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研究生: Girum Getachew
Girum Getachew
論文名稱: 親水性多功能奈米平台之開發及鑑定與其生醫應用
Synthesis, Characterizations, and Evaluations of Aqueous-based Multifunctional Nanoplatform for Biomedical Applications
指導教授: 張家耀
Jia-Yaw Chang
口試委員: 蔡伸隆
Shen-Long Tsai
李振綱
Cheng-Kang Lee
Huang, Chih-Ching
Huang, Chih-Ching
Chang, Jungshan
Chang, Jungshan
學位類別: 博士
Doctor
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2022
畢業學年度: 110
語文別: 英文
論文頁數: 238
中文關鍵詞: 量子點光學性能活性氧光熱療法共聚焦成像
外文關鍵詞: Quantum dots, Optical performance, Reactive oxygen species, Photothermal therapy, Confocal imaging
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在第一篇研究中,我們提出了一種簡單合成高效 (NH4)xCs1-xPbBr3量子點的方法,藉由調控氨的比例,合成出極佳量子產率的 (NH4)xCs1-xPbBr3 量子點。此外,使用以一步合成法將量子點與具光熱性質的染料(IR780)結合,據我們所知,此研究是第一篇嘗試將鈣鈦礦量子點與光治療分子整合的研究(簡寫為 (PQD-IR780),此複合物展現了良好的水溶性及高達57.85% 的光熱轉換效率。而體外細胞也證實細胞攝取PQD-IR780後,在HeLa, B16F1, HepG2細胞內展現出極高的螢光亮度。結果指出HeLa細胞攝取PQD-IR780後,其機制需依賴能量,且為小窩蛋白內噬作用。體外細胞存活率檢測和光熱療法可以顯現PQD-IR780具優異生物相容性,且可以在雷射照射下致使高溫,達到殺死癌細胞的效果。
第二部分,透過一步微波的方式摻雜鎂(Mg)離子,製備出發紅光的CsMgxPb1-xI3量子點。與未摻雜的CsPbI3相比,CsMgxPb1-xI3量子點具有優秀的光穩定性及更高的量子產率高達89%。接著,通過疏水端相互作用將疏水性CsMgxPb1-xI3 QDs封裝至釓共軛pluronic 127 (PF127-Gd) 微胞中 (PQD@Gd) ,提高了其生物利用度。PQD的光學性質及Gd存在可賦予PQD@Gd螢光和MRI成像以及光療特性。因此,通過採用T1和T2證明了PQD@Gd的MRI對比效果,這證實PQD@Gd奈米粒子具有優異的MRI對比效果(r2/r1比值為1.38) 。PQD@Gd奈米粒子表現出優異的生物相容性,在671 nm雷射照射下也能有效地在癌細胞中產生具有細胞毒性的活性氧,誘導細胞死亡。此外,PQD@Gd奈米試劑在可見光激發下對有機污染物也表現出優異的光催化活性。多功能奈米複合物PQD@Gd不僅具有雙重成像、光動力治療及優異的光催化特性,也可用於有機污染物或細菌去除,因此有機會成為癌症治療的選擇之一。
最後一部分,利用異原子(Cu, Cl離子)摻雜並結合碳點(CDs)的方式來調節活性氧(ROS)清除或生成的能力。起初CDs就具有良好的抗氧化及清除能力,而Cu和Cl共摻雜的CDs (CuCl-CDs) 不僅具有光動力療法 (PDT) 能力,且具有將過氧化氫(H2O2) 轉化為氫氧自由基 (•OH) 的仿生過氧化物酶活性。此外,具有生成ROS能力和仿生過氧化物酶特性的CuCl-CDs成功地與聚多巴胺 (PDA) 和葡萄糖氧化酶 (GOx)結合,以製備多功能GOx/CuCl-CD@PDA-PEG (GCP) 奈米複合材料。此新型GCP奈米複合物具有令人滿意的光熱轉換效率 (η=24.4%) ,並透過加入H2O2和雷射照射的方法產生大量的ROS。最後,GCP奈米複合物中的GOx可進行飢餓療法及催化連鎖反應,減少了細胞內的葡萄糖含量,並間接增強ROS生成能力。細胞實驗證實,GCP奈米複合材料具有良好的生物相容性,經由化療、光熱治療及飢餓療法,導致細胞死亡。


In the thesis work, we report a facile strategy for the highly efficient (NH4)xCs1-xPbBr3 QDs. By modulating the amount of ammonium, (NH4)xCs1-xPbBr3 QDs with higher quantum efficiencies were synthesized. Furthermore, (NH4)xCs1-xPbBr3 QDs was conjugated with a photothermal dye (IR780) via a one-pot reaction using poly(styrene-co-maleic anhydride) and IR780-MPTS. To the best of our knowledge, the present work is the first attempt integrating perovskite QDs and phototherapeutic molecules into one system (abbreviated as PQD-IR780), demonstrating good water dispersibility and high photothermal conversion efficiency of 57.85%. In vitro experiments performed to examine subcellular uptake showed high fluorescence brightness was observed in HeLa, B16F1, and HepG2 cancer cells cultured with PQD-IR780. The in vitro cell viability assays and photothermal therapy revealed that PQD-IR780 showed good biocompatibility and can induce hyperthermia upon laser irradiation.
High optical performance, red-emitting CsMgxPb1-xI3 QDs were prepared by doping with magnesium (Mg) ions into CsPbI3 QDs via a one-pot microwave pyrolysis technique. Next, the bioavailability of as-synthesized hydrophobic CsMgxPb1-xI3 QDs is improved by encapsulating them into Gadolinium conjugated pluronic 127 (PF127-Gd) micelles through hydrophobic interactions (PQD@Gd). The optical properties of PQD and the presence of Gd could endow the PQD@Gd with fluorescence and MRI imaging, and phototherapeutic properties. The MRI contrasting effects of PQD@Gd nanoagents are demonstrated by employing T1 and T2 studies which validated that PQD@Gd nanoagent had a superior MR contrasting effect. The PQD@Gd nanoagents have exhibited excellent biocompatibility, and efficiently produced cytotoxic reactive oxygen species (ROS) in the cancer cells under 671 nm laser illumination, and thereby induced cell death. The multifunctional PQD@Gd nanoagents developed in this study could be the potential choice of components for cancer therapy due to dual-modal imaging and photodynamic therapeutic properties.
The ROS scavenging or generation ability of the carbon dots (CDs) was regulated by incorporating with heteroatoms (Cu and Cl ions). The pristine CDs were found to be powerful anti-oxidants to scavenge ROS, whereas Cu and Cl co-doped CDs (CuCl-CDs) possessed not only ROS generation ability upon laser irradiation for photodynamic therapy (PDT), but also peroxidase-mimic activity that generates oxidative •OH from hydrogen peroxide (H2O2) for chemodynamic therapy (CDT). Furthermore, CuCl-CDs were successfully integrated with polydopamine (PDA) and glucose oxidase (GOx) to fabricate multifunctional GOx/CuCl-CD@PDA-PEG (GCP) nanocomposites. These novel GCP nanocomposites possessed satisfactory photothermal conversion efficacies (η = 24.4%) and gave a high yield of ROS via the combination of H2O2 and laser irradiation. Moreover, the presence of GOx in GCP nanocomposites enables these compounds to decrease the intracellular glucose levels for starvation therapy and the enzymatic cascade activity for enhanced ROS-mediated therapy. In vitro studies and confirmed that these GCP nanocomposites displayed good biocompatibility, and induced cells death via the cooperative effects of CDT, phototherapeutic effect, and starvation therapy.

中文摘要……...…………………………………………….……………………………………..I ABSTRACT IIIII ACKNOWLEDGEMENT IV TABLES OF CONTENTS……………………………………………………………………...VII LIST OF FIGURES XI LIST OF SCHEMES XXIIX LIST OF ABBREVIATIONS XXII CHAPTER ONE….……………………………………………………………………………….1 INTRODUCTION ………………………………………………………………………………..1 1.1 Background…………………………………………………………………………...…...2 1.2 Objectives of the study…………………………………………………………………….6 1.2 Compositions of the dissertation…………………………………………………………..7 CHAPTER TWO……………………………………………….....................................................9 LITERATURE REVIEW…………………..…………………………………………..................9 2.1 Nanoparticles…………………………………………………………………………….10 2.2 Carbon dots………………………………….…………………………………………...11 2.2.1 Synthetic approaches for carbon dots…………………….………………………..14 2.3 Perovskite quantum dots……...……….…………………………………………………21 2.3.1 Synthetic strategies for the perovskite quantum dots…………………………...…28 2.3.2 Surface stabilizations techniques for perovskite quantum dots……..……………..36 2.3 Biomedical applications of quantum dots………………………………………………..41 2.3.1 ROS-mediated therapy…………………………………………………………..…41 2.3.2 Photothermal therapy………………………………………………………………43 2.3.4 Bioimaging tools…………………………………………………………………...47 Chapter THREE.………………………………………………………….……………………...54 Brightly luminescent (NH4)xCs1-xPbBr3 quantum dots for in vitro imaging and efficient photothermal ablation therapy…………………………………………………………………...54 3.1 Introduction…...…………………………………………………………………………55 3.2 Experimental methods 57 3.2.1 Chemicals and reagents 5957 3.2.2 The preparation of (NH4)xCs1-xPbBr3 and CsPbBr3 QDs 58 3.2.3 The preparation of alloyed (NH4)xCs1-xPbX3 (X= Cl, Br, I or mixture) QDs……………………………………………………………58 3.2.4 Preparation of IR780-MPTS…….………………………………………………..59 3.2.5 Preparation of PQD@PSMA QDs……………………………………………..…59 3.2.6 Synthesis of PQD-IR780 nanocomposite……………………….………………..59 3.2.7 Evaluation of photothermal efficiency….………………………………………...59 3.2.8 Cell culture and biocompatibility test of PQD-IR780 nanocomposite……….…..60 3.2.9 In vitro photothermal test……………………………………………...………….61 3.2.10 Cellular uptake of PQD-IR780 nanocomposites……………………...………….61 3.2.11 Endocytosis pathway studies…………………………………………………….62 3.2.12 Characterization techniques………………………….…………………………..62 3.3 Results and discussion …………………………………………………………………..64 3.3.1 Synthesis and characterization of (NH4)xCs1-xPbBr3 QDs………………………..64 3.3.2 Synthesis of water-soluble phototherapeutic nanocomposites……………………74 3.3.3 In vitro cytotoxicity and photothermal toxicity…………………………………..78 3.3.4 Cell uptake and endocytosis pathways of PQD-IR780 nanocomposites…………81 3.4 Summary ...83 CHAPTER FOUR..........…………………………………………………………………………85 Highly Luminescent, Stable, and Red-Emitting CsMgxPb1–xI3 Quantum Dots for Dual-Modal Imaging-Guided Photodynamic Therapy and Photocatalytic Activity…………………………..85 4.1 Introduction 86 4.2 Experimental section…………………………………………………………………….90 4.2.1 Chemicals and reagents…………………………………………………………...90 4.2.2 Synthesis of CsPbI3 and CsMgxPb1-xI3 QDs……………………………………...90 4.2.3 Synthesis of PF127-Gd carriers…………………………………………………..91 4.2.4 Encapsulation of CsMgxPb1-xI3 into PF127-Gd carrier.…………………………..91 4.2.5 Examination of reactive oxygen species (ROS) generation……………………...92 4.2.6 Characterization of generated ROS………………………………………………92 4.2.7 Photocatalytic activity of PQD@Gd nanoagent………………………………….93 4.2.8 Detections of active species ……………………………………………………...94 4.2.10 Evaluation of PDT mediated cell toxicity……………………………………….94 4.2.11 Analysis of in vitro MR imaging………………………………………………...95 4.2.12 Examination of Intracellular imaging……………………………………………96 4.2.13 Endocytosis trafficking assay……………………………………………………96 4.2.14 Intracellular ROS ………………………………………………………………..96 4.2.15 Characterization techniques……………………………………………………...97 4.3 Results and discussion…………………………………………………………………..98 4.3.1 Preparation and characterization of CsPbI3 and CsMgxPb1-xI3 QDs……………...98 4.3.2 Photophysical properties of the CsPbI3 and CsMgxPb1-xI3 QDs….……………..105 4.3.3 Synthesis and characterization of PQD@Gd nanoagent………………………...111 4.3.4 Photodynamic therapy of PQD@Gd nanoagent………………….…………..…116 4.3.5 Photocatalytic evaluation of PQD@Gd nanoagent……………………………...119 4.3.6 MRI measurement of PQD@Gd nanoagent…………………………………….124 4.3.7 In vitro demonstration of PQD@Gd nanoagent ………………………………..127 4.3.8 In vitro fluorescence imaging …………………………………………………..129 4.3.9 Intracellular ROS detection ……………………………………………………..132 4.4 Summary ………………………………………………………………………………134 CHAPTER FIVE…………………………………………………………………………….…135 ROS generation/scavenging modulation of carbon dots as phototherapeutic candidates and peroxidase mimetics to integrate with polydopamine nanoparticles/GOx towards cooperative cancer therapy……………………………………………………………………………...…...135 5.1 Introduction 139 5.2 Experimental section…………………………………………………………………...143 5.2.1 Chemicals and reagents………………………..………………………………...140 5.2.2 Synthesis of CDs and CuCl-CDs………………………………………………..141 5.2.3 Preparation of PDA and PDA-PEG nanoparticles………………………………141 5.2.4 Synthesis of GCP nanocomposites……………………………………………...142 5.2.5 ROS scavenging assay of CDs and CuCl-CDs……………………………….…142 5.2.6 Photodynamic activities of the GCP nanocomposites…………………………..142 5.2.7 Characterization of ROS.………………………………………………………..143 5.2.8 Photothermal measurement of GCP nanocomposites…………………………...144 5.2.9 GOx-like enzymatic activity of GCP nanocomposites………………………….144 5.2.10 Peroxidase-like activity of GCP nanocomposites………………………………145 5.2.11 Biocompatibility of GCP nanocomposite in vitro studies...……………………145 5.2.12 In vitro cell viability through PDT and PTT effect……………..………………146 5.2.13 In vitro cell viability through starvation effect…………………………………147 5.2.14 Cellular imaging experiment……………………………………………………147 5.2.15 Intercellular assessment of ROS………………………………………………..148 5.2.16 Characterization techniques…………………………………………………….148 5.3 Results and discussion…………………………………………………………………152 5.3.1 Synthesis and characterization of CDs and CuCl-CDs………………………….152 5.3.2 ROS scavenging/generation activities of CDs and CuCl-CDs………………….160 5.3.3 Synthesis and characterization of GCP nanocomposites………………………..164 5.3.4 Photothermal properties of GCP nanocomposites………………………………168 5.3.5 Enzymatic cascade activity of GCP nanocomposites…………………………...169 5.3.6 In vitro evaluation of GCP nanocomposites…………………………………….174 5.4 Summary ……………………………………………………………………………....175 CHAPTER SIX…………………………………………………………………………………176 Conclusion and future perspectives.……………………………………………………………176 6.1 Conclusion 181 6.2 Future perspectives…………………………………………………………………….183 REFERENCE…………………………………………………………………………………...181 APPENDIX.……………………………………….……………………………………………201 PUBLICATIONS……………………………………………………………………………….219

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