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作者(中文):曾雅晴
作者(外文):Tseng, Ya-Ching
論文名稱(中文):具還原響應與酸鹼響應的含雙硫代馬來醯亞胺之雙性嵌端共聚物所形成之高分子液胞用於藥物控制釋放
論文名稱(外文):Reduction- and pH-Sensitive Polymersomes Formed from Dithiomaleimide-Linked AB2-Type Amphiphilic Block Copolymers for On-Demand Drug Release
指導教授(中文):彭之皓
指導教授(外文):Peng, Chi-How
口試委員(中文):陳俊太
陳韻晶
口試委員(外文):Chen, Jiun-Tai
Chen, Yun-Ching
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學系
學號:104023701
出版年(民國):107
畢業學年度:106
語文別:中文
論文頁數:86
中文關鍵詞:⾼分⼦液胞酸鹼響應還原響應
外文關鍵詞:polymersomepH-responsereduction-response
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⾼分子液胞 (polymersome) 為一種類似於脂質體的結構,藉由雙性高分子以親
水/疏水作用力 (hydrophilic-hydrophobic interaction) 於水相自組裝 (self-assemble) ,形成由雙層疏水薄膜包覆中央親⽔區域的載體,具有⾼機械強度與化學穩度性;⽽在藥物傳輸系統裡,高穩定性的高分⼦液胞往往存在低藥物釋放效率之問題。本研究設計AB2-type 之雙嵌端共聚物 PEG113-Mal-(PDPA30)2,自組裝形成高分⼦子液胞同時攜帶阿黴素 (doxorubicin, Dox),藉由酸鹼刺激響應 (pH-response) 與還原刺激響應 (reduction-response) 提升化療藥物之藥物釋放效率。此⾼分子系統以⾺來醯亞胺(malemide) 作為刺激響應之中⼼點,馬來醯亞胺之2 號位以及3 號位之碳硫鍵結與PDPA 疏⽔鏈段,分別具有還原響應與酸鹼響應;以此雙嵌段共聚物形成之高分子液胞攜帶阿黴素,以雙重刺激響應與單種刺激響應進⾏行阿黴素釋放,24 ⼩時阿黴素之累積釋放率於含有glutathione (GSH, 10 mM) 之 pH= 5.0 環境下為84.9%,而於未含有GSH (10 mM) 之 pH= 5.0 環境下為73.0%,顯⽰此高分子載體於雙重刺激響應以及單種刺激響應下,具有良好藥物釋放效率,且雙重刺激響應之藥物釋放效率更勝於單種刺激響應之藥物釋放效率。
Polymeric vesicles, also known as polymersomes, with the structure like liposome, are generally self-assembled from amphiphilic diblock copolymer to form an aqueous interior surrounded by a polymeric bilayer membrane, and exhibit good stability, such as mechanical and chemical stability. However, in drug release system, polymeric vesicles may have low drug release efficiency in cancer cells due to their compact structure. Herein, diblock copolymer of AB2-type PEG113-Mal-(PDPA30)2 had been developed to enhance the drug release efficiency by dual-stimuli. Maleimide was served as stimulative central moiety,
connecting hydrophilic poly ethylene glycol (PEG) and hydrophobic pH-sensitive poly(2-(diisopropylamino) ethyl methacrylate) (PDPA) segment, which is capable of carrying on structure disturbed by dually sensitized from pH-response and redox-response of PDPA segment and 2,3-position on maleimide. Enhanced drug release efficiency achieved up to 84.9% by dual-stimuli (pH = 5, GSH 10 mM) and 73.0% by single stimulus (pH = 5).
............................................................................................................................... I
Abstract ........................................................................................................................ II
謝誌............................................................................................................................. III
⽬目錄............................................................................................................................ IV
圖⽬目錄...................................................................................................................... VIII
表⽬目錄........................................................................................................................ XI
式⽬目錄....................................................................................................................... XII
附錄.......................................................................................................................... XIII
第⼀一章 ⽂文獻回顧......................................................................................................... 1
1-1 癌症 (Cancer) 與其治療⽅方式 .................................................................... 1
1-1-1 癌症的成因與⽣生物特性 .............................................................................. 1
1-1-2 癌症常⾒見之治療⽅方式 .................................................................................. 3
1-1-3 標靶藥物治療 (Targeted Therapy) ............................................................. 5
1-2 奈⽶米級藥物載體應⽤用於癌症治療 .............................................................. 7
1-2-1 增強通透與滯留效應 (Enhanced Permeability and Retention Effect, EPR)
V
...................................................................................................................... 7
1-2-2 腫瘤微環境 (Tumor Microenvironment) ................................................... 8
1-2-3 核內體逃脫 (Endosomal Escape) ............................................................. 10
1-3 刺激響應型⾼高分⼦子液胞奈⽶米載體 (Stimuli-Responsive Polymersomeric
Nanocarrier) ................................................................................................ 12
1-3-1 酸鹼響應型⾼高分⼦子液胞奈⽶米載體 (pH-Responsive Polymersomeric
Nanocarrier) ................................................................................................ 13
1-3-2 氧化還原響應型⾼高分⼦子液胞奈⽶米載體 (Redouction-Responsive
Polymersomeric Nanocarrier) ..................................................................... 15
1-3-3 其他響應型⾼高分⼦子液胞奈⽶米載體 (Other Responsive Polymersomeric
Nanocarrier) ................................................................................................ 16
1-4 2,3-⼆二溴⾺馬來醯亞胺 (2,3-Dibromomaleimide) ....................................... 18
1-4-1 可逆之蛋⽩白質⾼高效率選擇性 .................................................................... 18
1-4-2 功能性修飾之螢光現象 ............................................................................ 19
1-4-3 ⾼高分⼦子修飾 ................................................................................................ 20
1-5 原⼦子轉移⾃自由基聚合反應 (Atom Transfer Radical Polymerization,
ATRP) ......................................................................................................... 22
1-6 研究動機 .................................................................................................... 26
第⼆二章 實驗⽅方法與步驟........................................................................................... 27
2-1 AB2-type 雙嵌段共聚物之合成與鑑定 .................................................... 27
VI
2-1-1 聚⼄乙⼆二醇甲醚-疊氮化物 (PEG113-N3) 之合成 ....................................... 27
2-1-2 N-propargyl-2,3-dibromomaleimide 之合成 ............................................ 28
2-1-3 PEG113-Mal 藉由鍵擊反應之合成 ............................................................ 29
2-1-4 雙[2-(2'-溴代異丁醯氧基)⼄乙基]⼆二硫化物(Bis[2-(2'bromoisobutyryloxy)
ethyl] disulfide, BiBOEDS) 之合成 ........................................................ 30
2-1-5 以雙頭引發劑 (BiBOEDS) 合成聚甲基丙烯酸⼆二異丙氨基⼄乙酯
(PDPA30-SS-PDPA30, Poly(diisopropylaminoethyl-methacrylate)) ....... 31
2-1-6 聚甲基丙烯酸⼆二異丙氨基⼄乙酯 (PDPA30-SS-PDPA30) 以還原劑三苯基膦
(PPh3) 進⾏行雙硫鍵還原效率測試 .......................................................... 32
2-1-7 以加成-消去反應 (addition-elimination reaction) 合成AB2-type 之雙嵌
端共聚物 PEG113-Mal-(PDPA30)2 ........................................................... 33
2-2 PEG113-Mal-(PDPA30)2 之材料特性與刺激響應分析 .............................. 35
2-2-2 酸鹼性質滴定 ............................................................................................ 35
2-2-3 ⾼高分⼦子對酸鹼值之靈敏度測定 ................................................................ 35
2-2-4 ⾼高分⼦子對還原響應之動⼒力學測定 ............................................................ 36
2-2-5 以穀胱安肽進⾏行⾼高分⼦子之硫醇置換 ........................................................ 36
2-3 PEG113-Mal-(PDPA30)2 奈⽶米液胞製備與響應分析 .................................. 37
2-3-1 ⾼高分⼦子奈⽶米液胞製程 ................................................................................ 37
VII
2-3-2 ⾼高分⼦子奈⽶米液胞粒徑分析 ........................................................................ 37
2-3-3 ⾼高分⼦子奈⽶米液胞酸鹼響應分析 ................................................................ 38
2-3-4 ⾼高分⼦子奈⽶米液胞表⾯面電位量測 ................................................................ 38
2-4 以PEG113-Mal-(PDPA30)2 雙嵌段共聚物包覆及釋放阿黴素 ................. 39
2-4-1 製備包覆阿黴素之⾼高分⼦子奈⽶米液胞與分析 ............................................ 39
2-4-2 製作阿黴素於不同pH 磷酸緩衝液之減量線 ......................................... 40
2-4-3 包覆阿黴素之⾼高分⼦子奈⽶米液胞在酸鹼與還原響應下之釋放 ................ 41
第三章 結果與討論................................................................................................... 42
3-1 實驗設計 .................................................................................................... 42
3-2 PEG113-Mal-(PDPA30)2 之材料特性與刺激響應分析 .............................. 44
3-2-2 PEG113-Mal-(PDPA30)2 之酸鹼性質 .......................................................... 45
3-2-3 PEG113-Mal-(PDPA30)2 對於酸鹼性變化之靈敏度 .................................. 46
3-2-4 PEG113-Mal-(PDPA30)2 對於還原響應之動⼒力學測定 .............................. 49
3-2-5 PEG113-Mal-(PDPA30)2 與穀胱安肽之硫醇置換反應 .............................. 50
3-3 PEG113-Mal-(PDPA30)2 奈⽶米液胞之粒徑分析與刺激響應分析 ............ 51
3-3-1 AB2-type 之PEG113-Mal-(PDPA30)2 液胞性質與穩定性 ......................... 51
3-3-2 酸鹼值對PEG113-Mal-(PDPA30)2 液胞粒徑之影響 ................................. 53
VIII
3-4 以 PEG113-Mal-(PDPA30)2 奈⽶米液胞攜帶阿黴素 .................................... 56
3-4-1 包覆阿黴素之PEG113-Mal-(PDPA30)2 液胞 ............................................. 56
3-4-2 包覆阿黴素之PEG113-Mal-(PDPA30)2 液胞於不同酸鹼值下釋放阿黴素
之情形....................................................................................................... 58
3-4-3 包覆阿黴素之PEG113-Mal-(PDPA30)2 液胞於相同酸鹼值下添加穀胱安
肽釋放阿黴素之情形............................................................................... 59
3-5 結論 ............................................................................................................ 59
第四章 藥品與儀器................................................................................................... 60
第五章 參考⽂文獻....................................................................................................... 66
圖⽬目錄
圖1-1. 阿黴素引發細胞凋亡,與造成⼼心臟毒性之機制 .......................................... 4
圖1-2. 爾必得舒®之作⽤用機制 ................................................................................... 6
圖1-3. 增強通透與滯留效應 (EPR effect) ⽰示意圖 .................................................. 8
圖1-4. 腫瘤微環境⽰示意圖 ........................................................................................... 9
圖1-5. 核內體逃脫之⽰示意圖 .................................................................................... 10
IX
圖1-6. 質⼦子海綿效應 (proton sponge effect) 之⽰示意圖 ......................................... 11
圖1-7. PTTAMA 上之縮醛官能基因酸解使⾼高分⼦子液胞轉為親⽔水性⽰示意圖 ........ 14
圖1-8. 具有可離⼦子化之官能基為疏⽔水端包覆藥物,送於細胞內之途徑 ............ 14
圖1-9. 包覆阿黴素之⾼高分⼦子微胞以細胞內之榖胱⽢甘肽釋放阿黴素之途徑 ........ 16
圖1-10. 包覆阿黴素之⾼高分⼦子微胞以紫外光 (λ = 365 nm) 觸發藥物釋放之途
徑.................................................................................................................. 17
圖1-11. 2,3-⼆二溴⾺馬來醯亞胺之含硫蛋⽩白與硫醇置換效率⽰示意圖 ....................... 19
圖1-12. 以RAFT 進⾏行可控制之⾃自由基聚合的產物應⽤用 ...................................... 21
圖1-13. 以GSH 刺激含maleimide 之奈⽶米載體釋放CPT 之⽰示意圖 .................... 21
圖1-14. 原⼦子轉移⾃自由基聚合反應 (ATRP) 的反應機構 ..................................... 22
圖1-15. 利⽤用不同之引發劑與 CuIX/ TPMA (X = Br, Cl),於⼄乙腈下所測量原⼦子
轉移⾃自由基聚合反應之平衡常數.............................................................. 23
圖1-16. 利⽤用不同之含氮配位基與溴化亞銅形成錯合物,以α-溴異丁酸⼄乙酯
(EBiB) 作為引發劑,於⼄乙腈下所測量原⼦子轉移⾃自由基聚合反應之化
速率常數...................................................................................................... 24
圖1-17. 藉由原⼦子轉移⾃自由基聚合於⾼高分⼦子之活性位置進⾏行功能性化學修
飾.................................................................................................................. 25
X
圖2-1. PEG113-Mal-(PDPA30)2 之1H NMR (400 MHz, CDCl3) 圖譜 ...................... 34
圖2-2. PEG113-Mal-(PDPA30)2 之GPC 圖 ................................................................. 34
圖2-3. ⾼高分⼦子液胞之製程⽰示意圖 ............................................................................ 37
圖3-1. 同時運⽤用酸鹼響應與還原響應之藥物傳輸系統⽰示意圖 ............................ 43
圖3-2. ⾼高分⼦子構型與載體包覆阿黴素之⽰示意圖 .................................................... 43
圖3-3. 包覆芘之⾼高分⼦子液胞其I373/I383 對⾼高分⼦子濃度作圖 (λex= 337 nm) .......... 45
圖3-4. PEG113-Mal-(PDPA30)2 之滴定曲線 ............................................................... 46
圖3-5. 探討酸鹼性質響應與還原性質響應之對照組⽰示意圖。︒。 ............................ 48
圖3-6. 包覆芘之⾼高分⼦子液胞其 I383/I373 對 pH 值作圖 (λex= 337 nm) ............... 48
圖3-7. PEG113-Mal-(PDPA30)2 以苯硫酚進⾏行加成-消去反應之螢光光譜趨圖
...................................................................................................................................... 50
圖3-8. 以穀胱安肽10 mM 與(a) PEG113-Mal-(PDPA30)2 (b) PEG113-b-(PDPA40)2
溶於甲醇溶劑中進⾏行硫醇置換之GPC 圖 .................................................. 50
圖3-9. PEG113-Mal-(PDPA30)2 之粒徑⼤大⼩小與分散性 ............................................... 52
圖3-10. PEG113-Mal-(PDPA30)2 液胞粒徑於 pH = 7.4 之穿透性電⼦子顯微鏡影像 52
圖3-11. (a) ⾼高分⼦子液胞之粒徑與散射光強度隨著 pH 值變化情形 .................... 54
(b) ⾼高分⼦子液胞之散射光強度分佈隨著pH 值變化情形 ......................... 54
圖3-12. ⾼高分⼦子液胞之表⾯面電位隨著pH 值變化情形 .......................................... 55
XI
圖3-13. (a) 包覆阿黴素前後之⾼高分⼦子液胞粒徑分佈 ............................................. 57
(b) 包覆阿黴素之⾼高分⼦子液胞溶液與同濃度之阿黴素螢光光譜圖
(λex = 480 nm)。︒。 ............................................................................................ 57
圖3-14. 攜帶阿黴素之 PEG113-Mal-(PDPA30)2 ⾼高分⼦子液胞於 pH= 7.4、︑、6.0 以及
5.0 之磷酸緩衝溶液 (ionic strength = 0.14 M) 中釋放阿黴素之情形 ... 59
圖3-15. 攜帶阿黴素之 PEG113-Mal-(PDPA30)2 ⾼高分⼦子液胞於pH = 5.0 含有穀胱
安肽10 mM、︑、pH = 7.4 含有穀胱安肽10 μM 與pH = 7.4 的磷酸緩衝溶
液 (ionic strength = 0.14 M) 中釋放阿黴素之情形 ................................. 60
表⽬目錄
表2-1. PEG113-Mal-(PDPA30)2、︑、PDPA30-SS-PDPA30、︑、PDPA30-SS-Ph 及
PEG113-Mal 之分⼦子量及分⼦子量分佈指數 .................................................... 34
表3-1. 包覆阿黴素之PEG113-Mal-(PDPA30)2 ⾼高分⼦子液胞之粒徑、︑、粒徑分布、︑、藥
物包覆效率與藥物包覆含量.......................................................................... 57
XII
式⽬目錄
式2-1. 聚⼄乙⼆二醇甲醚-疊氮化物 (PEG113-N3) 之合成步驟 ................................... 28
式2-2. N-propargyl-2,3-dibromomaleimide 之合成步驟 ....................................... 29
式2-3. PEG113-Mal 之合成步驟 ............................................................................ 30
式2-4. 雙[2-(2′-溴代異丁醯氧基)⼄乙基]⼆二硫化物之合成步驟 ............................. 30
式2-5. 聚甲基丙烯酸⼆二異丙氨基⼄乙酯 (PDPA30-SS-PDPA30) 之合成步驟 ....... 31
式2-6. 聚甲基丙烯酸⼆二異丙氨基⼄乙酯之雙硫鍵還原測試⽰示意圖 ...................... 32
式2-7. AB2-type 之雙嵌端共聚物 PEG113-Mal-(PDPA30)2 之合成步驟 .............. 33
式 2-8. 藥物包覆效率之計算⽅方式與意義 ............................................................... 40
式 2-9. 藥物包覆含量之計算⽅方式與意義 ............................................................... 40
式 2-10. 累積釋放百分⽐比之計算⽅方式與意義 ......................................................... 41
XIII
附錄
附錄1. 聚⼄乙⼆二醇甲醚-疊氮化物之1H NMR (400 MHz, CDCl3) 圖譜 ................. 77
附錄2. 聚⼄乙⼆二醇甲醚-疊氮化物 (PEG113-N3) 之紅外線光譜圖 .......................... 77
附錄3. N-propargyl-2,3-dibromomaleimide 之1H NMR (400 MHz, CDCl3) 圖譜 . 78
附錄4. N-propargyl-2,3-dibromomaleimide 之紅外線光譜圖 .................................. 78
附錄5. PEG113-Mal 之1H NMR (400 MHz, CDCl3) 圖譜 ....................................... 79
附錄6. PEG113-Mal 之紅外線光譜圖 ........................................................................ 79
附錄7. 雙[2-(2′-溴代異丁醯氧基)⼄乙基]⼆二硫化物之1H NMR (400 MHz, CDCl3)
圖譜................................................................................................................ 80
附錄8. 聚甲基丙烯酸⼆二異丙氨基⼄乙酯之1H NMR (400 MHz, CDCl3) 圖譜 ....... 80
附錄9. 聚甲基丙烯酸⼆二異丙氨基⼄乙酯進⾏行還原斷鍵之GPC 圖 .......................... 81
附錄10. 阿黴素溶於⼆二甲基甲醯胺/四氫呋喃 (v/v=1/1) 之濃度對應其吸收強度
減量線.......................................................................................................... 81
附錄11. 阿黴素於不同pH 之磷酸緩衝液 (0.14 M) 的減量線 ............................ 81
附錄12. PEG113-Mal-(PDPA30)2 於甲醇溶劑 (10 mg/mL) 之紫外線吸收強度與螢
光放射強度.................................................................................................. 82
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