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研究生:劉旭珮
論文名稱:澳洲茄胺及其毫微粒子製劑對人類肝癌細胞毒殺作用之研究
論文名稱(外文):Cytotoxicity Evaluation of Solasodine and its Polyisobutylcyanoacrylate Nanoparticles in Human Hepatoma Cell
指導教授:蔡東榮蔡東榮引用關係
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:藥學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:103
中文關鍵詞:澳洲茄胺毫微粒子細胞毒殺作用
外文關鍵詞:solasodinenanoparticlescytotoxicity
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中文摘要
毫微球粒子的發展在近些年來已取代微脂粒而成為熱門的研究方向。本實驗採用水難溶性藥品Solasodine作為模式藥,應用固體分散系的製法,並添加PEG (polyethylene glycol)作為載體,以PIBCA (polyisobutylcyanoacrylate)作為聚合用之聚合物,探討Solasodine溶液、Solasodine併用Cyclosporin A、Solasodine製備成毫微球粒子及毫微球膠囊之外觀及其對人類肝癌細胞 (Hep3B與HepG2) 代謝情形及DNA合成之影響。
由穿透式電子顯微鏡觀察的結果顯示,以Solasodine所製備的處方其平均粒徑均達所求,約在150 ~ 200nm之間且形狀皆接近圓形。另外,應用固體分散系方式製備的毫微球粒子處方,其溶解度依不同的處方相較於Solasodine solution增加20% ~ 200%。藥品在毫微球膠囊中依不同的處方其包埋率經HPLC分析約在30 ~ 75%,其中以毫微球膠囊的包埋率最佳,因其可將藥品均勻分佈於油相中。將製備之Solasodine處方投予人類肝癌細胞 (Hep3B及HepG2)測定其細胞毒性(cytotoxicity);結果顯示Solasodine所製備的毫微球粒子及毫微球膠囊等處方對於人類肝癌細胞株 (human hepatocarcinoma cell, Hep3B及HepG2) 均具有細胞毒性 (IC50約在3~4μg/mL,依不同處方而異);而單純Solasodine solution之細胞毒性並不是很強,但若添加p-glycoprotein抑制劑-Cyclosporin A,則對人類肝癌細胞的毒性有顯著地增加 (IC50<0.1μg/mL)。為進一步了解其作用機轉,在肝癌細胞經過以上處方的作用後,分別以[3H]-thymidine incorporation及流體細胞分析儀 (flow cytometry) 分析其DNA含量的變化及DNA合成作用的影響,其中sub-G1細胞週期在投予處方後,於低濃度時即表現出,隨著濃度的增加,其sub-G1比例亦趨增加;相對地,G2/M細胞週期的比例降低,而G0/G1細胞週期的比例並未明顯改變,此因Solasodine製備成的毫微球膠囊及併用Cyclosporin A等處方可能係抑制細胞由G2/M期進入G0/G1期,並直接由G2/M期時細胞直接進行程式性死亡(apoptosis)之機轉造成細胞死亡。另外,將製備之Solasodine毫微球膠囊及Solasodine solution直接以腹腔注射進入老鼠體內測其血液與肝臟中的藥物濃度,可觀察出投予毫微球膠囊之老鼠體內血液與肝臟中的藥量較Solasodine solution高出許多,顯示製備成毫微球製劑在生物體內具有提高組織中濃度及器官標的的作用。

Abstract
The polymeric nanoparticles have been used in various pharmaceutical preparations for the improvement of cytotoxicity. In this study, we used solasodine as a model drug for preparation in solid dispersion. Solasodine isolated from the fruit of solanum aviculare L. that was the precursor of steroid and has been used for the treatment of hepatotoxicity. The water insoluble property of solasodine result in low efficiency of encapsulation. Polyethylene glycol and polysorbate 80 prepared the carrier of solid dispersion of solasodine and polyisobutylcyanoacrylate was applied for the emulsified polymerization. In order to investigate the cytotoxic mechanism and DNA synthesis of various solasodine formulations in human hepatoma cells (Hep3B and HepG2), the solasodine solution, solasodine add with cyclosporin A, polymeric nanoparticles of solasodine and nanocapsule formulation of solasdoine were used in the studies.
The physical characteristics of solasodine nanoparticle and nanocapsule formulations were examined by transitional electron microscopy. The results indicate that shape of these particles goes toward cycle and the average diameter was approximate 150 ~ 200 nm. The solubility of solasodine was increased around 20 ~ 200 % while nanoparticles was prepared by the method of solid dispersion. The efficiency of encapsulation of solasodine in various nanoparticles was approximating 30 ~ 70 %. The higher solasodine content of nanocapsule was the main cause of solasodine even distribution in the oil phase of the formulation.
The cytotoxicity of solasodine solution, solasodine add with cyclosporin A solution, nanoparticle formulations were determined by MTS assay , [3H]-thymidine incorporation and flow cytometry. The results indicate that the cytotoxicity of solasodine nanoparticle and nanocapsule formulations was great on human hepatoma cells. To define the cytotoxic mechanism of solasodine add with cyclosporin solution and nanocapsule (drug-loaded) formulation on human hepatorna cells, the change of DNA content in the cells after these formulations treatment was studied.
The significant cell death of Hep3B cell was observed with solasodine nanoparticle formulation (3~5μg/mL). However, the death of HepG2 cell was observed with nanoparticle formulation (4~5μg/mL) significantly. This suggested that the effect of solasodine nanoparticle formulation on Hep3B and HepG2 cell was irreversible. The sub-G1 feature analyzed by flow cytometry appeared in 24-h of solasodine combine cyclosporin solution, nanoparticle formulation and nanocapsule (drug-loaded) formulation treatment. In contrast to the cells in G2/M phase drastically shifting to apoptotic sub-G1 phase, no significant change of Hep3B at G0/G1 phase was detected. This implied that solasodine add with cyclosporin solution, nanocapsule formulation and nanocapsule (drug-loaded) formulation might arrest cell cycle at G0/G1 phase and drove the cells at G2/M phase to apoptosis.

目錄
中文摘要
英文摘要
本文
壹、緒論
一、生物可分解性的高分子毫微球粒子(biodegradable polymeric nanoparticles) 作為藥物輸送系統之概述---------------- 1
二、Solanaceae 科植物介紹--------------------------- 4
三、Solasodine結構及生物活性介紹--------------------- 5
四、Solid Lipid Nanoparticles (SLN)之概述--------------9
五、Solid dispersion之概述---------------------------10
六、細胞傷害死亡方式---------------------------------12
貳、實驗材料與方法
一、藥品與試劑---------------------------------------15
二、常用培養液及緩衝液之配製-------------------------15
三、儀器設備-----------------------------------------16
參、研究方法
一、毫微球粒子的製備及物化性質-----------------------18
二、Solasodine的HPLC分析條件-------------------------20
三、檢量線之製作-------------------------------------20
四、定量方法之確效-----------------------------------20
五、藥品在毫微粒膠囊中之包埋率測定-------------------21
六、溶解度之測定-------------------------------------21
七、Cytostatics--------------------------------------22
肆、結果與討論
一、藥品之定量分析-----------------------------------26
二、藥品溶解度之測定---------------------------------31
三、Solasodine製備成毫微球粒子以穿透式電子顯微鏡觀察
其外觀-----------------------------------------------32
四、藥品在毫微粒膠囊中之包埋率測定-------------------41
五、難溶性藥品Solasodine製備成Solid dispersion 後其溶
解度之測定-------------------------------------------43
六、Solasodine製備成Nanoparticle及Nanocapsule drug-
loaded formulation對於人類肝癌細胞(Hep3B及HepG2)
代謝情形之探討---------------------------------------45
七、Solasodine製備成Nanoparticle及Nanocapsule drug-
loaded formulation其作用時間對於Hep3B 及 HepG2
肝癌細胞生長的影響-----------------------------------57
八、Solasodine製備成Nanoparticle及Nanocapsule drug-
loaded formulation對人類肝癌細胞 (HepG2) DNA合成
之影響-----------------------------------------------63
九、利用細胞流體分析儀 (FACScan) 分析細胞週期
(cell cycle) 及定量細胞程式性死亡的變化情形----------68
伍、結論 --------------------------------------------94
陸、參考資料 ----------------------------------------95

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