臺灣博碩士論文加值系統

本篇論文主要探討，以化學改質的幾丁寡醣為起始材料，將聚合之單甲基聚己內酯(m-PCL)與甲氧基聚乙二醇(m-PEG)分子鏈分別與幾丁寡醣之羥基反應接枝(Graft)，而形成聚己內酯-幾丁寡醣-聚乙二醇(PCL-g-COS-g-PEG, PCP) 接枝共聚合物，利用直接形成法(Direct dissolution method)製備成為PCP微胞(Micelle)。以不同處理方法對粒子表面進行修飾，並探討其對奈米粒子特性與對細胞標的化能力及藥物釋放之影響。
結果顯示，本研究製備之PCP聚合物形成微胞之臨界膠體濃度(Critical Micelle Concentration, CMC)為0.0107 wt % ( ~ 1.0 μ M)。微胞之尺寸分佈約為40 - 120 nm。表面電位值會隨著PH值而改變，於PH7.4~PH3.0，測量結果為-3.23±1.26∼13.87±4.41mv。另外，由於PCP結構中含有COS，利用COS上含有的胺基，對此一特性對微胞加以應用研究。其一：於微胞形成後利用自由胺基做化學交聯(Cross-link)探討微胞包覆Doxorubicin(DOX)於不同PH值做藥物釋放測試；其二：在自由胺基上接枝具貼附細胞之因子– (Arg-Gly-Asp, RGD)，作細胞uptake之試驗。其結果顯示：1.透過化學交聯後所形成之交聯PCP微胞，在PH7.4、37℃條件下結果顯示交聯較未交聯之微胞於24小時內有有抑制突釋現象，從37.6 % 降至22.0 %，並且有效延緩釋放從96小時(73.4 %) 至192小時(69.4 %)；而於PH5.0時仍可延緩釋放至96小時(70.8%)。2.將鍵結上RGD之RGD-PCP微胞利用PC-12細胞作uptake試驗，結果發現有鍵結上RGD之微胞明顯的比一般的PCP微胞被PC-12細胞uptake為快。在本研究中所發展的PCP微胞結構中，保留許多的自由胺基，此官能基可以提供了許多不同的功能需求，而此形式微胞於藥物釋放系統中將有助於藥物傳遞的穩定性以及藥物之標地(Target)功能。

A new tri-block amine-group containing copolymer and micelle consisting of poly (��-caprolactone) - b - chitooligosaccharide - b – poly (ethylene glycol) (PCL-b-COS-b-PEG, PCP), was synthesized and characterized for applications of drug delivery. The characteristics of the PCP copolymer were investigated by Fourier-transform infrared spectrometry (FT-IR) to examine the containing amine and ester groups of COS and PCL of the copolymer, respectively, 1H nuclear magnetic resonance (1H NMR) to investigate the structure of PCP copolymer (e.g., PCL and PEG blocks grafted onto COS block), and gel permeation chromatography (GPC) to determine number average molecular weight of the block copolymer (Mn), 11340 Da/mole. The PCP copolymer can self-assemble to form micelles at the critical micelle concentration (CMC) of 0.0107 wt % (or ~ 1.0 μ M) determined by the UV-VIS absorption spectra. The mean diameter of the PCP micelles is 90 nm, as measured by a dynamic light-scattering (DLS) analyzer. Since the PCP micelles containing amine groups, the copolymer grafted with Arg-Gly-Asp (RGD) tri-peptide to form a RGD-PCP copolymer and then characterizes the micelle behaviors of the copolymer. The PCP-RGD micelles are uptake by PC-12 cells much faster than PCL-g-PEG and PCP ones. Moreover, the stability and release the release of doxorubicin from PCP micelles with genipin crosslinked is sustained(e.g., 8 days) longer than the micelles without crosslinked(e.g., 4 days) and micelles reported by other investigators. In conclusion, a new tri-block amine group containing PCP copolymer is synthesized that can self-assemble to form micelles which might be suitable for applications in drug delivery.

目錄
§第一章 緒論
§1-1材料簡介：…………………………………………………………………… 1
§1-1-1聚己內酯(Poly ε-carprolactone, PCL)………………………………………1
§1-1-2-1幾丁寡醣(chitosan-oligosaccharide) …………………………………………2
§1-1-2-2 幾丁物質於生物醫學上之應用…………………………4
§1-2-1藥物控制釋放傳遞系統………………………………………………………5
§1-2-2.奈米粒子的表面改質…………………………………………………………6
§1-3-1 聚合物微胞……………………………………………………………………9
§1-3-2 微胞的結構與型態………………………………………………………… 10
§1-3-3 微胞於醫學上之應用………………………………………………………12
§1-4精胺酸-甘胺酸-天門冬胺酸(RGD)……………………………………………14
§1-5奈米粒子搭載抗癌藥DOX之應用……………………………………………15
§1-6天然交聯劑-綠梔子素(genipin)…………………………………………………17
§2-1研究動機與實驗目的………………………………………………………… 18
§第二章實驗設備與方法
§2-1實驗藥品………………………………………………………………………19
§2-2 實驗儀器………………………………………………………………………22
§2-3 實驗整體流程…………………………………………………………………24
§2-4實驗方法和步驟………………………………………………………………25
§2-4-1 PCP共聚物材料合成………………………………………………………26
§2-4-1-1幾丁寡醣之改質(Pth-COS)…………………………………………………27
§2-4-2-1單甲基聚己內酯之聚合 …………………………………………………28
§2-4-2-2 m-PCL-NCO預聚合………………………………………28
§2-4-3 m-PEG-NCO預聚合………………………………………28
§2-4-4 PCL-g-Pth-COS-g-PEG接枝聚合…………………………30
§2-4-5 N-Phthaloylation還原…………………………………………….…………32
§2-4-6 PCP純化………………………………………………… 33
§2-5 PCP 材料性質分析
§2-5-1 核磁共振光譜 …………………………………………33
§2-5-2-1紅外線光譜………………………………………………………………34
§2-5-3-1凝膠滲透層析…………………………………………35
§2-6 微胞之製備與分析………………………………………………………….37
§2-6-1 PCP聚合物微胞製備……………………………………37
§2-6-2 臨界微胞濃度分析………………………………………37
§2-6-3 PCP聚合物微胞粒徑分析…………………………………………………38
§2-6-3-1 雷射粒徑分析儀…………………………………………………………38
§2-6-4 聚合物微胞表面電位量測…………………………………………………39

§2-6-5 TEM拍攝聚合物微胞………………………………………………………40
§2-7 PCP微胞之化學交聯………………………………………42
§2-7-1 以未交聯及交聯PCP微胞包覆DOX於不同PH值下作體外釋放……42
§2-7-2生醫工程之應用--- PC-12細胞標的化測定………………………………44
§2-7-2-1製備RGD-PCP micelles…………………………………………………44
§2-7-2-2微胞於PC-12細胞標的化測定…………………………………………46
§第三章 結果與討論
§3-1-1 PCP共聚物官能基鑑定……………………………………………………48
§3-1-1-2 以磁共振光譜儀分析……………………………………………………50
§3-1-1-2 以凝膠滲透層析分析分子量……………………………………………56
§3-2-1 臨界膠體濃度(CMC)分析…………………………………………………57
§3-2-2 以雷射粒徑儀量測其平均粒徑…………………………………………….59
§3-2-3表面電位 (zeta potential)分析………………………………………………59
§3-3 PCP微胞之化學交聯…………………………………………………………62
§3-3-2 RGD-PCP微胞細胞uptake試驗……………………………………………66
§3-3-2-1以及HPLC測試接枝率……………………………………………………66
§3-3-2-2以及FTIR對官能基作鑑定…………………………………………………66
§3-3-2-3 RGD-PCP微胞細胞uptake實驗……………………………………………67
















表索引
表3-1-1-2-1 COS Proton………………………………………………51
表3-1-1-2-2 PCP Proton……………………………………………….53
表3-1-1-2 Pth-COS、COS-PCL。PCP 分子量………………………56













圖索引

圖1-1-1 PCL/PEO/PCL之奈米微胞結構示意圖……………………………………2
圖1-1-2 CS coating PCL奈米粒子對epithelium貼附之細胞螢光圖…………………2
圖1-1-3(a) 幾丁質……………………………………………………………………3
圖1-1-3(b) 幾丁聚醣…………………………………………………………………3
圖1-2-1血小板與內皮細胞中常見之接受器(receptor)示意圖………………………8
圖1-3-1聚合物微胞示意圖…………………………………………………………10
圖1-3-2不同溶液系統中微胞之結構………………………………………………11
圖1-3-3EPR效應示意圖……………………………………………………………13
圖1-4 RGD tri-peptide amino acid sequences…………………………………………14
圖1-5-1 doxorubicin結構式…………………………………………………………15
圖1-5-2 DOX在體內不同部位，時間對濃度的分佈圖……………………………16
圖1-6 Genipin的分子結構式………………………………………………………18
圖2-4-1-1 m-PCL-NCO之反應式…………………………………………………27
圖2-4-2-2 m-PCL-NCO之反應式…………………………………………………29
圖2-4-2-3PEG-NCO之反應式…………………………………………………… 29
圖2-4-4-2PEG-Pth-COS-PCL之反應式………………………………………….……31
圖2-4-5 N-Phthaloylation幾丁寡醣之還原反應…………………………………….32
圖2-5-3-1 多孔性凝膠粒子層析管柱的分離效果…………………………………36
圖2-6-5 負染色原理說明圖…………………………………………………………40
圖2-7-1為包覆DOX微胞交聯示意圖………………………………………………44
圖2-7-2 RGD-PCPmicelles反應示意圖………………………………………………45
圖3-1-1-1以2 cm-1解析度掃描16次之ATR-FTIR分析圖………………………49
圖3-1-1-2-1COS之13C-1H NMR圖譜…………………………………………… 51
圖3-1-1-2-2 PCP之13C-1H NMR圖譜…………………………………………… 52
圖3-1-1-2-3 COS 1H NMR圖譜………………………………………………… 54
圖3-1-1-2-4 PCP 1H NMR圖譜……………………………………………………55
圖3-2-1-1不同濃度PCP聚合物加入DPH 水溶液之UV-VIS圖譜……………57
圖3-2-1-2合物CMC值量測圖……………………………………………………58
圖3-2-2 -1P micelles粒徑分佈圖PDI=0.35)………………………………………59
圖3-2-2-2下微胞之表面電位 (n=3)………………………………………………60
圖3-2-2-310倍CMC值濃度形成微胞TEM圖(X120K)…………………………62
圖3-3-1以genipin交聯及未交聯PCP微胞包覆DOX………………………… 64
圖3-3-1-2PH7.4、5.0、4.0、3.0下包含DOX之未交聯之PCP micelles累積釋圖…65
圖3-3-1-3 PH7.4、5.0、4.0、3.0下包含DOX之genipin交聯之PCP micelles累積釋放圖………………………………………………………………………………… 66.
圖3-3-2-2PCP與RGD-PCP copolymer FTIR分析圖……………………………… 67
圖3-3-2-3 PC-12細胞. Uptake of RGD-PCP micelles…………………………………68
圖3-3-2-4 PC-12細胞uptake RGD-PCP螢光微胞之螢光強度變化………………69

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