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研究生:曾詣文
研究生(外文):I-WEN TZENG
論文名稱:奈米幾丁聚醣藥物載體之製備及應用研究
論文名稱(外文):Preparation and Application of Nano Chitosan Drug Carriers
指導教授:凃耀國凃耀國引用關係
指導教授(外文):YAWO-KUO TWU
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
中文關鍵詞:幾丁聚醣藥物載體噴霧乾燥藥物釋放
外文關鍵詞:chitosandrug carriersspray dryingdrug release
相關次數:
  • 被引用被引用:3
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本研究目的在製備出奈米級的幾丁聚醣藥物載體,首先將市售幾丁聚醣溶於鹽酸中,並調整成適當之酸鹼值,乾燥後便得可溶於水的幾丁聚醣鹽酸鹽。再將幾丁聚醣鹽酸鹽混和不同濃度之架橋劑-綠梔子素與消炎止痛藥-待克菲那配製成溶液,進行噴霧乾燥得到奈米幾丁聚醣/待克菲那藥物載體,並對產物作粒徑及物性分析、細胞毒性、穿透實驗及藥物釋放之評估。
由場發射電子顯微鏡觀察,各樣本平均粒徑約在300 nm。利用富立葉紅外線光譜儀偵測發現,樣本中含有藥物時,在1456 cm-1處出現特徵吸收,且當架橋劑濃度增加時,在1540 cm-1之胺基吸收峰減小,顯示幾丁聚醣鹽酸鹽顆粒中有藥物待克菲那的存在,與架橋劑有架橋反應生成。將藥物載體與腸道上皮細胞(Caco-2)進行細胞毒性及穿透評估,發現藥物載體在濃度 100 ug/mL以下對Caco-2 細胞無明顯毒性。而由TEER實驗結果發現,樣本D有較佳的穿透能力達50 %,若樣本保存不善造成吸濕,顆粒變大則無法順利通過細胞間隙,細胞經過trypan blue染色後發現,樣本並未對細胞造成傷害。由藥物釋放研究發現,在前1小時小於50 %附著於表面的待克菲那會先被釋放出來,且隨時間增長藥物濃度也逐漸上升,因此確認幾丁聚醣藥物載體具有緩釋藥物之能力。
The objective of this study was to prepare nano chitosan drug carriers. First, chitosan was dissolved in HCl and the pH of solution was adjusted. Chitosan hydrochloride was obtained after dehydration. The solutions of chitosan-HCl with diclofenac sodium and genipin were prepared. Spray-drying was used to prepare nano chitosan drug carriers containing diclofenac sodium. To evaluate characteristics of these carriers, FESEM, cytotoxicity, permeability, and drug release tests were conducted.
The FESEM indicated that the size of carriers had an average size of 300 nm. The FTIR indicated that the samples appeared characteristic absorption peak when containing drugs. When increased the concentration of cross-linking agent in these carriers, the IR absorption peak of amide was decrease. MTT data showed that these carriers with a concentration below 100 ug/mL were non-toxic to Caco-2 cells. In TEER results, sample D has a penetration greater than 50 %. When samples containing moisture, aggregation happened and the size of samples become larger. Hence samples can’t penetrate cell junction successfully. The results of trypan blue assay showed that samples won't harm the cells. The drug release behaviors showed that less than 50 % diclofenac sodium could be release in one hour. With prolonging releasing time, the drug concentration was increasing. Chitosan drug carriers have potential in the application of controlled release.
封面內頁
簽名頁
授權書iii
中文摘要iv
英文摘要v
誌謝vi
目錄vii
圖目錄x
表目錄xii

1. 研究目的1
2. 文獻回顧2
2.1 幾丁質與幾丁聚醣簡介2
2.2 幾丁聚醣鹽類2
2.3 架橋劑綠梔子素(genipin)簡介4
2.4 待克菲那(diclofenac sodium)簡介7
2.5 幾丁聚醣於藥物控制釋放9
2.5.1 幾丁聚醣藥物載體的特性11
2.6 奈米化藥物載體13
2.6.1 噴霧乾燥法14
2.7 口服藥物傳輸模式16
2.7.1 Caco-2單層膜細胞16
2.7.2 幾丁聚醣物質於腸道穿透之研究17
2.8 細胞毒性評估20
2.8.1 細胞毒性試驗方法20
2.9 藥物釋放評估22
2.9.1 分光光度計偵測法22
2.9.2 HPLC定量法22
3. 材料與方法23
3.1 實驗架構23
3.2 實驗材料24
3.2.1 藥品24
3.2.2 耗材25
3.2.3 儀器設備26
3.2.4 細胞株27
3.3 研究方法28
3.3.1 幾丁聚醣鹽酸鹽製備28
3.4 幾丁聚醣鹽酸鹽藥物載載體製備28
3.4.1 噴霧乾燥法製備奈米藥物載體30
3.5 產物分析30
3.5.1 場發射電子顯微鏡(FESEM)30
3.5.2 富立葉紅外線光譜儀試驗(FTIR)31
3.6 細胞毒性之研究31
3.6.1 細胞復甦31
3.6.2 繼代培養32
3.6.3 MTT毒性試驗34
3.7 細胞穿透之研究34
3.7.1 TEER試驗35
3.7.2 Trypan blue細胞存活率試驗35
3.8 藥物釋放試驗37
3.8.1 標準品配製37
3.8.2 藥物釋放分析37
4. 結果與討論38
4.1 幾丁聚醣鹽酸鹽及載體之製備38
4.2 場發射電子顯微鏡觀察39
4.3 富立葉紅外線光譜儀試驗(FTIR)52
4.4 細胞試驗57
4.4.1 Caco-2細胞培養觀察57
4.4.2 細胞毒性試驗(MTT分析)58
4.4.3 Tranepithelial electrical resistance(TEER)分析59
4.4.4 Trypan blue存活率試驗61
4.5 藥物釋放試驗68
5. 結論74
參考文獻76

圖目錄

圖2.1 幾丁質及其衍生物之結構式3
圖2.2 幾丁聚醣與綠梔子素架橋後之結構式5
圖2.3 待克菲那之化學結構7
圖2.4 藥物釋放模式9
圖2.5 藥物釋放濃度與時間關係圖10
圖2.6 細胞間接合示意圖18
圖2.7 MTT反應機制21
圖3.1 實驗流程圖23
圖3.2 細胞電阻測定儀36
圖4.1 樣本A之(a)FESEM圖、(b)粒徑分析41
圖4.2 樣本B之(a)FESEM圖、(b)粒徑分析42
圖4.3 樣本C之(a)FESEM圖、(b)粒徑分析43
圖4.4 樣本D之(a)FESEM圖、(b)粒徑分析44
圖4.5 樣本E之(a)FESEM圖、(b)粒徑分析45
圖4.6 樣本F之(a)FESEM圖、(b)粒徑分析46
圖4.7 樣本G之(a)FESEM圖、(b)粒徑分析47
圖4.8 樣本A之表面結構圖48
圖4.9 樣本B ~ G之表面結構圖49
圖4.10 七種樣本之平均粒徑圖50
圖4.11 三個月後樣本G之形態與表面結構圖51
圖4.12(a)待克菲那與(b)架橋劑之FTIR圖譜53
圖4.13(a)待克菲那與(b)架橋劑之FTIR圖譜(指紋區)54
圖4.14 各樣本之FTIR圖譜55
圖4.15 各樣本之FTIR圖譜(1000 ~ 1800 cm-1)56
圖4.16 Caco-2細胞培養於flask之觀察62
圖4.17 各樣本MTT試驗(n=4)63
圖4.18 Caco-2細胞培養於transwell insert之觀察64
圖4.19 4種樣本之TEER試驗65
圖4.20 各樣本之TEER試驗66
圖4.21 Trypan blue試驗之鏡檢圖67
圖4.22 待克菲那之檢量線69
圖4.23 幾丁聚醣鹽酸鹽藥物載體之藥物釋放70
圖4.24 樣本B、C、D之藥物釋放百分比71
圖4.25 樣本E、F、G之藥物釋放百分比72

表目錄

表2.1 幾丁聚醣與架橋劑6
表2.2 幾丁聚醣包覆待克菲那之相關研究8
表2.3 幾丁聚醣製成藥物載體之方式與粒徑大小12
表2.4 噴霧乾燥法製備幾丁聚醣微粒15
表2.5 幾丁聚醣物質對於Caco-2單層膜細胞電阻值下降之影響19
表3.1 樣本之組成29
表3.2 Caco-2細胞之培養基33
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