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研究生： 邱顯庭
Chiu, Hsien-Ting
論文名稱： 發展新穎治療型奈米藥物平台：核殼結構之白蛋白質／金奈米棒奈米劑型於癌症應用
Development of Novel Therapeutic Nanoplatform: Albumin-Gold Nanorods Based Core-Shell Nanoagent for Cancer Application
指導教授： 黃郁棻
Huang, Yu-Fen
口試委員: 江啟勳
Chiang, Chi-Shiun
何佳安
Ho, Ja-an
胡尚秀
Hu, Shang-Hsiu
陳韻晶
Chen, Yun-ching
陳冠宇
Chen, Guan-Yu
學位類別： 博士
Doctor
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： 2018
畢業學年度： 106
語文別： 英文
論文頁數： 144
中文關鍵詞： 白蛋白 、金奈米棒 、巨噬細胞 、藥物傳遞 、腫瘤治療 、結合治療 、蛋白吸附
外文關鍵詞： albumin, gold nanorod, macrophage, drug delivery, cancer therapy, combination therapy, protein adsorption
相關次數： 點閱：3  下載：0 
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    • 近年在白蛋白(albumin)與金奈米棒(gold nanorods)之奈米系統快速發展下，兩系統於腫瘤之相關研究上已分別取得了相當豐碩的成果，然而，兩奈米系統的結合在現階段的研究中仍需要面對許多的挑戰，例如：如何使新結合的複合材料保持原有的穩定性與製程的再現性是目前普遍認為的核心問題之一。因此，以材料穩定性、再現性與製程便利為主要出發點，本研究成功地開發出戊二醛(glutaraldehyde)交聯法與有機溶劑沉降法(dissolvation)以製備出具備高載體穩定性與再現性的白蛋白與金奈米棒之複合材料，前者稱GTA載體；後者則稱EM載體。兩製成之複合奈米載體雖然在載體大小、表面電位與殼核構型等特徵非常相似，然而，本研究發現相較於EM載體，GTA載體經交聯後仍能保存部份白蛋白結構，降低表面環境蛋白質吸附，進而減少巨噬細胞辨認或增加癌細胞的吞噬量；另一方面，交聯後的白蛋白相較於EM載體的白蛋白被發現除了能增加材料於抗癌藥物(doxorubicin)的包覆量增加，還能提升載體在雷射光照後的穩定性，以幫助包覆在奈米載體內的抗癌藥物(doxorubicin)釋藥效率提升；此外，本研究發現交聯後的白蛋白能提升金奈米棒的光聲(photoacoustic)強度，以利於顯影劑的開發。接著，利用上述EM載體相較於GTA載體較容易被巨噬細胞吞噬的特點為主軸，本研究將此結果延伸至細胞傳遞的應用(cell-based delivery)。然而，由於過去諸多在癌症相關之文獻已發現不論是靜脈注射法或是原位注射法，兩者皆碰到了藥物於腫瘤微環境之擴散效率與分佈位置等問題，使得治療流程所需要的藥物劑量非常高，但治療效果有時仍不盡理想。因此有別於過去一般關於細胞傳遞的文獻注重於追求最佳化的注射劑量與相對應的治療療效，本研究針對藥物在腫瘤的分佈與滯留時間兩特點於癌症治療的重要性，藉由EM載體與EM載體結合之細胞傳遞兩系統做統整性的研究與討論。本研究發現EM載體結合之細胞傳遞系統相較於單一EM載體系統或甚至單一小分子抗癌藥物系統(free drug)在原位注射至腫瘤後，能夠有效地提升藥物於腫瘤的分佈與滯留的時間，經雷射光照後能更有效地抑制腫瘤生長；相對地，單一EM載體系統因藥物分佈不均勻，經雷射光照後產生的熱或載體攜帶之藥物無法有效地傳遞與分佈至腫瘤各處，導致產生的熱能量與載體攜帶的藥物僅累積於原藥物注射位置，此結果不但未能有效抑制腫瘤生成，過度的熱能量與藥物濃度造成該局部區域嚴重的潰傷等副作用，更使得該傷口無法復原；單一小分子抗癌藥物系統則因為在腫瘤的滯留時間太短，大幅度地減低腫瘤治療的效果。此結果證實如何提升藥物於腫瘤的分佈與滯留的時間在奈米傳遞系統的設計上是需要被更加地關注。綜合上述兩部份之結果，本系統成功地開發出殼核構型之白蛋白與金奈米棒之奈米複合系統(GTA和EM)，分別對於材料上的特徵、性質與癌症的相關應用上做一系列完整的討論，針對於EM載體系統，本研究進一步以結合細胞傳遞的概念，探討藥物分佈與滯留時間在癌症治療的重要性。希望這一系列在奈米載體於癌症治療的研究上，從材料製成、藥物包覆、影像能力、材料於細胞與動物環境所造成的影響 (例如：環境蛋白吸附)與藥物傳遞效率等不同的面向切入，未來能帶給其他研究者不同的認知與啟發，以幫助癌症的相關研究更加邁進了一步。

    Recently, the rapid development of albumin- and gold nanorod- based nanoplatforms has individually offered promising solutions for addressing numerous difficulties in cancer research. However, fabricating a hybrid nanocomposite comprising both albumin and gold nanorod with high quality, homogeneity and dispersity remains a great challenge nowadays. In this study, two robust and one-step synthesis, a direct glutaraldehyde (GTA) cross-linking and a new desolvation method were provided for the fabrication of a uniform core-shell gold nanorod/serum albumin nanoplatform (NR@SA). Interestingly, despite they have similar particle size, morphology and surface charge, it is surprisingly found that their behaviors are totally different in many perspectives. The cross-linked nanoparticles (NR@SAs, GTA) compared with denatured nanoparticles (NR@SAs, EM) preserves half-native characterizations, resulting in reducing protein corona formation, macrophage phagocytosis and enhancing tumor cell internalization; the other half-artificial properties strengthen the capability of drug loading/release and thermal transduction for photoacoustic imaging. The results provide the forefront and fundamental information at the interface of protein shell control for cancer theranostics. Furthermore, by conversely utilizing the aforementioned result of preference macrophage phagocytosis of NR@SAs (EM), nanoparticles-laden macrophage delivery system was successfully established. Instead of finding the best solution for chasing the maximum therapeutic efficacy, the bio-combination is aimed to dig in more basic investigations and discuss the importance of intratumoral drug homogeneity and retention ability in cancer therapy, because not just for those intravenous nano-drug delivery systems, nano/micro-particles for intratumoral therapy is also facing numerous difficulties such as uncontrollable injection site, uneven drug distribution and inefficient drug transportation, and the large dose requirement that is occasionally along with side effect, particularly in many clinical case. Our results demonstrated that the movable and drug-loading bio-reservoir compared with the pristine counterparts exerts the higher tumor coverage and prolongs drug retention time that can efficiently improve the therapeutic efficacy and minimize the possible adverse effect. By contrast, similar to the summaries in many clinical studies using intratumoral chemotherapy, injecting with the high dose of pristine nanoparticles not only displays limited therapeutic effect, but also leads to unhealable wounds and impair the quality of life. Overall, the two core-shell nanoparticles are devoted to a wide range study and understanding from fundamental research in biomaterial science to in vivo drug delivery application against cancer. Hopefully, this new discovery and related discussions will bring promising benefits in nanoparticle development for cancer theranostics.

    摘要 II Abstract IV Acknowledgement VI Table of Contents VIII Table of Figures X Chapter Ⅰ: Introduction 1 1.1 Gold nanoparticles 1 1.1.1 Gold nanoparticles: Introduction 1 1.1.2 Synthesis of gold nanorods (Au NRs) 2 1.1.3 Functionalization and modification of Au NRs 3 1.1.4 Advanced design of gold nanorods for cancer treatment 5 1.1.5 Gold nanorods as a contrast imaging agent 9 1.2 Albumin 11 1.2.1 Introduction for albumin 11 1.2.2 Market approved albumin technologies 11 1.2.3 Versatile albumin nanocarriers for drug delivery and biomedical imaging 14 1.2.4 Albumin-based hybrid nanoplatform 15 1.3 Nanoparticles for cancer therapy 17 1.3.1 What are the difficulties we are facing with? 17 1.3.2 An opportunity: Specific surface modification for drug delivery 19 1.3.3 An opportunity: External stimuli for an augment of EPR effect 20 1.3.4 An opportunity: Neutral cell-mediated delivery system 22 1.3.4 What are the other things regarding cancer therapy we should think of? 24 1.4 Objective and purpose of this study 27 Chapter Ⅱ: Experimental Sections 31 2.1 Materials 31 2.1.1 Chemicals 31 2.1.2 Buffer 32 2.2 Laboratory apparatus and equipment 33 2.3 Experimental Methods 34 2.3.1 Preparation of Au NRs, NR@SAs and NR@DOX:SAs. 34 2.3.2 Characterization of NR@SAs and NR@DOX:SAs. 35 2.3.3 Analysis of protein adsorption. 37 2.3.4 In vitro cell culture. 40 2.3.5 In vitro studies for investigation of GTA/EM system in the first section (3.1). 40 2.3.6 In vitro studies for investigation of nanoparticle-laden macrophage delivery system in the second section (3.2). 42 2.3.7 In vivo animal experiments. 43 2.3.8 In vivo animal investigation of GTA system in the first section. 43 2.3.9 In vivo animal investigation of EM system in the second section. 44 2.3.10 Photoacoustic microscopy system. 46 2.3.11 Statistical Analysis 47 Chapter Ⅲ. Results and Discussions 48 3.1 GTA system: bioprosthesis of a half-native and half-artificial nanohybrid for cancer theranostics 48 3.1.1 NR@SAs core-shell nanoplatform by a high extent of cross-linked strategy or a new desolvation method 48 3.1.2 Bioprosthetic albumin shell reduced free protein adsorption and phagocytosis by macrophages but enhanced tumor cell uptake 54 3.1.3 Bioprosthetic albumin shell reduced protein corona formation by blood proteins 61 3.1.4 Artificial characteristics: Crosslinking effect for high drug loading capacity of NR@SAs (GTA) 68 3.1.5 Hard and soft protein shells for in vitro drug release 72 3.1.6 Characterization of cross-linked SA shell: Amplifying the enhancement of photoacoustic signals over pristine Au NRs. 79 3.1.7 NR@SA nanoplatform for cancer theranostics 82 3.1.8 Combinational photothermal- and chemo-therapy. 84 3.2 EM system: The importance of drug distribution and retention ability for combined therapy. 88 3.2.1 Designing NR@SAs (EM) core-shell nanoplatform for cell-based delivery strategy 88 3.2.2 NIR light-activated drug release from payload-laden macrophages. 90 3.2.3 In vitro estimation of the feasibility of macrophage delivery system for in vivo studies 96 3.2.4 Cell-mediated drug delivery system improves drug coverage and distribution and enhances retention ability. 99 3.2.5 The more homogeneous photothermal drug delivery was achieved by combinational therapy through multiple intratumoral injections. 102 3.2.6 What happened when localized photothermal drug delivery was achieved? 106 3.2.7 The more homogeneous chemodrug and photothermal delivery by the cell-mediated delivery system resulted in precise NIR-laser-activated drug release for improved antitumor effects. 111 3.2.8 In vivo tumor-tropic migration and specific accumulation in the hypoxic region. 113 Chapter Ⅲ. Conclusion 117 Chapter Ⅳ. Perspectives 119 Appendix: Reference 123

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