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研究生:陳美瑾
研究生(外文):Mei-Chin, Chen
論文名稱:膠原蛋白包覆Sirolimus之藥物釋放型血管支架研究
指導教授:宋信文
指導教授(外文):Hsing-Wen Sung
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:56
中文關鍵詞:血管支架藥物釋放生物可分解膠原蛋白
外文關鍵詞:stentdrug releasebiodegradablecollagen
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冠狀動脈硬化為心血管系統最常見的疾病,臨床上有數種不同的治療方法,其中最有效的就屬經皮冠狀動脈擴張術(PTCA)。但是大約有30~50﹪的病患,在PTCA手術後3至6個月內,會發生血管再狹窄的病變。血管再狹窄的原因,主要是由於PTCA手術後血管回彈(recoil)與血管內膜的過度增生(hyper-proliferation)所致。為了防止擴張後血管的回彈,PTCA搭配血管支架(stent)的置放可達到良好的效果。至於血管內膜的過度增生方面,本研究擬以生物相容性佳的膠原蛋白(collagen),包覆抗血管內膜增生的藥物(sirolimus)並塗佈至血管支架上,進行藥物的控制釋放,以有效的抑制血管內膜的過度增生、降低血管再狹窄的機率。
本研究主要分為三部分。第一部分為膠原蛋白塗佈實驗,主要目的為尋找膠原蛋白均勻塗佈於支架上的最佳製程,並分析塗佈後其表面形態、均勻度、塗佈厚度與對支架之附著力。確定塗佈膠原蛋白之製程後,我們以天然交聯劑genipin來交聯膠原蛋白,並尋找其最適當的交聯條件,使交聯後的膠原蛋白仍能維持一定的完整性與交聯度。最後,將交聯後的collagen-coated stent進行膨脹(stent expansion)測試。第二部分為包藥釋放實驗,採多層式噴塗法(multi-layer spray coating,膠原蛋白層與sirolimus層交替噴塗),主要目的為以膠原蛋白高分子包覆抗血管內膜增生藥sirolimus,再以genipin進行交聯,製備一新型的藥物釋放型血管支架(drug-eluting stent)。最後於PBS中進行in vitro的藥物釋放實驗,探討不同包藥量(low dose ~30 mg/stent;high dose ~65 mg/stent)及包藥模式(有無阻礙層, with or without topcoat)之藥物釋放動力學情形。第三部分為血液相容性與抗酵素分解實驗,前者之目的為探討不同交聯程度及接枝肝素(heparin)的膠原蛋白之血液相容性;後者為探討不同交聯程度的膠原蛋白,其抗酵素分解的能力。
第一部分的塗佈結果顯示,於室溫下噴塗(spray coating)弱酸性之膠原蛋白溶液(pH~4),可達到最佳的膠原蛋白附著率、均勻度與塗佈後的平整性。在定量分析方面,發現膠原蛋白的噴塗量與塗佈厚度間,呈現一線性關係。此外,由原子力顯微鏡(AFM)的結果顯示,噴塗的膠原蛋白與支架表面之附著力,至少大於1500 nN(已達AFM探針的偵測極限)。為了使交聯後的膠原蛋白能維持一定的完整性與交聯度,又要避免交聯時膠原蛋白因過度吸水膨潤而變形,我們選擇在含有5﹪genipin之70﹪酒精中進行交聯。最後,我們以氣球導管擴張術將交聯後的collagen-coated stent由直徑1.5 mm膨脹至直徑2.6 mm,結果並沒有發生膠原蛋白剝落或分離的情形,進一步證實膠原蛋白與支架表面有相當好的附著力。
在第二部分的實驗裡,我們利用多層式噴塗法,以少量多層的方式分別將膠原蛋白及sirolimus噴塗至支架上,再噴塗含有5% genipin之70% 酒精溶液進行交聯。由in vitro的藥物釋放結果顯示,高劑量且沒有添加阻礙層的藥物釋放型血管支架可持續地釋放sirolimus至少達14天。此外,由低劑量支架的釋放結果來看,with topcoat的包藥模式能延緩藥物的釋放速率,降低burst release的情形發生。
第三部分血液相容性的實驗結果顯示,經肝素接枝及交聯程度達65%以上的膠原蛋白,具有較佳的血液相容性。由抗酵素分解的實驗結果顯示,隨著交聯程度的增加,膠原蛋白抗酵素分解的能力也越好。綜合以上的結果顯示,經genipin交聯處理的collagen-coated stent,很有潛力發展成一良好的藥物釋放型血管支架。
Background: Many clinical trails have shown that drug-eluting stents are effective in reducing restenosis. These stents are commonly coated with nondegradable polymers as a drug reservoir. However, as the drug is completely eluted, the permanent presence of the nondegradable polymer may induce unfavorable effects. The study was to develop a novel drug eluting stent with a biodegradable collagen coating. Preparation of this stent and its in vitro characteristics and drug release profiles are reported.
Methods: A spray coating process was developed to prepare the stent coated with collagen. The force required to remove the collagen coating from the metal stent was investigated by AFM (atomic force microscopy) and a balloon-expansion device. Collagen and sirolimus were sprayed layer-by-layer alternatively onto the external surface of the stent and subsequently crosslinked by genipin, a naturally occurring crosslinking agent. The in vitro hemocompatibility and degradability of the collagen coatings with distinct degrees of crosslinking were evaluated (n = 5). Four forms of the sirolimus-loaded stents were examined in an in vitro elution experiment: a low dose of ~30 mg per stent (0.78 mg/mm2) with or without topcoat; and a high dose of ~65 mg per stent (1.70 mg/mm2) with or without topcoat (n = 3).
Results: The results obtained in the AFM examination and the balloon expansion test demonstrated that the collagen coating adhered tightly to the stent surface. The hemocompatibility and the resistance against enzymatic degradation of the genipin-crosslinked collagen increased significantly as its degree of crosslinking increased. The results of the in vitro drug elution study showed that release of sirolimus from the stent without topcoat was able to maintain more than 14 days. The drug release study for the stent with topcoat is currently underway.
Conclusions: The aforementioned results indicated that the drug eluting stent developed in the study has a tightly adhered multi-layer collagen coating and can be used as a drug reservoir to sustain release of sirolimus.
內容 頁數
摘要 I
英文摘要 III
目錄 IV
圖索引 VII
表索引 IX

第一章 緒論
1.1 前言 1
1.2 冠狀動脈疾病 1
1.3 經皮冠狀動脈血管擴張術(PTCA) 1
1.4 冠狀動脈血管支架(stent) 2
1.5 藥物釋放型血管支架(drug-eluting stent) 3
1.6 膠原蛋白(collagen) 6
1.7 交聯處理 6
1.8 Sirolimus(Rapamune) 8
1.9 研究動機與目的 9

第二章 膠原蛋白塗佈實驗
2.1 研究目的 13
2.2 膠原蛋白溶液之配製 13
2.3 膠原蛋白之塗佈方式 13
2.3.1 沉浸塗佈(dip coating) 14
2.3.2 噴塗(spray coating) 14
2.4 膠原蛋白塗佈之定量 16
2.4.1 膠原蛋白塗佈厚度之定量 17
2.4.2 膠原蛋白塗佈附著力分析 17
2.5 膠原蛋白之交聯方式 19
2.5.1 交聯指數(fixation index)分析21
2.6 支架膨脹(stent expansion)測試21
2.7 膠原蛋白塗佈方式之結果 23
2.7.1 沉浸塗佈(dip coating)結果 23
2.7.2 噴塗(spray coating)結果 24
2.8 膠原蛋白塗佈之定量結果 28
2.8.1 膠原蛋白塗佈量vs. 塗佈厚度之分析 28
2.8.2 附著力分析 28
2.9 交聯膠原蛋白之結果 31
2.9.1 交聯後之表面形態、塗佈厚度與附著力變化33
2.10支架膨脹(stent expansion)測試之結果34
2.11結論-35

第三章 藥物釋放實驗
3.1 研究目的--36
3.2 包藥方式--36
3.2.1 混合噴塗包藥法--36
3.2.2 多層式(multi-layer)噴塗包藥法--37
3.3 體外釋放藥物動力學實驗--37
3.4 包藥方式之可行性評估-38
3.4.1 混合噴塗包藥法--38
3.4.2 多層式(multi-layer)噴塗包藥法-39
3.5 體外釋放藥物動力學結果-40
3.6 結論---40
第四章 血液相容性與抗酵素分解實驗
4.1 研究目的---42
4.2 製備不同交聯程度的膠原蛋白膜----42
4.3 血液相容性實驗----------42
4.4 抗酵素分解28天(enzymatic degradation)---44
4.5 交聯指數分析結果-------46
4.6 血液相容性實驗結果-----46
4.7 抗酵素分解結果------47
4.8 結論------48

參考文獻----51
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