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研究生:許馨尹
研究生(外文):Hsin-Yin Hsu
論文名稱:以超高壓均質技術製備奈米微脂粒應用於藥物載體系統之評估
論文名稱(外文):Developing nanosomes as drug delivery systems using ultra high-pressure homogenization
指導教授:梁弘人梁弘人引用關係
指導教授(外文):Hong-Jen Liang
學位類別:碩士
校院名稱:元培醫事科技大學
系所名稱:食品科學研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
畢業學年度:103
語文別:中文
論文頁數:107
中文關鍵詞:奈米微脂粒超高壓均質機藥物載體系統微脂粒
外文關鍵詞:NanosomeUltra-high pressure homogenizationDrug delivery systemsLiposome
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本研究以簡單價廉之磷脂質磷脂醯膽鹼 (phosphatidylcholine, PC)和膽固醇 (cholesterol, Chol)為基本配方,發展最佳化超高壓均質技術 (Ultra high pressure homogenization method, UHPH)方法製備NS,評估作為高效率藥物載體及疫苗用佐劑應用之可行性。本研究針對UHPH最佳化配方之NS分別進行物理穩定性試驗 (儲存穩定性、粒徑、包覆率、生物相容性試驗)以及以NS作為佐劑包覆或吸附BSA (bovine serum albumin)抗原進行動物免疫試驗,評估NS作為蛋白質包覆載體之佐劑效果。穩定性結果顯示NS在三種不同的溫度 (4°C、25°C、37°C),具有高穩定性。以雷射散射法粒徑測定儀測定NS之粒徑平均約為59.92 nm 。而冷凍電子顯微鏡圖顯示多數NS為單層小微脂粒 (small unilameller vesicles, SUV)結構。在蛋白質及胜肽藥物包覆效率方面 ,NS包覆不同藥物 (Amylase、GHK-Cu、BSA)後,經由SDS-PAGE電泳分析NS對不同蛋白質及胜肽的包覆效率。結果顯示NS對不同蛋白質及胜肽Amylase、GHK-Cu、BSA,包覆率分別為63~75.2%、48~58.1%、86.59%,顯著高於超音波震盪製備方式之微脂粒。長期儲存釋放能力試驗結果顯示將NS儲存於冰箱4℃,儲存一個月後NS對BSA包覆率為87.9%、儲存二個月後包覆率為85.3%,而以傳統製備方式之微脂粒包覆BSA儲存一個月後,包覆率降為0%。生物相容性結果顯示BV2神經膠質細胞以MTT染色法來檢測NS 其細胞存活率達到95%以上。NS與紅血球混合1小時後,紅血球細胞未見破壞。NS包覆與吸附疏水性6-coumarin (螢光劑)後,證實無法被細胞吞噬之6-coumarin 可藉由NS包覆或吸附的方式帶入細胞內,而以包覆方式其螢光之情形較為明顯,其細胞吞噬效果較佳。最後,動物免疫試驗中以NS包覆與吸附BSA後注射小鼠,二、四週後其產生之血清抗體比單一BSA組可引起較高免疫反應,證實其簡單PC 與Chol配方之NS具有優良佐劑之效果。本研究結果顯示利用UHPH製備之NS具有相當大的潛力作為可應用於大量製備之藥物載體及疫苗佐劑。
The present study was to formulate a novel liposome in nanoscale (nanosome, NS) based on simple materials such as phosphatidylcholine (PC) and cholesterol (Chol) as a novel drug-delivery system and vaccine adjuvant using ultra-high pressure homogenization method (UHPH). The present study evaluated the efficacy of optimized formulated NS as a protein-like drugs delivery system through the physical stability tests (including storage stability, particle size distribution, encapsulation efficiency, biocompatibility tests), as well as the adjuvanticity of NS-encapsulated or adsorbed BSA (bovine serum albumin) antigen in the animal vaccination test. The physical stability results showed that NS stored at different temperatures (4 ℃, 25 ℃, 37 ℃) exhibited high stability compared to other formulated liposomes. The results of laser scattering measurement showed that the average particle size of NS was approximately 59.92 nm. The cryo-electron microscopy image showed most of NS with single small vesicles (small unilameller vesicles, SUV) structure. The results of protein and peptide-like drug encapsulated efficiency showed that the encapsulated efficiencies of NS encapsulated different protein or peptide such as amylase, GHK-Cu, and BSA were 63~75.2%, 48~58.1%, 86.59%, respectively through SDS-PAGE electrophoresis, significantly higher than that of conventional liposome prepared by ultrasonic method. In addition, The encapsulation efficiency of NS encapsulated with BSA stored at 4 ℃ for one month and two months was 87.9% and 85.3%, respectively, while that of conventional liposome was reduced to 0%. Biocompatibility results showed 95% survival rate of BV2 microglia cells ain the presence of NS through MTT assay. No significant damage was found in red blood cells after mixed with NS for one hour. NS encapsulated or adsorbed hydrophobic compound 6-coumarin (fluorescent agents) which are not endocytosed by cells, have been observed in BV2 cells through endocytosis. The NS encapsulated with 6-coumarin was showed more profound in BV2 cells than that of NS adsorbed with 6 coumarin. The results of animal vaccination experiments showed that mice vaccinated with NS-encapsulated or absorbed BSA exhibited higher serum antibody response 2 and 4 weeks after vaccination than that of vaccinated BSA alone, suggesting excellent adjuvanticity of NS composed of simple PC and Chol. The present study provided considerable potential of NS as a drug carriers and vaccine adjuvant which can be mass-manufactured with low cost.
致 謝 I
摘 要 II
Abstract IV
目錄 VI
圖 目 錄 IX
表 目 錄 XI
附 錄 XII
第一章、前言 1
1.1藥物載體系統 1
1.2微脂粒 (Liposome) 5
1.2.1 Liposome起源 5
1.2.2 Liposome的物理結構 6
1.2.3 Liposome的穩定性 9
1.2.4 Liposome作為載體系統之應用 10
1.2.5 Liposome製備方式 15
1.3奈米微脂粒 (nanosome, NS) 24
第二章、研究目的 26
第三章、材料與方法 27
3.1實驗材料與動物 27
3.2實驗方法 31
3.2.1 NS製備 31
3.2.2 NS物理穩定性試驗 34
3.2.3 NS包覆能力試驗 37
3.2.4 NS生物相容性試驗 43
3.2.5 NS細胞吞噬試驗 45
3.2.6 NS作為佐劑效果之評估 47
第四章 實驗結果 50
4.1最佳之UHPH磷脂質配方選擇試驗 50
4.2最佳之UHPH均質條件選擇試驗 51
4.3 NS物理性質試驗 52
4.3.1 NS物理穩定性 52
4.3.2 NS粒徑分析試驗 52
4.3.3 NS結構 53
4.4 NS包覆率試驗 54
4.4.1 NS包覆澱粉酶能力試驗 54
4.4.2 NS包覆/吸附BSA能力試驗 54
4.4.3 NS包覆/吸附藍銅胜肽能力試驗 55
4.4.4 NS長期儲存包覆BSA能力試驗 56
4.5 NS生物相容性試驗 57
4.5.1 NS對神經膠質BV2細胞存活率試驗 57
4.5.2 NS紅血球相容性試驗 57
4.6 NS細胞吞噬試驗 58
4.7 NS作為佐劑效果之評估 59
第五章 討論 60
5.1 NS物理性質之探討 60
5.2 NS包覆能力之探討 63
5.3 NS生物相容性試驗 65
5.4 NS細胞吞噬試驗 67
5.5 NS作為佐劑效果之評估 69
5.6不同配方對於liposome之影響 71
第六章 結論 74
第七章 參考文獻 75
第八章 附圖 81

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