臺灣博碩士論文加值系統

本研究係利用一具有酸鹼應答行為、生物相容性及生物可降解性之Poly(D,L-lactide)-g-Poly(N-vinylimidazole-co-N-vinyl-2-pyrrolidone)(PLA-g-P(NVI-co-NVP))接枝共聚物以及具有免疫隱蔽性及生物可降解性之methoxy poly(ethylene glycol)-b-poly(D,L-lactide)(mPEG-PLA)二團聯共聚物，利用自我組裝設計出一同時具有環境酸鹼應答行為、可逆開關行為及免疫隱蔽性之接枝/複合型奈米微胞。mPEG可隱蔽內核結構的強正電性與疏水特性進而增加於體內循環時之穩定性與細胞吞噬量；NVI可藉由其酸鹼應答行為，待藥物載體被細胞吞噬後，可因正電排斥力造成結構的膨潤進而釋放藥物;PLA則具有包覆輸水抗癌藥物的能力。

研究中我們首先探討接枝型奈米微胞之形成機制並得到製備時的最佳化條件，而後我們依PLA-g-P(NVI-co-NVP)與mPEG-PLA臨界微胞濃度之不同，分別以不同重量比製備出各種組合之複合型奈米微胞，並從中篩選出較佳的組成進一步探討NVI本身之酸鹼應答行為對微胞型態之影響，結果指出接枝/複合型奈米微胞皆有良好的On-Off酸鹼應答行為。除此之外，我們更利用TEM與AFM等電子顯微鏡証實奈米微胞於不同環境下之核殼結構與表面型態變化。

藥物載體相關研究方面，我們將抗癌藥物doxorubicin包覆於PLA疏水內核，並利用免疫隱蔽性之外殼與具有酸鹼應答之特性進行「適時」、「適地」之藥物控制釋放。其中我們探討接枝/複合型藥物微胞之藥物包覆最適化條件(藥物包覆率高達約40%)，並觀察藥物載體於不同酸鹼環境下之On-Off藥物釋放情形，發現於pH5.0可得到快速且穩定的藥物釋放曲線；而於pH7.4時卻可將藥物確實地包覆於疏水內核。進一步地我們將藥物載體分別與Hs68、HeLa、HepG2等正常/癌細胞共同培養，結果指出於IC50附近，材料不具有任何毒性可言。最後，我們以共軛焦電子顯微鏡證實藥物載體於細胞酸性胞器內進行藥物制放，而後藉由擴散作用至細胞核內將細胞毒殺。

A novel mixed micelle comprised of poly(D,L-lactide)-g-poly(N-vinylimidazole-co-N-vinyl-2-pyrrolidone) (PLA-g-P(NVI-co-NVP)) graft copolymer with methoxy poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-PLA) diblock copolymer was successfully developed for application in cancer therapy. The mixed micelle had an biocompatibility, biodegradab, pH-triggered inner core of P(NVI-co-NVP)-g-PLA to enable intracellular drug delivery and an extended hydrophilic outer shell of mPEG to hide the inner core. PNVI in backbone of graft copolymer exhibited pH-triggered property, when pH<6.0, the nanoparticle swelled (about 50%) depend on composition of NVI in backbone but not deformed its conformation. Otherwise, the conformation went back to initial state when pH>6.0, if micelle capsulated hydrophobic anticancer drug, this phenomenon would be
called as “On-Off” controlled drug release.

In this study, we investigated the effect of mixed micelle with different critical micellar concentration (CMC) of diblock copolymer on comicellization. The results indicated that the CMC of diblock copolymer decreasing, the stability of mixed micelles increasing. Furthermore, the average size and polydipersity index(PI) of graft/mixed micelle can be measure by dynamic light scattering with the sample in phosphate buffer saline (PBS) at pH 7.4, it exhibited uniform size(about 70~100 nm) and narrow distribution(about 0.8~1.2).Besides, the core-shell structure of graft/mixed micelle at pH5.0 or pH7.4 can be prove by transmission electron microscopy (TEM).

The hydrophobic doxorubicin(DOX) was capsulated into the inner core of graft/mixed micelle by hydrophobic segment PLA for application in cancer therapy, and the capsulated efficiency can be as high as approximately 40 wt%. Graft/mixed micelle both exhibited high releasing rate in the initial 24 hr and the releasing behavior remained constant after 168 hr in the acidic surroundings(pH 5.0).Furthermore, there were rare initial burst releasing of graft/mixed micelle in neutral surroundings(pH 7.4). It means that the “On-Off” controlled drug release had successfully developed by altered pH value. In addition to above study, the efficiency of screening feature of mixed micelle can be distinguished from graft micelle in BSA/PBS stabe test and cytotoxicity, it means that mixed micelle exhibited better drug activity and lower material cytotoxitity. Finally, the free DOX and DOX-graft/mixed micelle distribution in cancer cell can be easily confirmed by confocal laser scanning microscopy(CLSM).

摘 要 i
Abstract ii
目 錄 iii
表 目 錄 vii
圖 目 錄 viii


第一章、研究背景與動機 1

第二章、文獻回顧 5
2-1、高分子組成單體之材料性質及其應用 5
2-1-1、poly(ethylene glycol)之性質與應用 5
2-1-2、Poly(D,L-lactide)之性質與應用 6
2-1-3、Poly(N-Vinylpyrrolidone)之性質與應用 8
2-1-4、Poly(N-Vinylimidazole)之性質與應用 9
2-2、高分子奈米微胞之介紹 11
2-2-1、高分子奈米微胞之形成機制 11
2-2-2、高分子奈米微胞之包覆原理 14
2-2-3、免疫隱蔽性奈米微胞 18
2-2-4、生物可降解型奈米微胞 20
2-2-5、酸鹼應答型奈米微胞 23
2-2-6、開關型奈米微胞 26
2-3、複合型奈米微胞之介紹 28
2-3-1、高分子高分子複合型奈米微胞 28
2-3-2、高分子微脂粒複合型奈米微胞 31
2-4、腫瘤組織構造及其與奈米藥物載體之傳遞行為 34
2-4-1、腫瘤組織構造與藥物傳遞之關係 34
2-4-2、奈米藥物載體之藥物傳遞機制 35
2-4-3、奈米藥物載體之細胞吞噬機制 38

第三章、實驗方法 42
3-1、實驗藥品 42
3-2、實驗裝置 44
3-3、名詞對照 45
3-4、酸鹼應答型接枝共聚物PLA-g-P(NVI-co-NVP)之合成 46
3-4-1、PLA-HEMA之合成 46
3-4-2、PLA-g-P(NVI-co-NVP)之合成 46
3-5、雙性二團聯共聚物mPEG-PLA之合成 47
3-6、共聚合物之結構鑑定與分析 48
3-6-1、1H-NMR結構鑑定與數目平均分子量鑑定 48
3-6-2、FT-IR鑑定 48
3-6-3、GPC分子量分佈鑑定 48
3-6-4、臨界微胞濃度(critical micelle concentration, CMC)之鑑定 49
3-7、接枝型奈米微胞之製備 50
3-8、複合型奈米微胞之製備 50
3-9、接枝/複合型奈米微胞之粒徑分析 51
3-10、接枝/複合型奈米微胞之界面電位分析 51
3-11、接枝型奈米微胞之高分子聚集行為分析 51
3-12、接枝/複合型奈米微胞之酸鹼應答行為分析 52
3-13、接枝/複合型奈米微胞之On-Off應答行為分析 52
3-14、接枝/複合型奈米微胞之殼核結構分析(TEM and AFM) 52
3-15、接枝/複合型奈米微胞之安定性分析 53
3-16、接枝/複合型奈米微胞之藥物包覆測試及性質分析 53
3-17、接枝/複合型奈米微胞之體外藥物釋放模擬分析 54
3-18、接枝/複合型奈米微胞之藥物On-Off應答行為分析 55
3-19、接枝/複合型奈米微胞之細胞存活率與細胞毒殺分析 55
3-20、接枝/複合型奈米微胞之細胞內藥物分佈情形與內吞行為分析 58

第四章、實驗結果與討論 60
4-1、酸鹼應答型接枝共聚物PLA-g-P(NVI-co-NVP)之製備與鑑定 60
4-2、二團聯共聚物mPEG-PLA之製備與鑑定 66
4-3、臨界微胞濃度(critical micelle concentration, CMC)之鑑定 69
4-4、接枝型奈米微胞之製備與鑑定 74
4-5、接枝型奈米微胞之聚集行為探討 77
4-6、複合型奈米微胞之鑑定與分析 84
(1) CMCGraft << CMCB1 86
(2) CMCGraft < CMCB2 89
(2) CMCGraft ≒ CMCB3 91
4-7、接枝/複合型奈米微胞之核殼型態分析 95
4-8、接枝/複合型奈米微胞之酸鹼應答行為分析 98
4-9、接枝/複合型奈米微胞之On-Off酸鹼應答行為分析 104
4-10、接枝/複合型奈米微胞之藥物包覆 114
4-11、接枝/複合型奈米微胞之體外藥物釋放模擬 118
4-12、接枝/複合型奈米微胞之安定性分析 123
4-13、接枝/複合型奈米微胞之體外細胞毒殺測試 125
4-15、接枝/複合型奈米微胞之細胞內藥物釋放及分佈測試 132

第五章、結論 138

第六章、參考文獻 144

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