Exploring the Evolution and Unique Properties of Multi-Branched Gold Nanostructures in Biomedicine

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研究生：	普莉亞 Priya Vijayaraghavan Radhamma
論文名稱：	Exploring the Evolution and Unique Properties of Multi-Branched Gold Nanostructures in Biomedicine Exploring the Evolution and Unique Properties of Multi-Branched Gold Nanostructures in Biomedicine
指導教授：	黃國柱 Hwang, Kuo Chu
口試委員:	袁俊傑 Yuan, Chiun-Jye 吳淑褓 Wu, Shu-Pao 江啟勳 Chiang, Chi-Shiun 宋信文 Sung,Hsing-Wen
學位類別：	博士 Doctor
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	177
中文關鍵詞：	多分支型奈米材料、生醫藥物、光動力治療
外文關鍵詞：	Branched Gold nanostructures, Biomedicine, Photodynamic therapy
相關次數：	點閱：2 下載：0
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近年來，貴金屬奈米粒子備受關注，由於它們有良好的光學性質、電性及催化性質，因此在多種應用中具有淺力，其中包含：生物醫學、觸媒以及感測器等。各種形貌金奈米材料已被發表過，而多分支的金奈米材料尤其重要，因為其特殊的形狀，導致近紅外光區有寬的表面電漿共振吸收峰。在本論文第一章中，我們著重於使用晶種成長法來合成金奈米海膽（Au nanoechinus）以及在雙尾陽離子界面活性劑（DC14TAB）控制下成長機制的探討。在其後的幾個章節中，將金奈米海膽的特殊性質應用在生物醫學上，包含癌症的光動力/光熱治療以及多種顯影的應用。
近年來，由於光熱治療的非侵入性特點使得在癌症治療備受重視。光動力治療（PDT）以及光熱治療(PTT)是光療法主要的兩種，其原理是利用感光試劑吸收光以後分別產生活性氧物種(ROS)和熱來達到毒殺細胞的效果。為了要讓照射的光線達到更好的穿深度，感光試劑必須吸收近紅外光(NIR)，因為生物組織在波段有最小的吸收，第一個近紅外光視窗波長介於650nm到900nm之間，而第二個近紅外光視窗波長屆在1000nm到1350nm之間，在這兩個波段中有以下幾個特性：低散射、極佳的組織穿深度及微弱自體螢光。第二章中，我們利用金奈米海膽作為PDT的載體，使用兩個近紅外光波段的雷射（915nm& 1064nm）激發產生單重態氧（1O2），進而達到毒殺癌細胞/腫瘤。
癌症是主要人類死因之一，非侵入性治療深層腫瘤組織是目前臨床上的一大挑戰，許多研究中的治療都是為了要克服這問題，但卻只能達到部分腫瘤毒殺或是抑制腫瘤生長。在第三章中，我們將展示如何使用金奈米海膽的PDT以及靜默基因的技術來根除深層組織的腫瘤，在此我們使用分別位於第一跟第二紅外光視窗的低強度雷射作為光源（915nm, 340mW/cm2; 1064nm, 420mW/cm2），本研究為未來深層腫瘤的治療做新的鋪路。
為了要達到更先進治療技術，奈米材料能夠擁有生物顯影應用是非常重要的。生物顯影技術的重要性在於它能夠做深層細胞的研究、提供致命疾病的偵測、狀態及治療等等資訊。在最後一張，我們發表金奈米海膽其三種生物顯影的應用：近紅外光激發/放光的上/下轉換過程、光聲顯影。綜觀本論文探討了金奈米海膽的光學性質以及在癌症的診斷及治療的應用。

In the recent years, noble metal nanoparticles have considerable attention owing to their fascinating optical, electrical and catalytic properties which makes them as potential candidates for various applications including, biomedical, catalysis and sensors. Among various morphologies being reported, anisotropic branch shaped gold nanoarchitechures are of utmost importance due to their unique morphology with broad and tunable localized surface plasmon resonance (LSPR) absorption in the near infra-red (NIR) region of the electromagnetic spectrum. In the first chapter of the thesis, we have primarily focused on the seed-mediated surfactant directed synthesis and its mechanistic investigation of the formation of multi-branch shaped Au nanoechinus using a novel twin tailed cationic surfactant (DC14TAB). In the following chapters, we have also utilized the unique properties of Au nanoechinus in various biomedical applications such as photodynamic/ photothermal therapy of tumors as well as multimodal imaging in broad dimensions.
In the recent years, phototherapy has attracted considerable attention as a powerful technique for treating cancers as well as malignant tumors with minimal invasiveness. Photodynamic therapy (PDT) and photothermal therapy (PTT) are two major phototherapeutic approaches, which require absorption of incoming light by a photosensitizer/reagent to generate reactive oxygen species (ROS) and heat for killing cancer cells, respectively. In order to have a deeper penetration of the incoming light, it is mandatory for a phototherapeutic reagent to absorb the tissue transparent near infra-red (NIR) light, where the biological components have minimum absorbance. The first NIR biological window was located in the region, 650 – 900 nm and the second NIR biological window at 1000 – 1350 nm, which therefore provides low scattering, excellent tissue penetration depths and poor autofluorescence. In the second chapter, we have shown that the photosensitization of singlet O2 from Au NEs can be achieved upon NIR light excitation in both the biological windows (915 and 1064 nm), and subsequently can exert photodynamic therapeutic effects for the destruction of cancer cells/tumors.
Cancer is one of the major diseases leading to human deaths. Complete destruction of deep tissue-buried tumors using non-invasive therapies is a grand challenge in clinical cancer treatments. Many therapeutic modalities were developed to tackle this problem, but only partial tumor suppression or delay growths were usually achieved. In the third chapter, we have demonstrated that complete destruction of deep tissue-buried tumors can be achieved by the combination of gold nanoechinus (Au NEs)-mediated photodynamic therapy (PDT) and gene silencing under ultra-low doses of near infra-red (NIR) light irradiation (915 nm, 340 mW/cm2; 1064 nm, 420 mW/cm2) in the first and second biological windows. Our findings pave out a new direction for the therapeutic design to treat deeply seated tumors in future cancer treatments.
In order to accomplish the scenario of modern theranostics, it is better for the nanomaterials to serve the purpose of intrinsic bioimaging properties in addition to the therapeutic capabilities. Bioimaging is of ultimate importance to have detail study in the underlying complex cellular and pathological events occurring inside body which gives appropriate information about the detection, status and treatment of various fatal diseases. In the last chapter, we have explored that multi-branched gold nanoechinus can serve as a new class of triple modal bioimaging reagent for NIR-to-NIR up-and down-conversion processes as well as photoacoustic imaging. Taken altogether, we have thoroughly explored the optical properties of the multi-branched gold nanoechinus in various biomedical applications for diagnosis and treatment of cancers.

Chapter 1: Synthesis of Multi-Branched Gold Nanoechinus using a Dimeric Cationic Surfactant and its Application for Surface Enhanced Raman Scattering
1 Background and Introduction    1
1.1. Noble metal nanoparticles    1
1.2. Gold nanomaterials    2
1.3Anisotropic Gold nanomaterials.    3
2 Experimental Section    7
2.1  Materials    7
2.2 Synthesis of N1, N1, N2 N2-tetramethyl-N1, N2-ditetradecylethane-1, 2-diaminium bromide    8
2.3 Synthesis of Citrate coated Gold nano seeds    8
2.4 Synthesis of Au NEs    8
2.5 Characterization of Au NEs………………………………….……………..9
2.6 SERS measurements………………………………………………………...9 
3 Results and Discussion    10
3.1 Synthesis & Characterization of Au NEs    10
3.2 Effect of DC14TAB    14
3.3 Effect of Ascorbic acid concentration    16
3.4 Effect of AgNO3    18
3.5 Isolation of intermediates and growth mechanism    24
3.6 SERS Measurements    28
4 Conclusions    30
5 References    31
Chapter 2 : Designing Multi-branched Gold Nanoechinus for NIR light activated Dual Model Photodynamic and Photothermal Therapy in the Second Biological Windows 
1 Background and Introduction    37
1.1 Photodynamic and Photothermal therapy    37
1.2 Nanomaterials as phototherapeutic reagents    37
1.3 Organic photosensitizers versus Nanomaterials    38
2 Experimental Section    41
2.1 Synthesis of double chain cetyltrimethylammonium bromide (DC14TAB) surfactant    41
2.2 Synthesis of ~18 nm Au seeds    41
2.3 Synthesis of Au NEs    42
2.4 Preparation of lipid-coated Au NEs    42
2.5 Singlet oxygen phosphorescence measurements    43
2.6 Cell culture, materials and reagents    43
2.7 Cellular uptake studies using confocal laser scanning microscopy (CLSM) and flow cytometry    43
2.8 Cytotoxicity assays    45
2.9 ROS experiments    46
2.10 Singlet oxygen sensor green (SOSG) experiments    46
2.11 Heat-shock protein expression (HSP70) analysis    47
2.12 In vivo fluorescence imaging    47
2.13 In vivo tumor growth study    48
2.14 Tissue sectioning and caspase 3 staining    49
2.15 In vivo monitoring tumor temperature    49
2.16 Singlet O2 phosphorescence Quantum yield measurements    49
3 Results and Discussion    51
3.1 Photosensitization of Singlet Oxygen by Au NEs in NIR Biological Windows I and II    51
3.2 In vitro NmPDT and NmPTT experiments    59
3.3 In vivo NmPDT and NmPTT experiments at NIR biological windows I and II    ………………………………………………………………………………66
4 Conclusions    74
5 References    75
Chapter 3 : Complete Destruction of Deep-Tissue Buried Tumors via Combination of Gene Silencing and Gold Nanoechinus-Mediated Photodynamic Therapy 
1 Background and Introduction    83
1.1 Deep Tissue Buried tumors    83
1.2 Nanomaterials for treating deep tissue buried tumors    84
1.3 Limitations for curing deep tissue buried tumors    85
1.4 Gene therapy    86
2 Experimental Section    87
2.1 Preparation of lipid-coated Au NEs    87
2.2 Detection of singlet oxygen phosphorescence emission    88
2.3 Preparation of siRNA-lipid-coated Au NEs    88
2.4 Particle size and zeta-potential measurements    89
2.5 Cell culture procedures    89
2.6 In vitro cellular uptake experiments using confocal laser scanning microscopy (CLSM)    89
2.7 Isolation of total RNA from siRNA-lipid-coated Au NEs internalized HeLa cells    90
2.8 Polymerase chain reaction (PCR) of total RNA to complimentary DNA    91
2.9 Real-time quantitative PCR (RT-qPCR) experiment    92
2.10 Western blotting to detect the expression levels of SOD1 gene    93
2.11 Dark and phototoxicity experiments using MTT assay    95
2.12 Calcein AM and PI stainin    96
2.13 In vitro detection of reactive oxygen species (ROS)    97
2.14 In vitro apoptosis detection using Annexin V    97
2.15 In vitro detection of singlet oxygen using Singlet oxygen sensor green (SOSG)    98
2.16 In vitro assessment of mitochondrial membrane potential using JC-1    ………………………………………………………………………………99
2.17 Cell cycle analysis…    99
2.18 In vivo fluorescence imaging of siRNA-lipid-coated Au NEs)    100
2.19 In vivo tumor growth study    101
2.20 HE and caspase-3 staining    102
3 Results and Discussion    102
3.1 In vitro combination of Au NEs-mediated photodynamic therapy and SOD1 gene silencing    102
3.2 In vivo Au NEs-mediated photodynamic therapy and gene silencing    114
4 Conclusions    126
5 References    127

Chapter 4: Multi-Branched Gold Nanoechinus Based Triple Modal Bioimaging: An Efficient NIR-to-NIR Up- and Down-conversion Emission and Photoacoustic Imaging 
1 Background and Introduction………………………………………………133
1.1 Biomedical Imaging    133
1.2 Plasmonic nanoparticles for in vitro in vivo imaging    136
1.3 Upconversion Luminescence Imaging    138
1.4 NIR-to-NIR fluorescence Imaging    140
1.5 Photoacoustic Imaging    141
2 Experimental Section    142
2.1 Synthesis of double chain cetyltrimethylammonium bromide (DC14TAB) surfactant:    142
2.2 Synthesis of ~18 nm Au seeds    143
2.3 Synthesis of Au Nanoechinus    143
2.4 Preparation of folate conjugated Au NEs:    144
2.5 Cell culture, materials and reagents    144
2.6 Cytotoxicity assays    145
2.7 Cellular uptake studies using Multiphoton confocal laser scanning microscopy (CLSM).    145
2.8 Upconversion luminescence imaging     146
2.9 NIR fluorescence spectra and in vivo imaging    146
2.10 Photoacoustic Imaging using 1064 nm pulse laser    147

3 Results and Discussion    147
3.1 Characterization of Au NEs    147
3.2 Upconversion imaging by Au nanoechinus    150
3.3 NIR luminescence of Au nanoechinus    158
2.4 Photoacoustic imaging studies of Au NE    163
4 Conclusions    167
5 References    169

                                

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