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研究生：	鄭乃綺 Cheng, Nai-Chi
論文名稱：	利用金-亞甲基藍奈米複合材料促進傳遞光動力治療於有氧和無氧環境中的抗癌細胞與抗菌生長 Enhanced photodynamic therapy delivery of Au-methylene blue nanohybrids for efficient antibacterial and anti-cancer treatments in the normoxia and hypoxia environments
指導教授：	黃志嘉 Huang, Chih-Chia
學位類別：	碩士 Master
系所名稱：	理學院 - 光電科學與工程學系 Department of Photonics
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	99
中文關鍵詞：	金奈米粒子、亞甲基藍、光動力治療、常氧、缺氧、抗癌、抗菌
外文關鍵詞：	gold nanoparticle, methylene blue, Type I/II PDT, normoxia, hypoxia, anti-cancer, antibacterial
相關次數：	點閱：63 下載：1
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透過控制前驅物金鹽、單寧酸、亞甲基藍莫耳數以調節生成奈米粒子之尺寸及形狀，以製作出具有不同光學活性的奈米粒子。藉由簡便且綠色的合成方法製作出金奈米粒子作為藥物載體。靠著前驅物之間生成的作用力，以大量攜載亞甲基藍藥物於金奈米粒子表面。尺寸較大的金奈米粒子具有較優異的表面增強拉曼散射能力。合成中添加的亞甲基藍大部分可吸附在金奈米粒子上，如此，吸附上的亞甲基藍可同時具有單體及二聚體的型態以增進了單態氧(type II)及氫氧自由基的生成(type I)。相似的合成方法也被用來生成Au@MB-Ce6奈米粒子應用於生物螢光成像。除此之外，也分析了複合上亞甲基藍後的金奈米粒子光學特性，特別針對拉曼光譜進行研究。金奈米粒子有效提升亞甲基藍的拉曼訊號促使治療和診斷結合的可能性。亞甲基藍-金奈米粒子與穀胱甘肽水溶液培養後，仍可有效地生成氫氧自由基以改善缺氧環境下的光動力治療效果。經穀胱甘肽水溶液培養後，亞甲基藍釋放的情形可以透過拉曼以及螢光影像進行監控。亞甲基藍-金奈米粒子搭配660 nm的光照可以有效生成活性氧化物質，進而導致惡性癌細胞凋亡。藉由進一步修飾小分子於粒子表面可提升且加速粒子於惡性癌細胞的累積量，以達到提升光動力治療效果及生物影像品質。

關鍵字: 金奈米粒子、亞甲基藍、光動力治療、常氧、缺氧、抗癌、抗菌

In this work, we modulate the concentration of precursors (HAuCl4, TNA and MB) to control of particle size and shape becomes particularly important to offer the desirable applications which required optical functions (Au/TNA/MB assembles). We hope to manufacture gold nanoparticles (Au NPs) as carriers of MB, which could load a large amount of MB via molecular interactions between precursors. The size and shape of the Au@TNA/MB were controllable by adjusting the different mole ratio of the reagents. The smaller size of the Au NPs reduced by TNA generated weak SERS effect, while a promotion of SERS was relied on the formation of bigger size for Au NPs as a SERS enhancer. As an increase of MB concentrations co-assembled onto the surface of Au NPs, we found the improvement of the 1O2 generation (type II) as well as the yield of the OH radical (·OH) in type I process. The similar synthesis method could be extended to fabricate Au@MB-Ce6 NPs for fluorescent bioimaging. In contrast, the integration of Au@MB NPs on the field of SERS, the drug releasement under high concentration GSH in vitro, PDT, and ligand targeting was also investigated. The improvement of SERS feature was found in the Au@MB NPs, leading the combination of therapy and diagnostic. After incubated with GSH solution, MB molecules releasing from the surface of Au@MB NPs parts of MB molecules were estimated still be dimer form and could generate ·OH and eventually elevate the PDT efficiency under hypoxia. The distribution of MB molecules after treating with GSH was monitored via the simultaneous fluoresce and Raman mapping images. The ROS generation of Au@MB NPs with the explosion of 660 nm laser (125 mW/cm2) induced the cell toxicity, leading to the apoptosis and death of cancer cells. We further modified ligands on Au@MB NPs to enhance the accumulation of nanoparticles to the cancer cells in short time. Au@MB NPs were incubated with cancer cells and monitored via fluoresce and Raman images. With the longer incubation time, the Raman intensity would decrease and the red fluorescence of MB would increase by four times.

Key word: gold nanoparticle, methylene blue, Type I/II PDT, normoxia, hypoxia, anti-cancer, antibacterial

中文摘要	I
Abstract	II
致謝	IV
Contents	V
List of Figures	VIII
List of Tables	XIII
Motivation	XIV
Part 1: Self-assembly	1
Chapter 1 Introduction	1
1 Green synthesis	1
2 Advantage of tannic acid	2
3 Self-assembly of TNA	2
4 Self-assembly of nanoparticles	3
Chapter 2 Experimental	7
1 Materials	7
2 Equipment	7
3 Methods	7
3.1 Preparation of Au@TNA/MB complex nanostructures	7
Chapter 3 Results and Discussion	9
1 Synthesis and characterization of Au@TNA/MB tubes	9
2 Synthesis of Au@TNA/MB tubes with different concentration of TNA	9
3 Synthesis and characterization of Au@TNA/MB nanoassemblies	10
4 Synthesis of Au@TNA NPs with different concentration of TNA	12
5 The complex formation of MB and TNA	13
6 Synthesis of Au@TNA NPs with re-used TNA	14
Chapter 4 Conclusion	22
Part 2: Photodynamic Therapy of Au@MB NPs	23
Chapter 1 Introduction	23
1 Photodynamic Therapy	23
1.1 Methylene blue	24
2 Bladder cancer	26
Chapter 2 Experimental	28
1 Materials	28
2 Equipment	28
3 Methods	29
3.1 Preparation of Au@MB NPs	29
3.2 Preparation of Au@TNA NPs	29
3.3 Singlet oxygen detection in vitro (Type II)	29
3.4 Hydroxyl radical detection in vitro (Type I)	29
3.5 Detection of singlet oxygen after MB released	30
3.6 Hemoglobin Colorimetric Assay Kit	30
3.7 Cellular Reactive Oxygen Species Detection (DCFH-DA)	31
3.8 Cell viability assay	31
3.9 Trypan blue exclusion test	32
3.10 Atrophy of cell	32
3.11 Au@MB NPs modified with ligands	32
3.12 BSA targeting	33
3.13 Bacteria	33
Chapter 3 Results and Discussion	34
1 Characterization of Au@TNA and Au@MB NPs	34
2 Generation of Singlet oxygen test via reagents	37
3 The biocompatibility of Au@MB NPs	38
4 In vitro PDT efficacy of Au@TNA and Au@MB NPs	39
4.1 Cell toxicity of normoxic cells after PDT treatment	39
4.2 In vitro 1O2 generation capability of Au@MB NPs	39
4.3 Cell toxicity of Au@MB NP against to bladder cancer cell	40
4.4 Cell toxicity of Au@MB NP against human uroepithelial cells	42
5 Generation of hydroxyl radical test via reagent	42
6 Au@MB NP-mediated PDT efficacy under hypoxia	43
6.1 Cell toxicity of Au@MB NPs in HT29 cells under hypoxic condition	43
6.2 Generation of hydroxyl radical test after drug release	43
6.3 Antibacterial activity of Au@MB NPs against aerobic and anaerobic bacteria	45
6.4 TEM images of bacteria mixed with Au@MB NPs before and after PDT	46
Chapter 4 Conclusion	61
Part 3: Raman/Fluorescence dual-imaging	62
Chapter 1 Introduction	62
1 Surface-enhanced Raman scattering	62
1.1 Electromagnetic (EM) enhancement	62
1.2 Chemical enhancement (CE)	63
2 Theranostic	64
2.1 Fluorescent bio-image	64
2.2 Raman bio-image	65
Chapter 2 Experimental	68
1 Materials	68
2 Equipment	68
3 Methods	68
3.1 Preparation of Au@MB NPs	68
3.2 Preparation of Au@TNA NPs with various sizes	68
3.3 MB release in various solution (pH 4, 7, 10 and GSH)	69
3.4 Raman	69
3.5 BODIPY 581/591 C11 stain	69
3.6 MB release in vitro	70
Chapter 3 Results and Discussion	71
1 Raman scattering property of Au@MB NPs	71
2 Synthesis and Raman property of Au@TNA NPs with various size	72
3 MB release behavior in GSH and buffer solution	72
4 MB release behavior in GSH-GSH kit	74
5 Combination of fluorescence and Raman mapping images	75
Chapter 4 Conclusion	90
Summary	90
References	92
                                    

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