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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 | Li-Ting Kao | en |
dc.contributor.author | 高立庭 | zh_TW |
dc.date.accessioned | 2021-06-07T17:51:31Z | - |
dc.date.copyright | 2013-03-04 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-09-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15759 | - |
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LT6-FITC接枝至乳酸-甘醇酸-聚乙二醇二胺聚合物或奈米顆粒上。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 nm以下的奈米顆粒。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、SKOV3)與未過度表現的細胞株(HepG2、A549)進行奈米顆粒的吞噬實驗,並以共軛焦顯微鏡的實驗結果作對照。在藥物的包覆實驗方面,本實驗選用杜薩魯比辛(Doxorubicin)與太平洋紫杉醇(Paclitaxel)作為包覆的藥物。利用聚乳酸-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-LT6胜肽接枝聚合物以單一乳化法(Single emulsion method),於不同的藥物比例進行未標靶杜薩魯比辛奈米顆粒、未標靶紫杉醇奈米顆粒、未標靶雙重藥物奈米顆粒與LT6胜肽標靶雙重藥物奈米顆粒的製備,並以高效液相層析儀(High-Performance Liquid Chromatography, HPLC)與連續式多功能微孔盤偵測系統分析藥物的包覆率。此外,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。最後再進行細胞存活率試驗(MTT Assay),了解藥物與含藥奈米顆粒對EGFR過度表現的SKOV3細胞株之毒殺效果。 實驗結果顯示,在聚乳酸-甘醇酸-聚乙二醇二胺的實驗中,當聚乳酸-甘醇酸活化物與聚乙二醇二胺比例調升至1:5,聚乙二醇二胺的接枝率最佳,可達99.8%。而在LT6-FITC的接枝實驗方面,在當實驗條件為pH7擁有最高的LT6-FITC接枝率,可達88.29%。 在流式細胞儀的細胞吞噬實驗中可以發現,聚乳酸-甘醇酸-聚乙二醇二胺-LT6-FITC標靶奈米劑型在2、24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,其中在24小時的實驗,聚乳酸-甘醇酸-聚乙二醇二胺-LT6-FITC標靶奈米劑型在SKOV3與MDA-MB-468細胞株的細胞吞噬皆可達90%以上,但其對於EGFR未過度表現的細胞株並無如此顯著的效果。 至於在含藥奈米顆粒的製備,本實驗所製備出的奈米顆粒粒徑約在100 nm至200 nm之間,隨著藥物負載率的增加,粒徑有逐漸縮小的趨勢。而在藥物體外釋放方面,在pH4.0時奈米顆粒中藥品的釋放較pH7.4迅速,其中杜薩魯比辛的釋放速度又比紫杉醇快。細胞存活率實驗結果顯示,雙重藥物組的毒殺效果較使用單一藥物理想,而本實驗所製備的LT6胜肽標靶雙重藥物奈米顆粒相較於單一或雙重藥物組與未標靶雙重藥物奈米顆粒具有較佳的細胞毒殺效果,且多具有顯著差異。由本實驗的結果可以證實,LT6胜肽標靶奈米顆粒對於EGFR過度表現的細胞具有特殊標靶能力,且在遞送抗癌藥物至EGFR過度表現的細胞也具有良好的效果。 | 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 find a simple way 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 LT6-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 and confocal fluorescence microscope to perform cellular uptake study in EGFR overexpression cancer cell lines (e.g., MDA-MB-468 and SKOV3) and EGFR non-overexpression cell lines (e.g., HepG2 and A549). We chose doxorubicin and paclitaxel as the model anticancer drugs, and the doxorubicin and/or paclitaxel loaded non-targeted and LT6 targeted nanoparticles were prepared. The high-performance liquid chromatography (HPLC) and fluorescence spectroscopy were applied to analyze the dug loading in nanoparticles and in vitro drug release in pH4.0 and pH7.4 release media. Finally, we used MTT study to investigate the SKOV3 cell viability of drugs and nanoparticles. In PLGA-PEG synthesis, the PEG conjugation ratio was increased to 99.8% 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-LT6-FITC, and the LT6-FITC conjugation efficiency was around 88.29%. For the cellular uptake study, PLGA-PEG-LT6-FITC-NP had a better target ability to EGFR overexpression cancer cell lines than EGFR non-overexpression cell lines. In addition, PLGA-PEG-LT6-FITC-NP had higher cellular uptake than the PLGA-PEG-FITC-NP in EGFR overexpressed cell lines after incubation for 2 and 24 hours, but not in EGFR non-overexpression cell lines. The particle size of the drug loaded nanoparticles were around 100~ 200 nm and it decreased when the drug loading increased. The drug release in pH4.0 release medium was faster than in pH7.4 release medium, and the release of doxorubicin was faster than the release of paclitaxel. In cell viability MTT study, the doxorubicin and paclitaxel free drugs combination had lower cell viability than doxorubicin or paclitaxel free drug only. LT6 targeted dual drug-loaded nanoparticles had lower cell viability than free drugs and non-targeted drug-loaded nanoparticles in EGFR overexpression cell line. All of these results demonstrated that the LT6 targeted nanoparticle showed a promising targeting ability to EGFR overexpression cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-07T17:51:31Z (GMT). No. of bitstreams: 1 ntu-101-R99423006-1.pdf: 5300902 bytes, checksum: 5d48eb04fdc1698782515a2c09b965ee (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 口試委員會審定書 i
致謝 ii 中文摘要 iii Abstract v 表目錄 xii 圖目錄 xv 第一章 緒論 1 一、 聚乳酸-甘醇酸(Poly(lactide-co-glycolide), PLGA) 1 (一) 聚乳酸-甘醇酸基本結構與與降解機轉 2 (二) 聚乳酸-甘醇酸的合成方式 4 (三) 聚乳酸-甘醇酸奈米顆粒製備方法 5 (四) 聚乳酸-甘醇酸奈米顆粒之聚乙二醇修飾 6 二、 奈米劑型之遞送策略 7 (一) 被動型標的(passive targeting) 7 (二) 主動型標的(active targeting) 8 三、 表皮生長因子受體(epidermal growth factor receptor, EGFR) 10 四、 LT6胜肽與表皮生長因子受體 11 五、 紫杉醇(paclitaxel)與杜薩魯比辛(doxorubicin)的臨床使用與研究 15 第二章 試劑與材料介紹 21 一、 聚乳酸-甘醇酸 (Poly(lactide-co-glycolide), PLGA) 21 二、 聚乙二醇二胺(Poly(ethylene glycol) bis(amine), PEG diamine) 22 三、 LT6胜肽 22 四、 杜薩魯比辛(Doxorubicin) 24 五、 太平洋紫杉醇(Paclitaxel) 26 六、 焦油腦(Pyrene) 27 七、 MTT試劑 28 第三章 實驗動機與目的 29 第四章 實驗試劑與儀器 30 一、 藥品 30 二、 細胞實驗材料 32 三、 儀器 34 四、 耗材 36 五、 藥品溶液及緩衝溶液之配製 36 第五章 實驗方法 38 一、 PLGA-PEG之合成 40 (一) PLGA之活化產物製備 (Milane et al., 2011) 40 (二) PLGA-PEG之合成 (Milane et al., 2011) 41 二、LT6-FITC之細胞株篩選實驗 42 三、PLGA-PEG接枝LT6-FITC之合成與相關奈米劑型的製備 43 (一) PLGA-PEG接枝LT6-FITC之合成 43 (二) PLGA-PEG與PLGA-PEG接枝LT6-FITC奈米顆粒的製備 45 (三) PLGA-PEG奈米顆粒接枝LT6-FITC之劑型製備 47 四、 PLGA-PEG接枝LT6-FITC奈米顆粒之細胞吞噬實驗 48 五、 PLGA-PEG接枝LT6-FITC奈米顆粒與PLGA-PEG接枝NR7-FITC混和奈米顆粒之細胞吞噬實驗 49 六、 PLGA-PEG接枝LT6-FITC奈米顆粒細胞吞噬實驗之共軛焦螢光顯微鏡分析 51 七、 結構及物性測定 52 (一) 核磁共振光譜(NMR) 52 (二) 紅外線分光光譜儀(FT-IR) 52 (三) 分子量評估-膠體滲透層析(GPC) 52 (四) 臨界微膠粒濃度(CMC)測試 (Song et al., 2011) 53 (五) 奈米粒子的物性測試 54 (六) 奈米顆粒之穿透式電子顯微鏡拍攝 54 八、 包覆抗癌藥物之奈米劑型的製備 55 (一) 杜薩魯比辛鹽酸鹽脫鹽反應(Kim et al., 2008) 55 (二) 包覆抗癌藥物之PLGA-PEG與PLGA-PEG-LT6奈米劑型的製備 55 (三) 奈米顆粒中杜薩魯比辛與紫杉醇的定量方法(Ahmed et al., 2006; Wang et al., 2011; Xu et al., 2009) 56 九、 藥物體外釋放實驗 59 十、 細胞存活率試驗 64 第六章 實驗結果 69 一、聚乳酸-甘醇酸接枝聚乙二醇二胺之合成(PLGA-PEG合成) 69 (一) PLGA-NHS之製備 69 (二) PLGA-PEG之製備 72 二、 LT6-FITC之細胞株篩選實驗 81 三、 聚乳酸-甘醇酸-聚乙二醇二胺與聚乳酸-甘醇酸-聚乙二醇二胺接枝LT6-FITC之奈米顆粒製備 82 (一) PLGA-PEG接枝LT6-FITC之合成 82 (二) PLGA-PEG與PLGA-PEG接枝LT6-FITC奈米顆粒的製備 87 (三) PLGA-PEG奈米顆粒接枝LT6-FITC之劑型製備 91 四、 聚乳酸-甘醇酸-聚乙二醇二胺-LT6-FITC奈米顆粒之細胞吞噬實驗 92 五、 混和PLGA-PEG接枝LT6-FITC奈米顆粒與PLGA-PEG接枝NR7-FITC奈米顆粒之細胞吞噬實驗 97 六、 PLGA-PEG接枝LT6-FITC奈米顆粒細胞吞噬實驗之共軛焦螢光顯微鏡分析 99 七、 包覆抗癌藥物之奈米劑型的製備 102 (一) 杜薩魯比辛定量精確度試驗 102 (二) 紫杉醇定量精確度試驗 105 (三) 包覆抗癌藥物之PLGA-PEG與PLGA-PEG-LT6相關奈米劑型的製備 108 (四) 藥物體外釋放實驗 112 (五) 含藥標靶奈米顆粒之細胞存活率試驗 129 第七章 討論 151 一、 PLGA-PEG的合成實驗 151 (一) 核磁共振光譜(NMR)與紅外線分光光譜儀(FT-IR)之結構分析 151 (二) 接枝率、產率、分子量分析 152 (三) 臨界微膠粒濃度(CMC)之分析 153 二、 LT6-FITC之細胞吞噬實驗 153 三、 PLGA-PEG-LT6-FITC的合成與PLGA-PEG-NP接枝LT6-FITC的實驗 154 (一) PLGA-PEG-LT6-FITC接枝聚合物的合成 154 (二) PLGA-PEG-NP接枝LT6-FITC之奈米顆粒的製備 155 四、 PLGA-PEG與衍生奈米劑型之製備與分析 155 (一) PLGA-PEG奈米顆粒之條件測試 155 (二) PLGA相關衍生物之奈米顆粒製備 156 五、 標靶奈米劑型之細胞吞噬實驗 157 (一) LT6標靶奈米劑型之細胞吞噬實驗 157 (二) PLGA-PEG-LT6-FITC-NP與PLGA-PEG-NR7-FITC-NP混和奈米顆粒之細胞吞噬實驗 159 六、 包藥奈米劑型之製備與分析 160 (一) 紫杉醇與杜薩魯比辛藥物定量 160 (二) 包藥奈米劑型之分析與探討 161 七、 杜薩魯比辛與紫杉醇之體外釋離實驗 163 八、 藥物奈米劑型之細胞毒殺試驗 166 第八章 結論 171 參考文獻 174 | |
dc.language.iso | zh-TW | |
dc.title | 應用聚乳酸-甘醇酸接枝聚乙二醇二胺於奈米標的載體之研究 | zh_TW |
dc.title | Studies on Active Targeting Nano-carriers Prepared from Poly(lactide-co-glycolide) Conjugated Poly(ethylene glycol) bis(amine) | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃耀斌(Yaw-Bin Huang),廖嘉鴻(Jia-Horng Liaw) | |
dc.subject.keyword | 胜肽,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,杜薩魯比辛,紫杉醇, | zh_TW |
dc.subject.keyword | Peptide ligand,nanoparticle,PLGA-PEG,epidermal growth factor receptor,doxorubicin,paclitaxel, | en |
dc.relation.page | 183 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2012-09-13 | |
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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