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研究生：	高鉑淙 Kao, Po-Tsung
論文名稱：	光驅動氣體奈米載體在生物醫學應用 Light-triggered Gas Nanocarriers in Biomedical Applications
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：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	147
中文關鍵詞：	亞硝基谷胱甘肽、硫化銅奈米粒子、金奈米粒子、普魯士藍奈米粒子、高分子囊泡、氣體載體、一氧化氮、二氧化碳、共軛焦顯微鏡影像、光熱效應、血管擴張、藥物治療、腫瘤治療
外文關鍵詞：	GSNO, copper sulfide NPs, Au NPs, prussian blue NPs, polymersome, gas nanocarrier, nitric oxide, carbon dioxide, confocal microscopy image, photothermal effect, vasodilation, chemotherapy, tumor therapy
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近年來，氣體治療已被廣泛應用於生物醫學研究。而傳統的氣體治療方式仍存有無法有效傳送氣體至病理區的問題。因此，創造具有高生物相容性的氣體載體，並提供精確的傳送方式是有其必要性的。我們的研究將著重在製備出多功能氣體載體，並利用材料的特性應用於生物醫學領域上。
第一個研究主題，我們設計一個策略，透過利用照光方式來釋放一氧化氮。此策略利用雙層乳化方式進行高分子(聚(乳酸-共-乙醇酸))奈米囊泡合成，再透過不同步驟分別將水相藥物(GSNO)及油相材料(硫化銅奈米粒子)分別包入高分子奈米囊泡的核與殼層中。而在此研究中，硫化銅奈米粒子提供了光熱效應和與藥物反應進而產生一氧化氮這兩項重要的功能。當材料透過雷射光照射，利用硫化銅奈米粒子的光熱效應使高分子球膜表面破裂，此時核中的藥物在向外流出時會與鑲在殼層的硫化銅奈米粒子進行反應而產生一氧化氮。在細胞與斑馬魚動物實驗上，我們利用一氧化氮染劑進行標定。實驗結果顯示，在細胞與斑馬魚動物實驗中均可觀察到綠色螢光產生。而另一方面，在斑馬魚動物實驗中也可以觀察到明顯的血管擴張現象。
第二個研究主題中，S-亞硝基硫醇類化合物一直以來被認定是良好的一氧化氮前驅物，其化合物具有治療血管收縮與相關疾病的潛力。但它們在體液中的不穩定導致容易降解，大幅度限制了一氧化氮在臨床上的應用。在本篇中，我們延續前研究主題的設計，在高分子囊泡殼核結構中分別將金奈米粒子和水相藥物(GSNO)包覆在殼核中，延長了藥物穩定性，並且可透過照光方式來控制釋放一氧化氮，而我們稱這材料為“goldsome”。在低強度雷射光短暫照射後，高分子囊泡中的 Au NPs 吸光產熱後會破壞 goldsome 表面使其通透性增加，進而使藥物通過膜表面時與金奈米粒子反應形成金硫鍵而使一氧化氮釋放。在動物實驗中，我們利用斑馬魚做為實驗模型，利用光控制釋放一氧化氮來進行血管擴張以緩解缺氧引起的腦血管收縮。
第三個研究主題，腫瘤中的缺氧區一直以來都是藥物治療的一大問題。缺氧誘導因子在腫瘤區會因氧氣減少或缺氧而大量表現。而缺氧誘導因子表現量的上升也會伴隨著血管內皮增生因子過度表現，進而產生新的血管。另一方面，普魯士藍奈米粒子具有良好的生物相容性以及光熱轉換效率，且廣泛被應用在腫瘤治療研究。在此實驗設計中，我們將普魯士藍奈米粒子表面進行碳酸氫根修飾，且透過光熱效應釋放二氧化碳。利用二氧化碳在腫瘤區形成更嚴重的缺氧環境，進而產生新的血管來增強藥物治療的效果。在細胞實驗中，我們成功地在細胞內釋放二氧化碳進而刺激缺氧誘導因子與血管內皮增生因子大量表現。

In recent years, gas therapy have been widely established for the biomedical applications. The traditional gas therapies have some disadvantages including less efficiency, difficulty in delivery and poor targeting. It is necessary to create the gas carrier with higher biocompatibility to deliver the gas with spatio-temporal accuracy. In addition, the delivery of gas from the nanocarriers in response to a trigger is highly anticipated.
In chapter 2, since the discovery of nitric oxide (NO) as a vasodilator, numerous NO therapies have been attempted to remedy disorders related to pathological vasoconstriction such as coronary artery disease. Despite the advances, clinical applications of NO therapies remain limited mainly because of the low stability of molecular NO donors (and NO molecules), and concerns about the increased oxidative stress and reduced arterial pressure associated with the systemic administration of NO. Here we design a photo-responsive polymersome with nitrosothiols and Cu1.6S nanoparticles in its core and shell, respectively, and demonstrate the photo-triggered release of NO and its vasodilatory activity on zebrafish. Unlike conventional approaches, our design enhances the stability of NO donors and prospectively enables spatiotemporal regulation of NO release, thus minimizing the harmful effects associated with conventional NO therapies. We anticipate that such a strategy will open up new clinical applications of NO and help reveal the complex biological effects of NO in vivo.
In chapter 3, we report the development of a ‘goldsomes’, which comprise the degradable poly(lactic-co-glycolic acid) (PLGA) polymersomes with 2.5-nm Au nanoparticles (NPs) encapsulated in the membrane compartment and s-nitrosoglutathione (GSNO) in the interior core, have allowed to prolong the stability of GSNO and enable the spatiotemporal control of NO generation with light. Upon a brief illumination at low intensity of light, the degradation of PLGA, resulted from the photo-thermal heating of Au NPs, caused GSNO to infiltrate through the PLGA membrane and react with Au NPs therein. The small Au NPs of high catalytic capability triggered the release of NO from GSNO with the concomitant formation of the thiolated Au NPs. The photo-triggered generation of NO from goldsomes was demonstrated in vitro and in living zebrafishes as a model, leading to the dilation of the vasculature and the alleviation of the cerebral vasoconstriction induced by hypoxia.
In chapter 4, the hypoxia region in solid tumor site caused an inhibition of chemotherapy because of depleted amount of blood vessels. We demonstrated the preparation of multifunctional Prussian blue nanoparticles as gas nanocarrier for the application in cancer therapy by angiogenesis. Prussian blue is an U.S. FDA approved materials which can cause considerably higher photothermal effect by light irradiation. In this study, iron ions on the nanoparticle surface play an important role to conjugate bicarbonate, the CO2 donor, which can release CO2 due to heating from photothermal effect. The PEGylate modification on nanoparticle surface provided the higher biocompatibility. Following the strategy, the generation of HIF-1a and VEGF in nucleus and medium was successfully demonstrated through a photo-triggered intracellular release of CO2.

摘要 ……….I
Abstract ……….III 
誌謝 ……….V 
Content ……….VII 
Table content ……….XII 
Figure content……….XIII 
Chapter 1. Introduction……….1 
1 Traditional Gas therapy……….1 
2 Function of Nitric oxide……….2 
3 Function of Carbon dioxide ……….12 
4 Nanoparticles as the gas delivery system ……….17 
5 Photo-triggered gas release system ……….22 
6 General introduction to materials/components used in present research 
……….26 
6.1 Copper sulfide NP ……….26 
6.2 Poly(Lactic-co-Glycolic Acid) (PLGA) polymer structure……….29 
6.3 Prussian blue NPs ……….34 
Chapter 2. Controllable NO release from Cu1.6S nanoparticle decomposition of S-nitrosoglutathiones following photothermal disintegration of polymersomes to elicit cerebral vasodilatory activity ……….37 
1 Motivation ……….37 
2 Materials……….38 
3 Experiment methods ……….39 
3.1 Preparation of Cu1.6S NPs ……….39 
3.2 Preparation of Cu1.6S-PLGA polymersomes……….39 
3.3 Preparation of GSNO/Cu1.6S-PLGA polymersomes ……….40 
3.4 Preparation of GSNO-PLGA polymersomes……….41 
3.5 Temperature elevation profile upon laser irradiation ……….42 
3.6 NO release measurements from the reaction of Cu1.6S NPs with GSNO ……….42 
3.7 NO release from GSNO/Cu1.6S-PLGA polymersomes upon 633nm laser irradiation……….42 
3.8 Stability performance of GSNO and GSNO/Cu1.6S-PLGA polymersomes ……….43 
3.9 Cell culture and Cytotoxicity studies……….43 
3.10 Fluorescence detection of NO in MRC-5 cells ……….44 
3.11 Determination of the exogenous generation of NO from GSNO in living zebrafish and its vasodilatory activity on the cerebral blood vessel of zebrafish ……….45 
3.12 Demonstration of the vasodilatory activity of photo-induced release of NO from GSNO/Cu1.6S-PLGA polymersomes on the cerebral blood vessel of larval zebrafish……….45 
4 Results and discussion ……….46 
4.1 Characterization of Cu1.6S NPs……….46 
4.2 Characterization of GSNO/Cu1.6S-PLGA polymersomes ……….47 
4.3 Evidence of controllable NO release with the reaction of Cu1.6S NPs 
……….50 
4.4 Evidence of controllable NO release from polymersomes upon laser irradiation ……….52 
4.5 Evaluation of GSNO stability in polymersomes……….56 
4.6 Photo-triggered intracellular release of NO from polymersomes..59 
4.7 Biocompatibility of GSNO/Cu1.6S-PLGA polymersomes……….63
4.8 Cerebral vasodilation in zebrafish model ……….65 
5 Conclusion……….70 
Chapter 3. Light triggering goldsomes induced catalytic reaction of Au nanoparticles for NO-generation to alleviate pathological vasoconstriction .. 71 
1 Motivation ……….71 
2 Materials……….73 
3 Experiment methods ……….74 
3.1 Preparation of Au NPs ……….74 
3.2 Verification of the generation of NO from the reaction of GSNO with Au NPs ……….74 
3.3 Preparation of Au-PLGA polymersomes and goldsomes……….75 
3.4 Determination of the heating curve upon light illumination ……….76
3.5 Demonstration of light-triggered generation of NO from goldsomes 
……….76 
3.6 Assessment of the cytotoxicity and cardiotoxicity of goldsomes……….77
3.7 Verification of the light-triggered generation of NO in vitro……….78 
3.8 Local generation of NO in the cerebral region of zebrafish ……….78 
3.9 Demonstration of selective and local vasodilation on the cerebral vasculature of zebrafish ……….79 
3.10 Demonstration of the therapeutic activity of goldsomes to alleviate cerebral vasoconstriction induced by hypoxia……….80 
3.11 Statistics……….80 
4 Results and discussion ……….80 
4.1 Characterization of Au NPs……….80 
4.2 Characterization of goldsomes ……….84 
4.3 Verification of the rationale behind the design of goldsomes and light-triggered generation of NO……….88 
4.4 Cytotoxicity and cardiotoxicity of goldsomes ……….93 
4.5 Light-triggered generation of NO in vitro ……….95
4.6 Light-triggered local generation of NO in vivo ……….97 
4.7 Light-triggered generation of NO exerts vasodilatory activity selectively on the cerebral blood vessels of zebrafish……….101 
4.8 Light-triggered generation of NO alleviates cerebral vasoconstriction induced by hypoxia in zebrafish ……….104 
5 Conclusion……….107 
Chapter 4. CO2 induced angiogenesis related factor expression via NIR light triggered functionalized Prussian Blue Nanoparticles ……….108 
1 Motivation ……….108 
2 Materials……….111 
3 Experiment methods ……….111 
3.1 Preparation of PB NPs ……….111 
3.2 Preparation of PB-BC NPs……….112 
3.3 Preparation of PB-BC-PEG NPs ……….112 
3.4 Temperature elevation profile upon laser irradiation ……….112 
3.5 Quantitation of CO2 Release from PB-BC-PEG NPs……….113 
3.6 Stability performance of PB-BC-PEG NPs ……….113 
3.7 Cell culture and cytotoxicity studies……….114 
3.8 Fluorescence detection of HIF-1a in MES-SA cells……….114 
3.9 ELISA analysis of HIF-1a and VEGF in MES-SA cells ……….115 
4 Results and discussion ……….115 
4.1 Characterization of PB NPs……….115 
4.2 Evidence of controllable CO2 release upon laser irradiation……….120
4.3 The Stability of PB-BC-PEG NPs ……….123 
4.4 Evaluation for in vitro biocompatibility of PB-BC-PEG NPs ……….125 
4.5 Detection of HIF-1a upon the photo-triggered intracellular release of CO2……….. 127 
4.6 Evidence of CO2 induced HIF-1a and VEGF expression in cells. 
……….129 
5 Conclusion……….131 
Chapter 5. Summary of Findings ……….132
References ……….133 
Instrumentation……….143
Abbrevation ……….145
Appendix ……….147 

                                    

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