. Compared to GO and rGO, which have been well known for great photothermal effect, FeOxH–rGO nanocomposites exhibits much higher photothermal conversion efficiency; ~2.6 and 1.7 fold higher than that of GO and rGO, respectively. FeOxH–rGO also induced >7-fold formation of reactive oxygen species (ROS) under NIR irradiation relative to cell medium only. Furthermore, we demonstrated that FeOxH–rGO nanocomposites have much better phototherapy effects for mice bring tumors relative to rGO. Therefore, FeOxH–rGO nanocomposite has a great potential to develop a high efficacy and safety therapeutic agent of combinatorial PDT/PTT for cancer therapy.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010036017.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48759
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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