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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林文貞(Wen-Jen Lin) | |
dc.contributor.author | Wei-Hsuan Chien | en |
dc.contributor.author | 簡瑋萱 | zh_TW |
dc.date.accessioned | 2021-06-16T08:16:18Z | - |
dc.date.available | 2019-02-25 | |
dc.date.copyright | 2014-02-25 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-02-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58470 | - |
dc.description.abstract | 近年來,具有生物可分解性與生物可相容性的高分子材料在生醫領域與藥物遞送上的研究不勝枚舉。其中聚乳酸-甘醇酸(Poly(lactide-co-glycolide), PLGA)因為其無毒性的性質與在人體中良好的降解特性,已被廣泛的應用於醫學或藥學領域,而其相關衍生物的合成、藥物劑型製備的研究因此備受矚目。
本實驗以聚乳酸-甘醇酸作為藥物載體的骨架,在聚乳酸-甘醇酸上接枝聚乙二醇二胺(poly(ethylene glycol) bis(amine), PEG diamine)進行化學結構的修飾,並以核磁共振儀(1H-NMR)、傅立葉轉換紅外線光譜儀(FTIR)、膠體滲透層析儀(GPC)確認其化學結構與分子量。再利用EDC-NHS 活化的方式,將對表皮生長因子受體(Epidermal growth factor receptor, EGFR)有標靶(target)能力的胜肽配體GE11(2R)-FITC 接枝至乳酸-甘醇酸-聚乙二醇二胺聚合物上。在本實驗中,奈米顆粒的製備皆是以溶媒揮發法(Solvent evaporation method)將聚合物製備成粒徑200 -300 nm 左右的奈米顆粒。接下來,除了對此接枝胜肽的奈米顆粒作物性探討之外,更進一步的利用流式細胞儀對於EGFR 表現量較高的細胞株(MDA-MB-468、MDA-MB-231)與低表現量的細胞株(MCF-7)進行奈米顆粒的吞噬實驗。本實驗利用質體DNA帶負電荷的特性,先和帶正電荷的1,2-二油酰基-3-三甲基銨丙烷(1,2-dioleoyl-3-trimethylammonium-propane,DOTAP)以聚電解質複合反應(Polyelectrolyte complexation)形成奈米複合體,再將此複合體以單一乳化法(Single emulsion method)包覆於聚乳酸-甘醇酸-聚乙二醇二胺聚合物與聚乳酸-甘醇酸-聚乙二醇二胺-GE11(2R)胜肽接枝聚合物奈米顆粒中,並以PicoGreen利用螢光分析質體DNA的包覆率。本實驗還進行藥物在pH4.0 與pH7.4 的體外釋放試驗(In vitro drug release study),以確認奈米顆粒中藥物的釋放模式。最後再測試包覆質體DNA的奈米顆粒於癌細胞的轉染效率。 實驗結果顯示,在聚乳酸-甘醇酸-聚乙二醇二胺的接枝實驗中,當聚乳酸-甘醇酸活化物與聚乙二醇二胺莫耳比例為1:5,聚乙二醇二胺的接枝率最佳,可達99.92%。而在GE11(2R)-FITC 的接枝實驗方面,當實驗條件為pH7 擁有最高的GE11(2R)-FITC 接枝率,可達83.20 ± 7.48%。 以流式細胞儀偵測奈米顆粒的細胞吞噬實驗,聚乳酸-甘醇酸-聚乙二醇二胺-GE11(2R)-FITC 標靶奈米劑型在2小時的吞噬實驗中,相較於未標靶的奈米顆粒,其對於EGFR 過度表現的癌細胞具有較佳的標靶能力,在MDA-MB-231與MDA-MB-468 細胞株的細胞吞噬皆可達86%以上,但其對於EGFR 未過度表現的細胞株則無如此顯著的效果。 將奈米複合體形式質體DNA包覆於聚乳酸-甘醇酸-聚乙二醇二胺-GE11(2R)胜肽接枝聚合物奈米顆粒中,其製備的奈米顆粒粒徑為147 ± 15.5 nm。在體外釋放試驗(In vitro drug release study)方面,奈米顆粒在pH4.0環境釋放較pH7.4快速。而pDNA/DOTAP loaded PLGA-PEG-GE11(2R)-NP於癌細胞轉染效率較pDNA/DOTAP loaded PLGA-PEG-NP轉染效果為佳。經由實驗結果顯示,GE11(2R)胜肽標靶奈米顆粒對於EGFR高度表現的細胞具有顯著的標靶能力,且在遞送基因至EGFR高度表現的細胞具有良好的轉染效果。 | zh_TW |
dc.description.abstract | There are many studies on application of polymeric materials in biomedical and drug delivery recently. Among them, poly(lactide-co-glycolide) (PLGA) has been widely applied in medical science and pharmaceutics for its nontoxic and biodegradable characteristics.
The PLGA was used as the main polymer in our drug delivery system. This experiment was desinged to conjugate poly(ethylene glycol) bis(amine) with PLGA. The chemical structure and molecular weight of PLGA-PEG were confirmed by using nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC). The synthesized PLGA-PEG was further conjugated with GE11(2R)-FITC as an epidermal growth factor receptor (EGFR) targeting ligand. The nanoparticles were prepared using solvent evaporation method and the physical properties of nanoparticles were characterized. Furthermore, we used flow cytometer to perform cellular uptake study in EGFR overexpressed cancer cell lines (e.g., MDA-MB-468 and MDA-MB-231) and EGFR low-expressed cell line (e.g.,MCF-7). Since the negatively charged characteristic of plasmid DNA, it was complexed with positively charged DOTAP via polyelectrolyte complexation before loaded in the GE11(2R) peptide ligand conjugated nanoparticles. The transfection of pDNA/DOTAP loaded PLGA-PEG-GE11(2R)-NP was performed in EGFR overexpressed cancer cell lines. In PLGA-PEG synthesis, the PEG conjugation ratio was increased to 99.92% at the molar ratio of PLGA-NHS: PEG diamine 1:5. On the other hand, pH 7 was the best condition to synthesize PLGA-PEG-GE11(2R)-FITC, and the GE11(2R)-FITC conjugation efficiency was around 83.20 ± 7.48%. For the cellular uptake study, PLGA-PEG-GE11(2R)-FITC-NP had a better targeting ability to EGFR overexpressed cancer cell lines than EGFR low-expressed cell line. In addition, PLGA-PEG-GE11(2R)-FITC-NP had higher cellular uptake than the PLGA-PEG-FITC-NP in EGFR overexpressed cell lines after incubation for 2 hours, but not in EGFR low-expressed cell line. The possible mechanism was through clathrin-mediated endocytosis and macropinocytosis. The particle size of pDNA/DOTAP loaded PLGA-PEG-GE11(2R) nanoparticles were 147.0 ± 15.5 nm. The pDNA released from nanoparticles in pH 4.0 release medium was faster than in pH 7.4 release medium. These results demonstrated that the PLGA-PEG copolymer was degraded by the lipase and hydrolyzed in the acid buffer. It was implied that pDNA was stably encapsulated in the PLGA-PEG-GE11(2R) nanoparticles during systemic circulation (pH7.4) and released from PLGA-PEG-GE11(2R) nanoparticles after internalized into EGFR overexpressed cells. The pDNA/DOTAP loaded PLGA-PEG-GE11(2R) nanoparticles were then transfected in EGFR overexpressed cell lines and had higher transfection efficiency than pDNA/DOTAP loaded PLGA-PEG nanoparticles. All of these results suggested that GE11(2R) peptide conjugated PLGA-PEG nanoparticles could be a biocompatible and functionalized nanocarrier for gene delivery application. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T08:16:18Z (GMT). No. of bitstreams: 1 ntu-103-R00423015-1.pdf: 5053754 bytes, checksum: e2dfe6bca3f31c39fa178340b5cae4a7 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 口試委員會審定書 I
致謝 II 中文摘要 III Abstract V 目錄 VII 表目錄 XI 圖目錄 XIII 第一章 緒論 1 一、 基因療法(Gene Therapy) 1 (一) 基因療法載體 1 (二) 正電性分子載體 7 二、 奈米劑型之遞送策略 8 (一) 被動型標的 8 (二) 主動型標的 9 三、 聚乳酸-甘醇酸(Poly(lactide-co-glycolide), PLGA) 11 四、 聚乳酸-甘醇酸奈米顆粒之聚乙二醇修飾 11 五、 表皮生長因子受體(epidermal growth factor receptor, EGFR) 13 六、 GE11(2R)胜肽與表皮生長因子受體 15 第二章 試劑與材料介紹 17 一、 聚乳酸-甘醇酸 (Poly(lactide-co-glycolide), PLGA) 17 二、 聚乙二醇二胺(Poly(ethylene glycol) bis(amine), PEG diamine) 19 三、 GE11(2R)胜肽 20 四、 質體DNA(plasmid DNA pEGFP-N1) 22 五、 1,2-二油酰基-3-三甲基銨丙烷(1,2-dioleoyl-3-trimethylammonium-propane (chloride salt),DOTAP) 23 第三章 實驗動機與目的 24 第四章 實驗試劑與儀器 25 一、 藥品 25 二、 Plasmid DNA純化實驗 27 三、 Plasmid DNA定量實驗材料 28 四、 細胞實驗材料 28 五、 儀器 29 六、 耗材 31 七、 藥品溶液及緩衝溶液之配製 31 第五章 實驗方法 34 一、 PLGA-PEG 之合成 36 (一) PLGA 之活化(Milane et al., 2011) 36 (二) PLGA-PEG 之合成 (Milane et al., 2011) 37 二、 細胞株EGFR 表現量 (expression level) 篩選實驗(Klutz et al., 2011) 41 三、 GE11(2R)-FITC 之細胞株篩選實驗(高立庭, 2012) 42 四、 PLGA-PEG 接枝GE11(2R)-FITC 之合成與相關奈米劑型的製備 43 (一) PLGA-PEG 接枝GE11(2R)-FITC之合成(高立庭, 2012) 43 (二) PLGA-PEG-FITC與PLGA-PEG-GE11(2R)-FITC物性分析(高立庭, 2012) 45 (三) PLGA-PEG 與PLGA-PEG 接枝GE11(2R)-FITC奈米顆粒的製備(高立庭, 2012) 47 (四) 奈米顆粒的物性測試 48 (五) 奈米顆粒之穿透式電子顯微鏡拍攝 48 (六) PLGA-PEG-NP之安定性試驗(Fillafer et al., 2009) 48 五、 PLGA-PEG 接枝GE11(2R)-FITC奈米顆粒之細胞吞噬實驗(高立庭, 2012) 49 六、 PLGA-PEG-GE11(2R)-FITC 奈米顆粒進入細胞之途徑探討 50 Endocytosis inhibiton assay (Qhattal and Liu, 2011) 50 七、 包覆基因複合體之奈米劑型的製備 51 (一) 基因複合體的製備(Du et al., 2011) 51 (二) 洋菜凝膠電泳分析法(Ozgel and Akbuga, 2006) 52 (三) 包覆基因複合體之PLGA-PEG 與PLGA-PEG-GE11(2R)奈米劑型的製備(劉佳雯, 2012) 53 (四) 奈米顆粒的物性測試 53 (五) 奈米顆粒之穿透式電子顯微鏡拍攝 53 (六) 奈米顆粒中質體DNA的定量方法(Tahara et al., 2011) 54 (七) 包覆基因複合體之奈米顆粒中質體DNA完整性測試 55 (八) 紅血球凝集試驗(Hattori et al., 2013) 56 八、 質體DNA的體外釋放實驗 57 (一) 基因體外釋放實驗pDNA含量定量 57 (二) 質體DNA的體外釋放實驗(高立庭, 2012; 劉佳雯, 2012) 58 九、 包覆基因複合體之奈米劑型的細胞轉染實驗(Tahara et al., 2011) 62 十、 包覆基因複合體之PLGA-PEG 與PLGA-PEG-GE11(2R)奈米劑型細胞轉染實驗之共軛焦螢光顯微鏡分析(徐婉貽, 2012) 63 十一、 統計方法 63 第六章 實驗結果 64 一、 聚乳酸-甘醇酸接枝聚乙二醇二胺之合成(PLGA-PEG之合成) 64 (一) PLGA-NHS 之製備 64 (二) PLGA-PEG 之製備 67 二、 細胞株EGFR 表現量 (expression level) 篩選實驗 81 三、 GE11(2R)-FITC 之細胞株篩選實驗 83 四、 PLGA-PEG-GE11(2R)-FITC 之合成與相關奈米劑型的製備 84 (一) PLGA-PEG-GE11(2R)-FITC之合成 84 (二) PLGA-PEG 與PLGA-PEG-GE11(2R)-FITC奈米顆粒的製備 89 五、 奈米顆粒的細胞吞噬實驗 102 六、 PLGA-PEG-GE11(2R)-FITC 奈米顆粒進入細胞之途徑探討 107 七、 包覆基因複合體之奈米劑型的製備 109 (一) 基因複合體的製備 109 (二) 洋菜凝膠電泳分析法 110 (三) 包覆基因複合體之PLGA-PEG 與PLGA-PEG-GE11(2R)奈米劑型的製備 112 (四) 包覆基因複合體之奈米顆粒中質體DNA完整性測試 115 (五) 紅血球凝集試驗(Hattori et al., 2013) 117 八、 質體DNA的體外釋放實驗 118 (一) 基因體外釋放實驗pDNA含量定量 118 (二) 質體DNA的體外釋放實驗 120 九、 包覆基因複合體之奈米劑型的細胞轉染實驗 125 十、 包覆基因複合體之PLGA-PEG 與PLGA-PEG-GE11(2R)奈米劑型細胞轉染實驗之共軛焦螢光顯微鏡分析 127 第七章 討論 129 第八章 結論 147 第九章 參考文獻 151 | |
dc.language.iso | zh-TW | |
dc.title | 胜肽配體結合聚乳酸-甘醇酸接枝聚乙二醇二胺作為主動標的奈米載體之潛力 | zh_TW |
dc.title | The potential of peptide conjugated PLGA-PEG as an active targeting nanocarrier | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 沈麗娟,李美賢 | |
dc.subject.keyword | 胜?標靶,奈米顆粒,聚乳酸-甘醇酸-聚乙二醇二胺,表皮生長因子受體,細胞轉染, | zh_TW |
dc.subject.keyword | Peptide ligand,nanoparticle,PLGA-PEG,epidermal growth factor receptor,pDNA,transfection, | en |
dc.relation.page | 157 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-02-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥學研究所 | zh_TW |
顯示於系所單位: | 藥學系 |
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