Alginate Based Nanocomposites for In-Vivo Cancer Diagnosis and Therapy｜國立臺灣科技大學博碩士論文系統

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研究生：	KEFYALEW DAGNEW ADDISU KEFYALEW DAGNEW ADDISU
論文名稱：	Alginate Based Nanocomposites for In-Vivo Cancer Diagnosis and Therapy Alginate Based Nanocomposites for In-Vivo Cancer Diagnosis and Therapy
指導教授：	蔡協致 Hsieh-Chih Tsai
口試委員:	蔡協致 Hsieh-Chih Tsai 鄭智嘉 Chih-Chia Cheng 何明樺 Ming-Hua Ho 廖愛禾 Liao, Ai-Ho 鄭如忠 Ru-Jong Jeng 鍾次文教授 Tze-Wen Chung
學位類別：	博士 Doctor
系所名稱：	應用科技學院 - 應用科技研究所 Graduate Institute of Applied Science and Technology
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	185
中文關鍵詞：	海藻酸鈉、T1-磁共振成像（MRI）、腫瘤診斷、鑭系元素、聚多巴胺、類球面、斑馬魚、治療診斷學
外文關鍵詞：	Alginate, T1-Magnetic Resonance Imaging (MRI), Tumor diagnosis, Lanthanides, Polydopamine, Spheroids, Zebrafish, Theranostics
相關次數：	點閱：269 下載：0
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癌症是目前世界上最致命的疾病，預計在2030年會造成超過1150萬人死亡。常見的幾種癌症包括有：大腸癌、口腔癌、子宮頸癌、乳癌等，及早的發現並治療可以提高治癒率。目前有廣大的研究將奈米載體用於癌症的診斷與治療，海藻酸是天然存在的無支鏈、生物相容、無毒、非免疫原性和可生物降解的多醣之一，可用作奈米醫學的傳輸工具，海藻酸還具有許多游離的羥基和羧基，透過修飾這些官能基形成海藻酸衍生物，可以改變如溶解度、親疏水性、物理化學和生物學的特性，使其成為功能性化學材料的理想選擇。
本論文具有兩個獨立的研究系統：第一部分研究了兩種由鈣/錳離子複合海藻酸 - 聚多巴胺奈米凝膠或海藻酸 - 多巴胺奈米凝膠(AlgPDA(Ca/Mn)或AlgDA(Ca/Mn))的混合物。在這樣的系統中，Ca2+陽離子與Alg的羧酸進行離子相互作用，以增強奈米凝膠的穩定性，同時Mn陽離子的複雜性也增加了合成奈米凝膠的膠體穩定性。利用超導量子干涉儀裝置(SQUID)測量證實了製備CA的磁性，證明了潛在的順磁性。因此，T1弛豫率測量顯示，PDA複合合成奈米凝膠在7.0 T MRI圖像中顯示出強烈的正對比度增強，r1 = 12.54 mM-1∙s-1，而DA複合合成奈米凝膠顯示相對較低的T1弛豫效應，r1 = 10.13 mM-1∙s-1。此外，當與小鼠巨噬細胞（RAW 264.7）和HeLa細胞一起培養時，兩種合成的奈米凝膠在濃度高達0.25mM濃度時具有> 92％的細胞存活率，在體內分析中具有高的生物相容性。體內MRI測試顯示了合成的奈米凝膠在長時間內表現出了高的信噪比，這為腫瘤和解剖學成像提供了更長的圖像採集時間。此外，T1加權MRI結果顯示聚乙二醇化的AlgPDA(Ca/Mn)奈米凝膠顯著的增強肝臟和腫瘤組織的信號。本研究中使用低成本的一鍋合成法，利用高滲透長滯留效應(EPR)，提供了一種有前途和競爭的方法用於非侵入性腫瘤檢測和全面的解剖學診斷，顯著增強了體外和體內成像。
第二部分的研究，通過簡單的金屬 - 配體相互作用製備具pH敏感性鑭系元素多功能奈米粒子，用於進行癌細胞成像和藥物傳遞。首先，研究了兩種分別為铽/銪或鏑/氧化鉺奈米粒子(Tb/Eu @ AlgPDA和Dy/Er @AlgPDA NPs)複合的海藻酸 - 聚多巴胺的系統，然後用腫瘤靶向葉酸(FA)負載抗癌藥物阿黴素(DOX)修飾Tb/Eu @ AlgPDA，以形成FA-Tb / Eu @ AlgPDA-DOX奈米粒子。在這樣的系統中，除了主動靶向葉酸和葉酸受體之間的相互作用之外，還引入了PDA的貽貝特性以加強腫瘤靶向性和滲透性。Tb/Eu @ AlgPDA系統在光致發光效率優於Dy/Er @ AlgPDA，發出強烈和尖銳的螢光光譜。此外，通過MTT試驗與HeLa細胞孵育時，Tb / Eu @ AlgPDA奈米粒子細胞活力> 93.3％，達到0.50 mg / mL 優於Dy /Er @ AlgPDA 奈米粒子細胞活力(82.4％)；與斑馬魚的胚胎和成魚 (Danio rerio)動物實驗測試上則顯示其具有高生物相容性。在FA-Tb/Eu @ AlgPDA-DOX系統表現中表現出pH敏感和持續的藥物釋放；在HeLa細胞的球狀模型中，與游離的阿黴素相比，FA-Tb / Eu @ AlgPDA-DOX系統顯示出更好的穿透效率和抑制類球面生長。與斑馬魚胚胎一起孵育後，FA-Tb / Eu @ AlgPDA-DOX系統也展現出較好的抗腫瘤能力。所有這些結果表明FA-Tb / Eu @ AlgPDA-DOX 奈米粒子是一種具有腫瘤靶向和癌症治療的穿透能力的多功能奈米載體。

Despite improvements in diagnosis and therapy, cancer remains the world’s deadliest disease, with 11.5 million deaths forecast in 2030. Some of the most common types of cancer, including cervical, breast, oral and colorectal cancers, are more likely to cure if they are detected and treated early. A wide range of platforms are currently being investigated as nanocarriers for the diagnosis and treatment of cancer. Alginate is one of the naturally occurring unbranched, biocompatible, non - toxic, non-immunogenic and biodegradable polysaccharides which can be used as delivery vehicles for nanomedicine. Alginate also has a number of free groups of hydroxyl and carboxyl distributed along the backbone, making it ideal for chemical functionality. By forming alginate derivatives by functionalizing the hydroxyl and carboxyl groups available, properties such as solubility, hydrophobicity and physicochemical and biological characteristics can be altered. This dissertation is organized on the basis of two separate sections: the first section examined two systems of calcium/manganese cations complexed with either alginate-polydopamine or alginate-dopamine nanogels ((AlgPDA(Ca/Mn or AlgDA (Ca/Mn)) nanogels (NG). In such systems, Ca2+ cations form ionic interaction via carboxylic acids of the Alg backbone to enhance the stability of the synthetic NG. Likewise, the complexity of Mn cations also increased the synthetic nanogel 's colloidal stability. Magnetic property of prepared CAs was confirmed with superconducting quantum interference device (SQUID) measurements, proving the potential paramagnetic property. Hence, T1 relaxivity measurement showed that PDA-complexed synthetic NG reveals a strong positive contrast enhancement with r1=12.54 mM-1∙s-1 in 7.0 T MRI images while DA-complexed synthetic NG showed relatively lower T1 relaxivity effect with r1 =10.13 mM-1∙s-1 value. Furthermore, both synthetic NGs reveals negligible cytotoxicity with > 92% cell viability up to 0.25 mM concentration when incubated with mouse macrophage (RAW 264.7) and HeLa cells, and high biocompatibility under in vivo analysis. An in vivo MRI test indicate that the synthetic NG exhibits a high signal to noise ratio for longer hours, which provides a longer image acquisition time for tumor and anatomical imaging. Additionally, T1-weighted MRI results revealed that PEGylated AlgPDA(Ca/Mn) NGs signiﬁcantly enhanced the signals from liver and tumor tissues. Therefore, owing to enhanced permeability and retention (EPR) effect, significantly enhanced both in-vitro and in-vivo imaging, low cost and one-pot synthesis method, the Mn-based biomimetic approach used in the study provides a promising and competitive alternative for non-invasive tumor detection and comprehensive anatomical diagnosis. In section two, a pH-responsive mixed lanthanide-based multifunctional nanoparticles (NPs) were fabricated by simple metal-ligand interactions for simultaneous cancer cell imaging and drug delivery. First, two new systems of alginate-polydopamine complexed with either terbium/europium or dysprosium/erbium oxides NPs (Tb/Eu@AlgPDA and Dy/Er@AlgPDA NPs) were investigated. Tb/Eu@AlgPDA NPs were then functionalized with a tumor-targeting ligand folic acid (FA) and loaded anti-cancer drug, doxorubicin (DOX), to form FA-Tb/Eu@AlgPDA-DOX NPs. Under such systems, the mussel-inspired property of PDA was introduced to improve tumor targetability and penetration in addition to active targeting (interaction between FA and folate receptor). Photo-luminescent efficiency showed that the Tb/Eu@AlgPDA system is superior to Dy/Er@AlgPDA, presenting intense and sharp emission on fluorescence spectra. In addition, compared to Dy/Er@AlgPDA NPs (82.4%), Tb/Eu@AlgPDA NPs exhibits negligible cytotoxicity with > 93.3% cell viability up to 0.50 mg/mL when incubated with HeLa cells via MTT assay, and high biocompatibility when incubated with the embryo and larvae of zebrafish (Danio rerio). FA-Tb/Eu@AlgPDA-DOX system exhibit a pH-responsive and sustained drug release pattern. In a spheroid model of HeLa cells, FA-Tb/Eu@AlgPDA-DOX system showed better penetration efficiency and spheroid growth inhibitory effect as compared with free DOX. After incubated with zebrafish embryo, FA-Tb/Eu@AlgPDA-DOX system also exhibited improved antitumor efficacies rather than the other experimental groups in HeLa tumor-xenografted zebrafish. Therefore, all these results suggested that FA-Tb/Eu@AlgPDA-DOX NPs is a promising multifunctional nanocarrier with tumor targeting and penetration capacity for cancer therapy.

摘要i
ABSTRACT    iii
ACKNOWLEDGMENT    v
CONTENTS    vii
LIST OF TABLES    xii
LIST OF FIGURES    xiii
LIST OF SCHEMES    xviii
LIST OF ABBREVIATION    xix
CHAPTER  1    1
1    Introduction    1
2    Objectives of the study    6
CHAPTER  2    7
Literature Review    7
1    Cancer    7
1.1. Cancer treatment methods    7
2    Major nano-medicines types in the clinic    11
2.1    Polymeric based nanoparticles    13
3    The main mechanisms of nanoparticle preparation using polysaccharides    15
3.1    Polyelectrolyte complexation (PEC)    15
3.2    Self-assembly    16
3.3    Polysaccharide Nanoparticles by Crosslinking    16
3.4 Alginate    19
4    Types of biomedical imaging modalities    24
4.1    Magnetic resonance imaging (MRI)    25
4.2    Optical imaging (fluorescent imaging)    28
5    Influence of nano-based technology properties in cellular uptake    30
5.1    Size    30
5.2    Shape    31
5.3    Surface charge    31
6    Drug delivery modes for cancer treatment    32
6.1    Passive targeting    32
6.2    Active targeting    33
6.3    Triggered release    34
CHAPTER  3    35
Bioinspired, Manganese Chelated Alginate-Poly (Dopamine) Nanomaterials for Efficient In-Vivo T1_Weighted Magnetic Resonance Imaging (MRI)    35
1    Introduction    35
2    Experimental Section    39
2.1    Materials    39
2.2    Synthesis of Alginate-Dopamine (AlgDA) conjugates    39
2.3    Preparation of AlgDA(Ca/Mn) and AlgPDA(Ca/Mn) NGs    40
2.4    Characterization    41
2.5    T1 and T2   Relaxivity MRI studies    42
2.6    Cytotoxicity test (MTT assay)    42
2.7. Xylenol orange disodium salt for spectrophotometric determination of free manganese ions.    43
2.8.   PEG surface coating to form AlgPDA(Ca/Mn)-PEG preparation    43
2.7    In-vivo MR imaging    44
2.8    Histological and bio-distribution study    45
3    Results and Discussion    47
3.1    Synthesis and characterization of Alginate-polydopamine (AlgPDA) and Alginate dopamine (AlgDA) conjugates    47
3.2    Magnetic properties    60
3.3    T1 and T2   relaxation measurements    61
3.4    In vivo mice T1- MR Images    69
4    Conclusions    79
CHAPTER  4    80
Mixed Lanthanide Oxide Nanoparticles Coated with Alginate-Polydopamine as Multifunctional Nano-Vehicles for Dual-Modality: Targeted Imaging and Chemotherapy    80
1    Introduction    80
2    Experimental Section    85
2.1    Materials and Chemicals    85
2.2    Synthesis of AlgDA conjugates    85
2.3    Synthesis of Tb/Eu and Dy/Er oxide NPs    86
2.4    Preparation of AlgPDA coated Tb/Eu and Dy/Er oxide NPs.    87
2.5    Characterization    87
2.6    Cell Viability Assay of Tb/Eu@AlgPDA and Dy/Er@AlgPDA   NPs    88
2.7    Cellular uptake and in-vitro Cytotoxicity of FA-Tb/Eu@AlgPDA-DOX NPs.    88
2.8    Loading of DOX onto FA- Tb/Eu@AlgPDA NPs    89
2.9    In-vitro drug release study    90
2.10    Bio-toxicity evaluation and fluorescence imaging in Zebrafish    90
2.11    Zebrafish tumor-xenografts and compound treatment.    91
2.12    Cell culture and Tumor spheroid model formation    92
2.13    Spheroid growth inhibition study    92
2.14    NPs penetration and distribution study    93
3    Results and Discussion    94
3.1    Preparation of AlgPDA-coated mixed lanthanide oxide NPs    94
3.2    Optical properties    102
3.3    Biocompatibility and Targeted cancer cell imaging nanocomposite carriers    106
3.4    Drug loading and in-vitro release    109
3.5    Cellular uptake and in-vitro Cytotoxicity of FA-Tb/Eu@AlgPDA-DOX NPs.    111
3.6    Zebrafish in-vivo biocompatibility and imaging    116
3.7    Anti-tumor effects study in tumor-xenograft zebrafish models    120
3.8    Spheroid growth characterization and growth inhibition study    122
3.9    Distribution of DOX in 3D spheroids    126
4    Conclusions    131
CHAPTER  5    132
1    General Summary    132
2    Recommendations    134
REFERENCES    136
APPENDIX -A:  List of Publications    163
APPENDIX -B:  Conferences and Awards    164


                                

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