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研究生:羅郁茹
研究生(外文):Yu-Ju Lo
論文名稱:利用三特異性T細胞接合抗體(Anti-CD3∕PD-L1∕mPEG)修飾ganetespib磷脂質穩定化奈米微胞體應用於癌症免疫化學療法
論文名稱(外文):Trispecific T-cell engager antibody (Anti-CD3/PD-L1/mPEG) decorated ganetespib-loaded lecithin-stabilized micellar drug delivery system (LsbMDDS) for cancer immunochemotherapy
指導教授:何秀娥
指導教授(外文):Hsiu-O Ho
口試委員:林山陽陳怜均謝堅銘
口試委員(外文):Shan-Yang LinLing-Chun ChenChien-Ming Hsieh
口試日期:2020-07-15
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:藥學系(碩博士班)
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:127
中文關鍵詞:ganetespibT細胞活化三特異性抗體免疫檢查點磷脂質穩定化奈米微胞體
外文關鍵詞:ganetespibT cell activationtri-specific T cell engager antibodyimmune checkpointlecithin-stabilized micellar drug delivery system
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第一章 緒論 1
第一節、 癌症免疫療法(Cancer immunotherapy) 1
一、 免疫監測與免疫編輯(Immune surveillance and Immune editing) 1
二、 主動與被動式免疫治療(Active and passive immunotherapy) 4
2.1 被動式免疫治療 4
2.1.1 單株抗體(monoclonal antibody) 4
2.1.2 過繼性細胞療法(Adoptive cell therapy, ACT) 15
2.1.3 溶瘤病毒(Oncolytic virus, OV) 17
2.2 主動式免疫治療(Active immunotherapy) 18
2.2.1 癌症疫苗(Cancer vaccines) 18
2.2.2 免疫檢查點抑制劑(Immune checkpoint inhibitors, ICIs) 19
第二節、 免疫療法合併治療策略 22
第三節、 奈米藥物傳遞系統(Nano-drug delivery system) 24
一、 奈米載體的種類 25
1.1 脂質體(Liposomes) 27
1.2 微胞體(Micelles) 27
1.3 脂質聚合物混成奈米顆粒(Lipid-polymer hybrid nanoparticles, LPHNs) 28
二、 奈米藥物之遞送策略 30
2.1 被動運輸(passive delivery) 31
2.2 主動運輸(active delivery) 31
第四節、 模式藥物(Model drug)介紹 33
一、 Ganetespib 33
1.1 物理化學性質 33
1.2 藥理機轉 33
1.3 熱休克蛋白(Heat shock proteins, HSPs) 34
1.3.1 熱休克蛋白90(Heat shock protein 90, HSP90)抑制劑 37
第二章 研究目的與動機 40
第三章 試劑與材料 42
一、 實驗材料 42
二、 細胞實驗材料 43
三、 藥品溶液或細胞培養液之配置 43
四、 實驗儀器 44
第四章 實驗方法 46
第一節、 Ganetespib磷脂質穩定化奈米微胞體(GSP-LsbMDDS)之處方開發及其物理化學性質探討 46
一、 Ganetespib磷脂質穩定化奈米微胞體(GSP-LsbMDDS)處方開發 46
1.1 Ganetespib 溶解度測定 46
1.2 磷脂質穩定化奈米微胞體外相比例及超音波震盪條件選擇 46
1.3 Ganetespib磷脂質穩定化奈米微胞體之製備 47
1.4 Ganetespib磷脂質穩定化奈米微胞體之放大製程 47
二、 Ganetespib磷脂質穩定化奈米微胞體(GSP-LsbMDDS)之物理化學性質測量 49
2.1 奈米微胞體粒徑大小及分布 49
2.2 奈米微胞體表面電荷測定 49
2.3 奈米微胞體外觀測定 50
2.4 奈米微胞體藥物含量測定 50
2.4.1 Ganetespib之高效液相層析條件 50
2.4.2 Ganetespib之檢量線標準品溶液配製 51
2.4.3 藥物包覆率及負載率 52
2.5 奈米微胞體安定性試驗 52
第二節、 三特異性抗體修飾ganetespib磷脂質穩定化奈米微胞體(GSP-LsbMDDS-TsAb)之體外細胞試驗與體內動物試驗 53
一、 三特異性抗體修飾ganetespib磷脂質穩定化奈米微胞體 53
1.1 奈米微胞體之甲氧基聚乙二醇與抗體之最適比例優化 53
1.2 估算磷脂質穩定化奈米微胞體表面修飾三特異性抗體數量 55
二、 藥物體外釋放試驗(in vitro drug release studies) 56
三、 體外細胞試驗(in vitro studies) 58
3.1 細胞培養(cell culture) 58
3.1.1 腫瘤細胞 58
3.1.2 免疫細胞 58
3.2 誘導PD-L1表現試驗 59
3.3 細胞吞噬試驗(cellular uptake studies) 60
3.4 細胞存活率試驗(cell viability studies) 61
3.4.1 癌細胞之細胞存活率試驗 61
3.4.1 T細胞介導(T cell-mediated)之細胞毒性試驗 63
四、 體內動物試驗(in vivo studies) 64
4.1 實驗動物 64
4.2 建立腫瘤異種移植原位模式小鼠(Xenograft orthotopic model) 64
4.2.1 腫瘤細胞培養 64
4.2.2 免疫細胞培養及染色 65
4.2.3 異種移植原位模式小鼠 65
4.3 腫瘤抑制試驗(Tumor inhibition studies) 66
4.3.1 活體螢光光學影像分析免疫細胞於體內分佈試驗 67
4.3.2 組織病理切片學評估 69
第五章 研究結果與討論 70
第一節、 Ganetespib磷脂質穩定化奈米微胞體(GSP-LsbMDDS)之處方開發及其物理化學性質探討 70
一、 Ganetespib磷脂質穩定化奈米微胞體(Lecithin-stabilized micellar drug delivery system, LsbMDDs)處方開發 70
1.1 Ganetespib 溶解度測定 70
1.2 磷脂質穩定化奈米微胞體外相比例及超音波震盪選擇 70
二、 Ganetespib 磷脂質穩定化奈米微胞體之物理化學性質測量 72
2.1 奈米微胞體粒徑大小及表面電荷測定 72
2.2 奈米微胞體外觀測定 75
2.3 奈米微胞體藥物含量測定 76
2.3.1 Ganetespib 溶液之高效液相層析分析方法及確效 76
2.4 奈米微胞體安定性試驗 79
第二節、 三特異性抗體修飾ganetespib磷脂質穩定化奈米微胞體(GSP-LsbMDDS-TsAb)之體外細胞試驗與體內動物試驗 81
一、 三特異性抗體修飾ganetespib磷脂質穩定化奈米微胞體 81
1.1 奈米微胞體之甲氧基聚乙二醇與抗體之最適比例優化 81
1.2 估算磷脂質穩定化奈米微胞體表面修飾三特異性抗體數量 83
二、 藥物體外釋放試驗(in vitro drug release studies) 84
三、 體外細胞試驗 86
3.1 誘導PD-L1表現試驗 86
3.2 細胞吞噬試驗(cellular uptake studies) 87
3.3 細胞存活率試驗 93
3.3.1 癌細胞之細胞存活率 93
3.3.2 T細胞介導(T cell-mediated)之細胞毒性試驗 96
四、 體內動物試驗 100
4.1 腫瘤抑制試驗 100
4.2 活體螢光光學影像分析免疫細胞於體內分佈試驗 105
4.3 組織病理切片學評估 114
第六章 結論 119
第七章 參考文獻 120
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