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研究生:陳貽華
研究生(外文):I-Hua Chen
論文名稱:磷酸鈣奈米微粒作為非病毒基因遞送載體研究
論文名稱(外文):Study of calcium phosphate nanoparticles as non-viralvectors for gene delivery system
指導教授:李源弘
指導教授(外文):Yuan-Haun Lee
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:材料科學與工程學研究所
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:106
中文關鍵詞:磷酸鈣奈米微粒基因治療奈米結構自組裝
外文關鍵詞:nanoparticlecalcium phosphategene therapyself-assemble
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摘要
基因治療的重要關鍵在於一個良好且有效的基因遞送系統將具有功能性的基因送至細胞中表現。奈米微粒作為基因遞送載體具有可靶向和於體內長效循環以增加轉染效果的潛力。然而,適當的製程、粒徑大小和低毒性等性質是發展奈米微粒作為基因載體首要課題。本研究目的在於發展磷酸鈣奈米微粒作為非病毒基因載體和探討載體的材料特性,研究以water/AOT/hexane 之w/o 微乳化系統製備磷酸鈣奈米微粒,探討製程條件對粒徑分佈影響和材料性質分析,進而探討製備DNA-磷酸鈣奈米微粒複合物作為非病毒基因載體的適當條件。
研究以DLS 作粒徑分析並以TEM、FE-SEM 觀察奈米微粒型態,DNA-磷酸鈣奈米微粒複合物由TEM 觀察結果為球殼狀結構,且分散性良好,粒徑分佈集中於100nm 附近,平均粒徑隨Wo 值減少而減小。磷酸鈣奈米微粒擁有大於80%包覆基因的效率,且電泳結果證實DNA-磷酸鈣奈米微粒複合物能於DNaseI 環境下保護基因不被水解。由MTT 測試結果DNA-磷酸鈣奈米微粒複合物對細胞活性無負面影響。體外細胞實驗以293T 細胞進行轉染,DNA-磷酸鈣奈米微粒複合物能遞送基因並表現蛋白質,且轉染效果隨時間增加而增加,屬於緩慢釋放性質,符合非病毒載體適當製程、粒徑大小、低毒性、保護基因及基因表現等基本要求。
未來將有潛力經由顆粒表面修飾特定配位體有選擇性的遞送基因至
標的細胞,達到靶向效果,成為新的基因遞送系統選擇。
Abstract
In the post genomic era, gene therapy has become a major part of medical research. One of the major challenges of gene therapy is producing usable and reliable vectors to deliver the therapeutic gene. The vectors used for gene therapy need to deliver the therapeutic genes into the desired target tissues both efficiently and specifically. The aim of this study is producing novel non-viral calcium phosphate nanoparticles as a new vector for gene delivery. Calcium phosphate nanoparticles were prepared by water-in-oil microemulsion method with water to surfactant molar ratio, Wo = 2~10.
In this study, the particles size distribution of nanoparticles was determined by DLS and the morphology of nanoparticles was observed by TEM and FE-SEM. We report the design and synthesis of ultra-low size, highly monodispersed DNA doped calcium phosphate nanoparticles of size around 100 nm in diameter. The structure of DNA -calcium phosphate nanocomplex observed by TEM was displayed shell-like structure. In this study, we used pEGFP as reporter gene. The encapsulating efficiency to encapsulate DNA inside the nanoparticles was greater than 80%. In MTT test, both calcium phosphate nanoparticles and DNA-calcium phosphate nanocomplexes have no negative effect for 293T cells. By gel electrophoresis of free and entrapped pEGFP DNA, the DNA encapsulated inside the nanoparticles were protected from the external DNaseI environment. In vitro transfection studies in 293T cell-line, the DNA-calcium phosphate nanocomplex could be used safely to transfer the encapsulated DNA into the 293T cells and expression green fluorescent protein. The characteristic of DNA-calcium phosphate nanocomplexes to deliver DNA belongs to slow release. The properties of DNA-calcium phosphate nanocomplexes were fit in the requirement of non-viral vectors for gene delivery system.
In the future study, the surface of these nanoparticles can be suitably modified by some ligands to deliver gene to target cells under in vivo condition.
Calcium phosphate nanoparticles have potential to serve as efficient and
alternative DNA carriers for targeted delivery of genes.
目錄
摘要Ⅰ
英文摘要Ⅱ
目錄Ⅳ
圖目錄Ⅵ
表目錄Ⅷ
第一章 導論1
前言
1-1 奈米微粒3
1-1-1 奈米微粒基本特性3
1-1-2 奈米微粒於生物醫學應用-4
1-2 基因治療(Gene therapy)11
1-2-1 基因遞送系統11
1-2-2 病毒載體(Viral vector)12
1-2-3 非病毒載體(Non-viral vector)16
1-3 奈米微粒材料及製備方法選擇21
1-4 研究目的26
第二章 理論基礎28
2-1 微乳化系統 28
2-2 微胞幾何形狀33
2-3 二微乳化沉澱法36
第三章 實驗方法40
3-1 實驗儀器40
3-2 實驗藥品41
3-3 製備溶液和實驗材料42
3-4 實驗方法及流程43
3-4-1 實驗架構43
3-4-2 磷酸鈣奈米微粒製備45
3-4-3 DNA-磷酸鈣奈米微粒複合物製備47
3-5 材料分析與測試49
3-5-1 TEM分析49
3-5-2 FE-SEM分析49
3-5-3 DLS 粒徑分析50
3-5-4 DNA 包覆效率測試50
3-6 電泳分析52
3-7 MTT 測試54
3-8 體外細胞實驗56
3-8-1 DNA-磷酸鈣奈米微粒複合物轉染實驗56
3-8-2 磷酸鈣法56
3-8-3 微脂粒對照組57
第四章 結果與討論58
4-1 磷酸鈣奈米微粒製備58
4-1-1 DLS 粒徑分析和TEM 粒徑型態58
4-1-2 FE-SEM 粒徑型態59
4-1-3 材料定性分析63
4-1-4 製程條件對粒徑分佈影響66
4-2 DNA-磷酸鈣奈米微粒複合物的製備69
4-2-1 DLS 粒徑分析和TEM粒徑型態69
4-2-2 FE-SEM 粒徑型態69
4-2-3 製程條件對粒徑分佈影響73
4-2-4 載體型態76
4-3 成核模型83
4-4 分散安定性86
4-5 包覆效率87
4-6 電泳分析88
4-7 MTT 測試90
4-8 體外細胞實驗92
第五章 結論97
第六章 未來展望99
第七章 參考文獻100
圖目錄
圖1-1 於CdSe外層披覆ZnS並修飾peptid示意圖6
圖1-2 奈米微粒作為DNA探針研究示意圖7
圖1-3 基因治療技術關聯圖10
圖1-4 基因遞送系統的分類12
圖1-5 物理法及化學法製備奈米微粒示意圖23
圖1-6 逆微胞系統中製備奈米微粒的方法25
圖2-1 界面活性劑的分類29
圖2-2 AOT界面活性劑構造29
圖2-3 臨界微胞濃度之微胞形成示意圖31
圖2-4 球型微胞及逆微胞構造32
圖2-5 各類微胞幾何形狀32
圖2-6 界面活性劑構形及微胞幾何形狀關係34
圖2-7 排列成六邊形堆積的柱狀微胞34
圖2-8 AOT/hexane/water 20℃三元相圖35
圖2-9 二微乳化法製備奈米微粒示意圖37
圖2-10 二微乳化法中逆微胞間液滴與液滴的溶質交換機38
圖3-1 實驗架構之流程圖43
圖3-2 磷酸鈣奈米微粒製備之流程圖45
圖3-3 DNA-磷酸鈣奈米微粒複合物製備之流程圖47
圖3-4 電泳分析流程圖52
圖3-4 MTT原理示意圖55
圖4-1 磷酸鈣奈米微粒TEM照片及DLS粒徑分佈圖60
圖4-2 磷酸鈣奈米微粒FE-SEM圖(a)Wo=10(b)Wo=5 61
圖4-2 磷酸鈣奈米微粒FE-SEM 圖(c)Wo=3.5(d)Wo=2 62
圖4-3 TEM 選區繞射分析 64
圖4-4 EDS 元素分析 65
圖4-5 磷酸鈣奈米微粒於不同Wo 粒徑分佈峰和標準差比較 67
圖4-6 磷酸鈣奈米微粒平均粒徑和Wo 值關係 68
圖4-7 磷酸鈣奈米微粒TEM照片及DLS粒徑分佈圖70
圖4-8 DNA-磷酸鈣奈米微粒複合物FE-SEM 圖(a)Wo=10(b)Wo=5 71
圖4-8 DNA-磷酸鈣奈米微粒複合物FE-SEM圖(a)Wo=3.5(b)Wo=2 72
圖4-9 DNA-磷酸鈣奈米微粒複合物於不同Wo粒徑分佈峰和標準差比較74
圖4-10 DNA-磷酸鈣奈米微粒複合物平均粒徑和Wo值關係75
圖4-11 DNA-磷酸鈣奈米微粒複合物TEM型態78
圖4-12 DNA-磷酸鈣奈米微粒複合物於FE-SEM下表面型態80
圖4-13 DNA-磷酸鈣奈米微粒複合物於超音波震盪後TEM型態81
圖4-14 超音波震盪後DNA-磷酸鈣奈米微粒複合物TEM和DLS粒徑分析
82
圖4-15 DNA-磷酸鈣奈米微粒複合物微小粒子聚集的TEM型態-84
圖4-16 DNA-奈米微粒複合物成核模型 85
圖4-17 Wo=10、5、3.5、2包覆效率圖 87
圖4-18 電泳分析結果 89
圖4-19 MTT 測試結果 91
圖4-20 48小時蛋白質表現 94
圖4-21 48小時轉染率比較 95
圖4-22 DNA-磷酸鈣奈米微粒於72小時不同位置蛋白質表現結果 96
表目錄
表1-1奈米材料之特性 2
表1-2 基因遞送系統一覽表 20
表1-3 常用奈米微粒製備方式 22
表2-1 界面活性劑參數與聚集體結構 33
表4-1 磷酸鈣奈米微粒平均粒徑、標準差和粒徑分佈67
表4-2 DNA-磷酸鈣奈米微粒複合物平均粒徑、標準差和粒徑分佈峰比例表 74
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