半導體的新鍊金術:銅硒奈米粒子於癌症協同治療之應用

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研究生：	馬諾肯德 Manoj Kandel
論文名稱：	半導體的新鍊金術:銅硒奈米粒子於癌症協同治療之應用 Harnessing Semiconductor Alchemy: Copper Selenide Nanoparticles for Synergistic Cancer Therapy
指導教授：	邱信程 Chiu, Hsin-Cheng
口試委員:	黃郁棻 Huang, Yu-Fen 謝明發 Hsieh, Ming-Fa 莊爾元 Chuang, Er-Yuan
學位類別：	碩士 Master
系所名稱：	原子科學院 - 生醫工程與環境科學系 Department of Biomedical Engineering and Environmental Sciences
論文出版年：	2024
畢業學年度：	112
語文別：	英文
論文頁數：	64
中文關鍵詞：	大腸癌、硫化氫、化學動力療法、光熱治療、癌症治療
外文關鍵詞：	Colorectal Cancer, Hydrogen Sulfide, Chemodynamic Therapy, Photothermal Therapy, Cancer Therapy
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大腸直腸癌 (CRC) 是一種嚴重影響全球健康的惡性腫瘤，在所有癌症中其死亡率高居第二，由於不良預後和高復發率，大部分傳統治療方案的效果不甚理想。在過去十年中，出現大量針對大腸直腸癌治療和控制復發的研究，但大多數研究結果只在少數病例中達到治療標準，且伴隨非常低的預期生存率。奈米粒子為對抗大腸直腸癌提供了一條有前景的途徑，能夠精確的靶向癌細胞進行藥物釋放以及重塑腫瘤微環境，從而提高治療效果，同時最大限度地減少副作用。目前已經廣泛嘗試各種奈米顆粒用於藥物運輸和實現癌症治療，包括固體脂質奈米粒子、攜帶藥物的奈米載體、細胞膜包覆的奈米粒子、有機以及無機奈米粒子等，這些載體克服傳統藥物低腫瘤靶向性和療效不佳的問題。在各式載體中，又屬能夠活化、催化腫瘤微環境改變的無機奈米粒子有做為臨床治療方案的潛力。腫瘤組織在體內會建構出與一般組織不同的環境特性，以促進癌細胞增殖、遷移和侵襲，因此重塑腫瘤微環境可能是阻止腫瘤生長的有效方法之一。抑制癌細胞增殖和釋放有毒化合物的協同效果可以達到消除腫瘤的目的。
在這項研究中，我們設計出在體內改變能帶間隙同時響應NIR-II的硒化銅奈米粒子用於癌症治療。透過一步水熱法合成銅位點缺陷的硒化銅奈米粒子（Cu2-xSe NPs），並以牛血清蛋白（BSA）進行表面修飾以提高材料的穩定性。作為首次假設奈米材料能帶間隙在體內產生變化的研究，Cu2-xSe NPs 能消耗結腸癌細胞中過度表現的 H2S，並反應形成 CuSSe NPs。此時奈米粒子的能帶間隙改變，從而增強光熱性能。含銅的奈米粒子有利於透過不同方式重塑腫瘤微環境。二價銅離子用於消耗穀胱甘肽 (GSH) 並產生羥自由基 (·OH)，透過凋亡途徑促進癌細胞死亡。本文製備的 Cu2-xSe NPs 尺寸為 56 nm，具有優異的光熱性能，可作為 H2S 消耗劑並催化 GSH 氧化，並在後續實驗中使用結腸癌細胞（CT 26）分析奈米材料在體外和皮下腫瘤模型中癌症催化治療的有效性，實現協同光熱治療以及內源性能帶間隙改變的癌症治療方案。

Colorectal cancer (CRC), a widespread malignancy affecting a significant global population presents treatment challenges with conventional approaches. Extensive research has been carried out in the past decade for colon cancer treatment and control recurrence with most of the results being up to par in few cases with very low survival predictability. Nanoparticles offer a promising avenue for CRC therapy, enabling precise cancer cell targeting drug release and remodeling cancer microenvironment, thus improving treatment effectiveness while minimizing side effects. The therapeutic modalities that have been under extensive research include solid lipid nanoparticles, nanoparticles loaded with drugs, cellular membrane coated micro/nanoparticles, and inorganic nanoparticles which overcome the poor tumor targeting and efficacy of conventional drugs. Among them inorganic nanoparticles with tumor microenvironment activable catalytic property possesses potential to be translate to clinical practice. Cancer microenvironment with its unique feature facilitates cell proliferation, migration and invasion. Therefore, remodeling cancer microenvironment can be an effective way to stop cancer growth. Controlling cell proliferation combined with toxic compounds can work together to eliminate tumors.
In this work we report in vivo band gap changing NIR-II responsive copper selenide nanoparticles for synergistic cancer therapy. Copper deficient copper selenide nanoparticles (Cu2-xSe NPs) were synthesized by one step hydrothermal process and surface modified with bovine serum albumin (BSA) for colloidal stability. Cu2-xSe NPs reacts with and deplete overexpressed H2S in colon cancer cells forming CuSSe NPs. While doing so band gap of nanoparticles is changed and subsequently the photothermal property is enhanced. Potential of endogenous change in the band gap of nanomaterials is postulated for the first time in this work. Copper containing nanoparticles is advantageous in remodeling cancer microenvironment in different ways. Bivalent copper is used to deplete the level of glutathione (GSH) and to produce hydroxyl radical (•OH) that promotes cell death via apoptosis. Cu2-xSe NPs prepared herein is 56 nm in size and possess excellent photothermal property, acts as a H2S depleting agent and catalyze GSH oxidation. It’s effectiveness in cancer catalytic therapy were analyzed using CT26 colon cancer cells in vitro and subcutaneous tumor model. Our study presents endogenously band gap changing nanoparticles for synergistic cancer therapy.

Abstract
Table of contents List of figures
Table of contents
Chapter 1 Introduction .................................................................................................................. 1
Chapter 2 Literature Review and Theory...................................................................................... 5
1 Introduction to colorectal cancer ........................................................................................ 5
2 Nanoparticles in colorectal cancer ...................................................................................... 7
3 H2S in colon cancer ........................................................................................................... 10
4 Copper in cancer ................................................................................................................ 13
5 Photothermal Therapy ....................................................................................................... 16
6 Chemodynamic therapy ..................................................................................................... 18
7 Bandgap changing semiconductor nanomaterials. ......................................................... 21
Chapter 3 Research motivation ................................................................................................... 22
Chapter 4 Materials and Methods ............................................................................................... 23
1 Materials............................................................................................................................. 23
2 Methods .............................................................................................................................. 23
2.1 Synthesis of Cu2-xSe@BSA nanoparticles .................................................................. 23 4.2.2 Characterization of Cu2-xSe@BSA nanoparticles...................................................... 23 4.2.3 Photothermal assay ..................................................................................................... 24
2.4 GSH depletion assay ................................................................................................... 24
2.5 H2S depletion assay ..................................................................................................... 24
2.6 ROS concentration detection ...................................................................................... 24
2.7 Cell Uptake Study............... 25
2.8 Cell viability assay....................................................................................................... 25
2.9 Cell migration assay.................................................................................................... 25
2.10 Intracellular ROS concentration detection.............................................................. 25
2.11 In-vitro H2S level detection ....................................................................................... 26
2.12 Intracellular GSH consumption............................................................................... 26
2.13 Cleaved caspase 3...................................................................................................... 26
2.14 In vivo antitumor analysis and biosafety ................................................................. 26
Chapter 5 Results and Discussion ............................................................................................... 28
1 Characterization of Cu2-xSe@BSA nanoparticles............................................................. 28
1.1 DLS size, zeta potential and TEM images of the nanoparticles................................ 28
1.2 HR-TEM and XRD analysis ....................................................................................... 29
1.3 XPS analysis................................................................................................................ 30
1.4 Band Gap study ........................................................................................................... 32
2 Properties of Cu2-xSe@BSA nanoparticles........................................................................ 33
2.1 UV-Vis absorbance...................................................................................................... 33
2.2 Photothermal properties ............................................................................................. 34
2.3 EPR measurement....................................................................................................... 36
2.4 GSH Depletion ............................................................................................................ 37
2.5 H2S depletion assay ..................................................................................................... 38
3 In vitro experiments ........................................................................................................... 40
3.1 Cell uptake study ......................................................................................................... 40
3.2 Cell viability assay....................................................................................................... 41
3.3 In-vitro H2S depletion ................................................................................................. 42
3.4 Cell migration assay.................................................................................................... 43
3.4 In-vitro ROS generation ............................................................................................. 44
3.6 Cleaved caspase 3........................................................................................................ 45
3.7 In-vitro thiol degradation (GSH depletion)................................................................ 46
4 In-vivo experiments............................................................................................................ 47
4.1 In vivo therapeutic efficacy of Cu2-XSe@BSA NPs.................................................... 47
4.2 In vivo enhanced photothermal property ................................................................... 49 5.4.3 In vivo H2S depletion .................................................................................................. 50
4.4 CD 31 proliferation marker ........................................................................................ 51
4.5 In-vivo immunostaining.............................................................................................. 52
4.6 Blood biochemistry evaluation ................................................................................... 53
4.7 In vivo biodistribution................................................................................................. 54
4.8 Hematoxylin and eosin (H&E) staining ............... 55
Chapter 6 Conclusions................................................................................................................. 57
References .................................................................................................................................... 58
                                

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