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  • 學位論文

發展一定性定量方法分析協同包覆微脂粒系統中之血紫質及艾黴素

Qualitative and quantitative analysis of hematoporphyrin and doxorubicin co-encapsulated in the liposomes

指導教授 : 蔡翠敏

摘要


Doxorubicin (DOX)為目前臨床上常見之癌症用藥,其對心臟所產生之不可逆反應為目前劑型發展之首要議題,現今市面包覆DOX的長效型微脂體商品,可延長藥物在血液中的滯留時間,並提高藥物本身的穩定性以降低藥物使用劑量;除了改變藥物劑型外,可利用光照的方式給予特定波長激發累積在腫瘤部位的微脂粒提高藥物釋放速率,亦能降低藥物對其他正常組織的傷害,另可控制光照時的波長、強度與劑量,為一極具有潛力的癌症治療方式。先前本實驗室發展出在脂雙層包覆光感物質—血紫質(hematoporphyrin;Hp)及在核心親水層包覆DOX的光觸發型微脂粒仍處於初步階段,為延續此研究並瞭解其穩定性與觸發效果,本實驗探討主要有三大目標,首先藉由紫外光/可見光光譜儀與螢光光譜儀分析瞭解free及liposomal form的Hp和DOX的吸收及螢光光譜的變化;第二部分則以高效液相層析儀(HPLC)結合螢光偵測器發展能同時分離及定量Hp與DOX的最佳條件;第三部分則是利用固相萃取方式分離free及liposomal form藥物後,由HPLC分析以瞭解劑型在不同儲存條件與在細胞培養基中的穩定性,藉此得知在一溶液中微脂粒釋放出多少free form的藥物。實驗結果顯示free與liposomal form的Hp或DOX的吸收光譜及散射光譜皆不盡相同,Hp及DOX同時包覆在微脂粒的螢光強度皆會比單一物質包覆在微脂粒中要來得弱;本研究所發展之HPLC分析方法,移動相為50 mM NaH2PO4 (pH 2.09±0.01):Acetonitrile:1-propnaol= 30:15:2、流速1.2 ml/min,可在15分鐘內將Hp及DOX從管柱中沖提出,在10~2500 ng/ml之間具有線性關係;固相萃取分離liposomal及free form藥物部分,藥物的總回收率小於40%,低回收率的原因可能來自於在固相萃取時,破壞微脂粒的界面活性劑與微脂粒成分中的PEG形成微胞,此新形成的微胞可能也會包覆疏水性的藥物並在微胞的外層吸附親水性的藥物,導致固相萃取的回收率不高。

並列摘要


The commercial liposome formulations of doxorubicin (DOX) have the advantage of slowly releasing DOX while long-term circulating in the blood vessel. Light-activation is a potential method of triggering and accelerating the drug release rate at the tumor location. Light-activation also provides a wide range of adjustable parameters (e.g., wavelength, duration and intensity) that can be optimized for a suitable condition. A light-sensitive liposomal formulation, co-encapsulated with hematoporphyrin (Hp) in the lipid bilayer and DOX in the core of liposome, was previously developed in our laboratory. However, the stability of the liposomal formulation in the cell culture medium and the release after phototriggering has not yet been understood. In addition, significant cell toxicity was observed even before light-triggering, indicating possible burst-release of DOX from the liposomes. The purpose of this study is to investigate the spectral difference between the liposomal and free form compounds and develop a simple and simultaneous quantification method for Hp and DOX utilizing the high-performance liquid chromatography (HPLC). The absorption and emission spectra of Hp and DOX varied between free form and liposomal form. The fluorescence intensity and emission peak of these two compounds in the co-encapsulated liposomes were lower than that in the single encapsulated liposomes. The two compounds could be separated and eluted out from the column in 15 minutes, and the detection limits were 10 ng/ml for both of them. The calibration curves were linear over a concentration range of 10-2500 ng/ml. A solid phase extraction method was applied to separate the liposomal and free form compounds in order to monitor the stability of the liposomes in various storage and incubation conditions. The total extraction recovery from the liposomes was less than 40%, and the low recovery may be due to the formation of new micelles. The liposome-disrupting surfactant and the existed PEG in the liposomes could have formed micelle complex during the solid extraction process. The newly formed micelles may encapsulate the hydrophobic compounds and adsorb the hydrophilic compounds, resulting in poor extraction recovery.

參考文獻


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