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研究生:宋政穎
研究生(外文):Zheng-Ying Sung
論文名稱:新型奈米顆粒應用於癌症治療之研究
論文名稱(外文):Novel Nanoparticles for Cancer Therapy
指導教授:劉澤英
指導教授(外文):Tse-Ying Liu
學位類別:碩士
校院名稱:國立陽明交通大學
系所名稱:生物醫學工程學系
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:93
中文關鍵詞:骨肉瘤奈米顆粒被動累積磁導航
外文關鍵詞:OsteosarcomaMagnetic nanovehicleCancer therapy
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目錄
中文摘要.............................I
Abstract............................II
本文目錄.............................III
圖目錄...............................VIII
表目錄...............................XI
第一章、緒論.........................1
第二章、文獻回顧.....................4
2-1、骨肉瘤 (Osteosarcoma; OS).......4
2-1-1、骨肉瘤簡介....................4
2-1-2、骨肉瘤肺轉移...................6
2-1-3、骨肉瘤肺轉移臨床診斷和治療手段...8
2-2、癌症轉移 (Cancer Metastasis).....12
2-2-1、奈米藥物及無機顆粒於癌症轉移之發展.12
2-2-2、癌症轉移之流程與機制.............15
2-2-3、細胞骨架與癌症轉移之關聯性........20
2-3、磁性奈米顆粒 (Magnetic Nanoparticles)..25
2-3-1、超順磁性氧化鐵之性質及臨床應用....25
2-3-2、摻和鑭系元素之超順磁性奈米顆粒.....30
2-4、血清白蛋白 (Serum Albumin; SA)......32
2-4-1、血清白蛋白與細胞之交互作用.........32
2-4-2、血清白蛋白奈米顆粒於生物醫學之應用...34
第三章、材料與方法.......................35
3-1、實驗設計...........................35
3-2、材料與儀器..........................36
3-2-1、材料合成之化學藥品................36
3-2-2、生物實驗試劑........................37
3-2-3、儀器..........................38
3-3、材料合成方法 (Preparation of Eu:SPIO@BSA)....39
3-3-1、摻合銪之超順磁性氧化鐵合成 (Pyrolysis Method).....39
3-3-2、牛血清白蛋白-摻合銪之超順磁性氧化鐵複合奈米顆粒製備 (Emulsion method)......................39
3-3-3、SPIO@FITC-BSA和Eu:SPIO@FITC-BSA螢光奈米顆粒製備.....................................40
3-4、材料性質分析 (Characterization of Eu:SPIO@BSA)..........................41
3-4-1、粒徑與表面電位分析 (Size & Zeta Potential)............................41
3-4-2、穿透式電子顯微鏡影像 (TEM).........41
3-4-3、掃描式電子顯微鏡影像與能量色散X射線譜分析 (SEM & EDX)....................................41
3-4-4、X射線光電子能譜分析 (XPS)............42
3-4-5、X光粉末及薄膜繞射分析 (XRD)..........42
3-4-6、磁性分析 (Magnetometric analysis)....42
3-4-7、熱重分析 (TGA).......................42
3-4-8、感應耦合電漿質譜分析 (ICP-MS).........43
3-5、體外試驗 (In vitro Test)...............44
3-5-1、小鼠前驅成骨細胞培養 (MC3T3-E1)........44
3-5-2、小鼠骨肉瘤細胞培養 (K7M2, K7M2-AS5w.FLuc)....44
3-5-3、細胞毒性試驗 (Cytotoxicity Assay).........45
3-5-4、細胞吞噬試驗 (Cellular Uptake Assay)........45
3-5-5、傷口癒合試驗 (Wound Healing Assay)..........46
3-5-6、細胞遷移試驗 (Transwell Migration Assay).......46
3-5-7、細胞侵犯試驗 (Transwell Invasion Assay).......47
3-5-8、細胞偽足形貌觀察 (Cell Pseudopodia Morphology)....47
3-5-9、免疫螢光染色法 (Immunofluorescence assay)......48
3-5-10、溶血實驗及血球形貌觀察 (Hemolytic assay).....49
3-6、活體試驗 (In vitro Test).....................50
3-6-1、小鼠原位骨肉瘤誘導 (Orthotopic Model).......50
3-6-2、小鼠骨肉瘤肺轉移治療 (Treatment for Osteosarcoma Pulmonary Metastasis)..........................50
3-6-4、組織累積與代謝實驗 (Biodistribution Analysis)....51
3-6-5、血液生化檢測 (Biochemical Test)............51
第四章、結果與討論..........................52
4-1、材料性質分析 (Characterization of Eu:SPIO@BSA nanoparticles).................................52
4-1-1、穿透式電子顯微鏡影像與動態光散射粒徑分析 (TEM Images and Size Distribution of Eu:SPIO@BSA).............52
4-1-2、掃描式電子顯微鏡影像與能量色散 X 射線譜分析 (SEM images and EDX analysis)..............................54
4-1-3、X射線光電子能譜分析 (XPS)...............56
4-1-4、X射線繞射光譜分析 (XRD)................58
4-1-5、熱重損失分析 (Thermogravimetric Analysis; TGA).....60
4-1-6、感應耦合電漿質譜分析 (ICP-MS).........62
4-1-7、磁性特徵分析 (Magnetic Analysis).......63
4-2、體外試驗 (In vitro Test).................65
4-2-1、細胞毒性試驗 (Cytotoxicity Assay)............65
4-2-2、細胞吞噬試驗 (Cellular Uptake Assay)........67
4-2-3、傷口癒合試驗 (Wound Healing Assay).......69
4-2-4、細胞遷徙與侵犯試驗 (Transwell Migration/ Invasion Assay)........................................71
4-2-5、細胞偽足形貌分析 (Cell Pseudopodia Morphology)....73
4-2-6、生物標誌物 (Immunofluorescence Staining Images of Biomarkers)..................................75
4-2-7、溶血實驗及血球形貌觀察 (Hemolytic assay and RBCs Morphology)................................77
4-3、動物試驗 (In vivo Test)....................79
4-3-1、裸鼠骨肉瘤自發性肺轉移模型建立 (Model of Pulmonary Metastasis from Osteosarcoma)...................79
4-3-2、腫瘤與組織累積試驗與肝功能檢驗 (Biodistribution and Biochemical Tests)............................81
4-3-3、骨肉瘤肺轉移治療 (In vivo Therapeutic Efficacy of Eu:SPIO@BSA NPs on Metastasis of OS)............................................84
第五章、結論.....................................86
第六章、參考文獻..................................87

圖目錄
Figure 2-1 Biologic growth pattern of osteosarcoma....5
Figure 2-2 Overexpression of transcription factors, oncogenes and dysregulation of tumor suppressor genes in osteosarcoma....5
Figure 2-3 Age distribution of patients with osteosarcoma 7
Figure 2-4 Schematic illustration of deep tumor penetrating strategies for anti-metastasis therapy....14
Figure 2-5 Schematic illustration of the metastatic cascade in osteosarcoma....18
Figure 2-6 Overview of different modes of cell migration....23
Figure 2-7 Mechanisms of cancer cell motility.....24
Figure 2-8 The different physiochemical properties of SPIONs affect their biokinetics and fate in vivo....27
Figure 2-9 Properties of magnetic nanoparticles (MNPs) for the development of drug delivery systems....29
Figure 2-10 Schematical illustration of the impurity doping routes in the host lattice.....31
Figure 2-11 The Gp60-mediated uptake of albumin–PTX NPs by the transcytosis....33
Figure 3-1 Graphical Abstract....35
Figure 4-1 TEM images of (A) SPIO, (B) Eu:SPIO, (C) SPIO@BSA, and (D) Eu:SPIO@BSA NPs. (E) Particle-size distribution of SPIO@BSA and Eu:SPIO@BSA were measured by dynamic light scattering (DLS).....53
Figure 4-2 SEM images and the corresponding EDX analysis of (A, B) SPIO@BSA and (C, D) Eu:SPIO@BSA NPs.....55
Figure 4-3 XPS survey and high resolution spectra of SPIO and Eu:SPIO NPs.....57
Figure 4-4 XRD patterns of (A) SPIO and Eu:SPIO NPs; (B) SPIO@BSA and Eu:SPIO@BSA nanoparticles.....59
Figure 4-5 TGA curves of SPIO, Eu:SPIO, SPIO@BSA, Eu:SPIO@BSA, and BSA.....61
Figure 4-6 Magnetization–magnetic field strength (M–H) curves of (A) SPIO and Eu:SPIO and (B) SPIO@BSA and Eu:SPIO@BSA, which were suspended in solution (inset) with the application of a magnetic field.....64
Figure 4-7 Effect of SPIO@BSA, Eu:SPIO@BSA, and EuCl3·6H2O on cell viability of K7M2 and MC3T3-E1 cells for (A-C) 24 h and (D-F) 48 h.....66
Figure 4-8 Cellular uptake images of K7M2 and MC3T3-E1 cells treated with fluorescent SPIO@BSA and Eu:SPIO@BSA nanoparticles for 24 h incubation.....68
Figure 4-9 Effect of SPIO@BSA, Eu:SPIO@BSA nanoparticles, and EuCl3·6H2O on cell migration of K7M2 cells determined by wound healing assay.....70
Figure 4-10 Effect of SPIO@BSA, Eu:SPIO@BSA NPs and EuCl3·6H2O on migration and invasion capacity of K7M2 cells examined by transwell chamber assays.....72
Figure 4-11 SEM images of K7M2 cells showed shape changes in cellular morphology after SPIO@BSA, Eu:SPIO@BSA NPs, and EuCl3·6H2O treatments for 24 h.....74
Figure 4-12 Immunofluorescence staining of matrix metalloproteinase 2 (MMP-2) and vinculin in K7M2 cells with SPIO@BSA, Eu:SPIO@BSA, EuCl3·6H2O treatment for 24 h.....76
Figure 4-13 Effect of SPIO@BSA, Eu:SPIO@BSA NPs, and EuCl3·6H2O on hemolytic activity determined by in vitro hemolytic assay. 78
Figure 4-14 Bioluminescent images of primary osteosarcoma model and pulmonary metastasis.....80
Figure 4-15 (A) MR images and (B) ICP-MS analyses of tissue accumulation, and (C) hemotaology tests after administering Eu:SPIO@BSA suspensions in tumor-bearing nude mice.....83
Figure 4-16 In vivo therapeutic efficacy of control, Eu:SPIO@BSA nanoparticles and EuCl3·6H2O on the growth and lung metastasis of osteosarcoma determined by IVIS images.....85

表目錄
Table 2-1 Chemotherapeutic Agents Used in the Treatment of Osteosarcoma....11
Table 2-2 Druggable molecular targets in the metastatic cascade....19
Table 4-1 The amount of Fe and Eu element weight content in the SPIO, SPIO@BSA, Eu:SPIO, and Eu:SPIO@BSA measured by ICP-MS.....62
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