臺灣博碩士論文加值系統

基於小尺寸效應，奈米材料展現特殊之化學及物理特性，於生醫領域已發展如藥物載體、生醫影像及生物感測器。然而，奈米材料對於生物體之損傷、毒性及其複雜作用關係，使奈米材料之安全性成為重要研究議題。因此，本論文主題將專注於開發多功能高治療效果之奈米藥物及探討奈米材料之毒性影響。奈米載體將藉由上轉換奈米粒子與金奈米材料所組成，上轉換奈米粒子具將高能量之光能轉低能量及多重放光之特性，可做為光轉換器及螢光顯影，而金奈米材料具強烈之表面電漿共振效應，可用以吸收上轉換之光能產生熱，抑或增強上轉換之放光或增強光敏劑吸收光之效能。此產生之熱能可用以光熱治療，熱量及分布將藉由光熱轉換效應探討並搭配模擬分析球狀及棒狀之金奈米材料差異，其光熱治療效果將照射980 nm雷射藉細胞毒性方法評估。於上轉換奈米複合體結構添加光敏劑－亞甲藍，其可產生活性氧物質用於光動力治療，並藉控制不同厚度之二氧化矽殼層來調控藥物包埋量，而金奈米棒可調整其表面電漿共振峰與亞甲藍疊合，將有效提升亞甲藍對於激發光之吸收截面，且金奈米棒具表面電漿效應，可提升活性氧物質之產量，增進其光動力治療效果。本論文亦將探討銅銦化硫量子點之低毒性成因，因尚未具明確研究解析其低毒性原因，於此將藉秀麗隱桿線蟲作為模式生物與銅銦化硫量子點培養不同時間，觀察其毒性變化，並藉由Ｘ光吸收近邊緣結構解析其銅銦化硫量子點於不同培養時間之氧化價數變化，進而分析其化學結構之穩定性。此外並對不同形狀及大小之二氧化鈦奈米材料對於人類肺及口腔細胞進行毒性評估，並藉細胞生長及細胞凋亡分析其二氧化鈦濃度梯度效應。故本研究乃致力於開發多功能奈米複合體應用於光熱及光動力治療，並探討銅銦化硫量子點及二氧化鈦奈米材料之毒性影響。

Nanomaterials (NMs) reveal unique chemical and physical properties based on small-sized effects, allowing their application as drug carriers, biosensors, and in bio-imaging. However, the safety of these NMs has also attracted attention because the complex interaction between NMs and organism can cause damage or cytotoxicity. This thesis focuses on developing a new type of highly efficient nanomedicine and investigating the cytotoxicity and safety of NMs. A multifunctional NM that consists of upconversion nanoparticles (UCPs) and Au NMs was fabricated for therapy and imaging. UCPs can convert light from high energy to low energy and serve as light source in multi-emission. Moreover, Au NMs generate heat when absorbing the light from UCPs through strong surface plasmon resonance (SPR). The difference in heat quality and distribution between sphere- or rod-shaped Au NMs was studied by photothermal effect and stimulation model. The efficiency of photothermal therapy (PTT) was tested through cell viability assay by irradiating with a 980 nm laser. Photodynamic therapy (PDT) was carried out by doping photosensitizer-methylene blue (MB) in the similar upconverting nanocomposites. MB was used to produce reactive oxygen species (ROS) in PDT to optimize the loading amount by changing the thickness of silica shell. In particular, the amount of ROS was further enhanced by conjugating with Au nanorods, which expectedly increased the absorption cross section of MB. The efficacy and mechanism among different SPR peaks were investigated and compared between sphere- and rod-shaped Au NMs. The low cytotoxicity of novel CuInS2 quantum dots (CIS QDs) was investigated because of the absence of contention. Caenorhabditis elegans was used as organism model with CIS QDs for toxicity study, and X-ray absorption near edge structure was employed to study the relationship between toxicity and chemical stability of CIS QDs under various treatment times. Moreover, TiO2 NMs with various sizes and structures were used to treat different human oral and lung cells to investigate the toxic effects. To determine the cellular response of cells to TiO2 NM treatment, we performed apoptosis assay and cell cycle analysis to identify the mechanism of cytotoxicity. Consequently, we successfully developed multifunctional NMs based on UCPs and Au NMs for PTT or PDT and bio-imaging. We also determined the factors that affected the low cytotoxicity for CIS QDs and the causes of damage from TiO2 NMs.

口試委員審定書 I
謝誌 II
摘要 III
Abstract IV
Content V
Figure contents XI
Table contents XXI
Abbreviation table XXII
Chapter 1. Introduction 1
1.1 Fundamental Knowledge of Nanomaterials 1
1.1.1 Surface, small scale, and quantum confinement effect 2
1.1.2 “Top-down” and “Bottom-up” approaches 7
1.2 Nanomaterials 8
1.2.1 Lanthanide-doped upconversion nanoparticles 8
1.2.2 Au nanomaterials 17
1.2.3 CuInS2 quantum dots 22
1.2.4 Titanium dioxide nanoparticles 24
1.3 Nanomaterials in Biological Research 26
1.3.1 Photothermal and Photodynamic therapy 28
1.3.3 Cytotoxicity effect 31
1.4 Research Motivations 33
1.5 References (chapter 1) 38
Chapter 2. Experimental Approaches and Techniques 46
2.1 Chemicals and Materials 46
2.2 Preparation of Nanomaterials 48
2.2.1 Fabrication of UCP@SiO2-NPs- FA and UCP@SiO2-NRs-FA 48
2.2.2 Fabrication of UCP@SiO2:MB-NRs-FA 51
2.2.3 Synthesis of CIS and CIS/ZnS QDs 52
2.2.4 Extraction of TiO2 NPs from sun-screening cosmetics 53
2.3 Instruments for Characterization 54
2.3.1 Transmission electron microscopy 54
2.3.2 X-ray diffractometer 57
2.3.3 UV/Vis spectrometer 60
2.3.4 Fourier transform infrared spectrometer 63
2.3.5 Photoluminescence spectrometer 67
2.3.6 Inductively coupled plasma mass spectrometry 70
2.3.7 X-ray absorption near edge structure 73
2.4 Instruments for Biological Analysis 75
2.4.1 Multimode microplate readers 75
2.4.2 Confocal microscopy 77
2.4.3 Flow cytometry 79
2.5 References (chapter 2) 81
Chapter 3. Plasmon-induced Hyperthermia: Hybrid Upconversion NaYF4:Yb/Er and Gold Nanomarterials for Oral Cancer Photothermal Therapy 83
3.1 Introduction 83
3.2 Experimental Section 85
3.2.1 Photothermal ability of UCP@SiO2-NP or UCP@SiO2-NR in solution 85
3.2.2 Simulative analysis 86
3.2.3 Cell viability assay and cellular uptake 86
3.2.4 PTT test 87
3.3 Results and Discussion 87
3.3.1 Characterization of UCP@SiO2-NPs-FA and UCP@SiO2-NRs-FA 87
3.3.2 Simulative model for the evaluation of the heating variation 99
3.3.3 UCP@SiO2-NPs-FA and UCP@SiO2-NRs-FA for PTT in vitro 103
3.4 Conclusions 106
3.5 References (chapter 3) 107
Chapter 4. Localized Plasmon-enhanced Reactive Oxygen Species Generation: Near infrared-triggered Upconversing NaYF4:Yb/Er Hybrid Au Nano-rods for Photodynamic Oral Cancer Therapy 111
4.1 Introduction 111
4.2 Experimental Section 114
4.2.1 Detection of ROS in aqueous solution 114
4.2.2 Detection of intracellular ROS 115
4.2.3 Cell viability assay for PDT 115
4.2.4 In vivo PDT treatment 116
4.2.5 Detection of mitochondria membrane potential 116
4.2.6 Caspase 3 activity assay 116
4.3 Results and Discussion 118
4.3.1 Characterization of UCP@SiO2:MB-NRs 118
4.3.2 Evaluation of ROS production by UCP@SiO2:MB-NRs 126
4.3.3 PDT efficacy of UCP@SiO2:MB-NRs-FA in vitro and in vivo 131
4.4 Conclusions 138
4.5 References (chapter 4) 139
Chapter 5. Evaluations of Chemical Stability and Cytotoxicity of CuInS2 and CuInS2/ZnS Core/Shell Quantum Dots 143
5.1 Introduction 143
5.2 Experimental Section 146
5.2.1 Cell viability test 146
5.2.2 In vitro confocal images 146
5.2.3 C. elegans maintenance 146
5.2.4 Obtaining synchronous cultures of C. elegans 147
5.2.5 In C. elegans confocal images and XANES analysis 147
5.3 Results and Discussion 148
5.3.1 Fabrication and characterization of CIS and CIS/ZnS QDs 148
5.3.2 Coating OCMCS at surface of CIS and CIS/ZnS QDs for biological studies 152
5.3.3 Cell viability and confocal images of QDs materials 154
5.3.4 Confocal images and XANES analysis of QDs materials in C. elegans 156
5.4 Conclusions 160
5.5 References (chapter 5) 161
Chapter 6. Evaluation of the Intracellular Uptake and Cytotoxicity Effect of TiO2 Nanostructures for Various Human Oral and Lung Cells under Dark Conditions 165
6-1. Introduction 165
6.2 Experimental Section 167
6.2.1 Cell culture 167
6.2.2 Intracellular uptake analysis 167
6.2.3 Cell viability analysis 168
6.2.4 Flow cytometric analysis of cell cycle 168
6.2.5 Flow cytometric analysis of apoptosis 169
6.2.6 Caspase-3/7 activity assay 169
6.2.7 Western blot analysis 169
6.3 Results and Discussion 170
6.3.1 Characterization of TiO2 nanomaterials 170
6.3.2 Investigation of intracellular uptake and cell viability for TiO2 nanomaterials 172
6.3.3 Investigation of cytotoxicity effect for TiO2 nanomaterials 175
6.4 Conclusions 180
6.5 References (chapter 6) 181
Chapter 7. Concluding Remarks 186
7.1 Conclusions 186
Publication in International Scientific Journals 188
Publication of Patents 189
Taiwan Patent 189
Publication in Conferences 190
Honor 190

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2.5 References (chapter 2)
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3.5 References (chapter 3)
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