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研究生： 范彥剛
Khang, Pham Yen
論文名稱： 近紅外光IIb激發上轉換奈米粒子作為腫瘤之光動力治療
NIR-IIb light-triggered upconversion nanoparticles for tumors photodynamic therapy
指導教授： 葉晨聖
Yeh, Chen-Sheng
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： 2021
畢業學年度： 109
語文別： 英文
論文頁數： 94
外文關鍵詞： Photodynamic therapy, upconversion nanoparticles, singlet oxygen, photosensitizer, rose bengal, chlorin e6, biological window, near-infrared IIb, 1550 nm laser, deep tissue penetration, pancreatic tumors
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    • Recently, the fluorescence imaging in the second near-infrared biological window b (NIR-IIb, 1500–1700 nm) has emerged as a promising strategy owing to high resolution and deeper tissue penetration. However, in marked contrast to the rapid development in the bioimaging field, hitherto no concrete study has been directed toward photodynamic therapy (PDT) therapeutic design following NIR-IIb region excitation. Therefore, the present study aims to design a 1550 nm (located in NIR-IIb window) light-responsive upconversion nanoparticles (UCNPs) to establish dual-PDT and applications to treat pancreatic tumors.
    Chapter 1 provides an overview of PDT and its Achille’s heel, NIR-IIb biological window, UCNPs, the dual functions of Er3+ ion, the concept of UCNPs-based PDT, and dual photosensitizer loading strategy. The NIR-IIb window's advantages, including near-zero autofluorescence, low light scattering, and deep tissue penetration, were highlighted. Besides, Er3+ ions can serve dual functions as activator and sensitizer are the great instinct enabling harvest the energy of 1550 nm excitation for effective upconversion process. The enhanced singlet oxygen (1O2) generation in dual-PDT applications also was introduced. The detailed experimental procedures were further shown in Chapter 2.
    Chapter 3 presents the main finding of this study. The core-shell structure of LiYbF4:30%Er@LiGdF4 (shell thickness of 8.4 nm) UCNPs was synthesized, which can be excited by a 1550 nm (NIR-IIb) laser. Then, the dual-photosensitizers (PSs), rose bengal (RB) and chlorin e6 (Ce6), were carried by the silica-coated core-shell LiYbF4:Er@LiGdF4 UCNPs via electrostatic attraction and covalently bonding, respectively, forming LiYbF4:Er@LiGdF4@SiO2/RB,Ce6. The UCNP's emission in both the green (∼548 nm) and red (∼666 nm) colors under 1550 nm laser excitation was fully utilized to simultaneously trigger RB and Ce6, respectively. Notably, the simultaneous activation of dual-PS generated abundant singlet oxygen (1O2) by 1550 nm laser irradiation. The water absorption around 1400–1500 nm, which may cause a heating-up effect, was overcome by performant a laser on-off switching sequence instead of continuous irradiation. By comparing the laser energy attenuation after penetration through various pork tissue thicknesses as well as the attenuation coefficient, we confirmed the deeper tissue penetration of 1550 nm laser over that of 808 nm laser. Subsequently, the in vitro experiments, including MTT assay, live/dead cell staining, and confocal imaging, demonstrated a synergistic effect with higher PDT efficacy from dual-PS than the single-PS-loaded nanocarriers under a single dose treatment. The outcome from in vivo treatment of pancreatic tumors also was consistent with in vitro results, showing an enhanced antitumor effect of dual-PDT relative to single-PDT.

    Abstract I Acknowledgments III Table of Contents IV List of Figures VII List of Tables XIII Chapter 1 Introduction 1 1.1 Photodynamic therapy (PDT) and the Achille’s heel in PDT 1 1.2. NIR-IIb biological window 2 1.2.1 Introduction to NIR-IIb biological window 2 1.2.2 The benefits of NIR-IIb biological window 3 1.2.3 Imaging in NIR-IIb biological window 6 1.3. Upconversion nanoparticles (UCNPs) 9 1.3.1 General concept of UCNPs 9 1.3.2 Characterization of electronic configurations of lanthanide ions 10 1.3.3 Mechanisms of the upconversion process 10 1.3.4 Host matrix, sensitizer, and activator 12 1.4. Erbium ion: the dual functions of sensitizer and activator 14 1.5. Upconversion nanoparticles‐based photodynamic therapy (UCNPs-based PDT) 18 1.5.1 General concept of UCNPs-based PDT 18 1.5.2 Available NIR lasers for UCNPs-based PDT 20 1.6. Photosensitizer 22 1.6.1 Selecting a photosensitizer 22 1.6.2 Organic and inorganic photosensitizer 22 1.6.3 Photosensitizer loading strategy 23 1.7 Dual photosensitizer loading strategy 24 1.8 Motivation and design strategy of nanoparticles and therapy 25 Chapter 2 Methodology 28 2.1. Materials and Instruments 28 2.1.1 Materials 28 2.1.2 Instruments 30 2.2. Experimental processes 32 2.2.1 Preparation of LiYbF4:Er UCNPs 32 2.2.2 Preparation of LiYbF4:30%Er@LiGdF4 core-shell UCNPs 33 2.2.3 Preparation of UCNP@SiO2/RB-NH2 34 2.2.4 Preparation of UCNP@SiO2/RB,Ce6 35 2.2.5 Preparation of PEGylated UCNP/RB,Ce6 36 2.2.6 Photosensitizer-loading capacity 36 2.2.7 Stability performance 37 2.2.8 Collection of upconversion emission 37 2.2.9 Temperature elevation profile upon laser irradiation 37 2.2.10 Evaluation of singlet oxygen generation 37 2.2.11 Cell culture 38 2.2.12 In vitro cytotoxicity evaluation 38 2.2.13 Cellular uptake study 38 2.2.14 Detection of intracellular singlet oxygen generation 39 2.2.15 Comparison of tissue penetration depth of 1550 nm and 808 nm NIR lasers 39 2.2.16 Photodynamic therapy in vitro: MTT assay 40 2.2.17 Photodynamic therapy in vitro: live/dead cell staining 40 2.2.18 Photodynamic therapy in vitro: confocal laser microscope imaging 40 2.2.19 In vivo biosafety evaluation 41 2.2.20 In vivo antitumor efficacy of PDT 41 2.2.21 Hematoxylin and Eosin (H&E) staining 42 2.2.22 Statistical analysis 42 Chapter 3 Results and discussions 43 3.1 Preparation and characterization of core UCNPs 43 3.2 Preparation and characterization of core-shell UCNPs 46 3.3 Dual photosensitizer loading and surface modification 51 3.4 Photosensitizer-loading capacity and stability performance 54 3.5 Energy transfer from UCNPs to the photosensitizer 58 3.6 Overcoming laser-induced heating effect 59 3.7 Evaluation of singlet oxygen generation 61 3.8 In vitro cytotoxicity evaluation and cellular uptake study 63 3.9 Intracellular singlet oxygen generation 65 3.10 Comparison of tissue penetration depth of 1550 nm and 808 nm NIR lasers 67 3.11 In vitro PDT treatment efficacy: MTT assay 68 3.12 In vitro PDT treatment efficacy: live/dead cell assay and laser confocal imaging 70 3.13 In vivo biosafety evaluation 72 3.14 In vivo antitumor efficacy of PDT 73 Chapter 4 Conclusions and further research directions 77 References 78 Abbreviations 93 Appendix 94

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