Investigation of dual-effect liposome n inhibiting tumor growth

癌症是由細胞不正常複製、增生，所形成的惡性腫瘤組織，會入侵並破壞正常組織器官，甚至轉移至身體其他部位，導致患者死亡。目前常使用之化學治療方式，因無法只針對癌細胞進行專一毒殺，使用上會對患者造成副作用。所以科學家利用微脂體 (liposome) 能專一累積於腫瘤組織之特性，將化療藥物 Doxorubicin (Dox)包覆其中，藉此改善 Dox毒性；但由於包覆於微脂體中，但由於藥物釋放速度過慢，而使 Lipo-Dox治療癌症效果不如預期。而近期一種癌症治療方式—光動力治療，則是針對腫瘤部位，利用光感物質被特定波長光源激發，產生單態氧及自由基，造成腫瘤細胞死亡之治療模式。本篇研究即是想結合光動力治療及化學治療兩種方式，同時將光感物質 Chlorin e6 (Ce6)與Dox包覆於微脂體中，除了藉由 Ce6之光動力效應，產生單態氧及自由基，造成腫瘤毒殺外，還能改變微脂體脂雙層之通透性，加速包覆於其中Dox之釋放，達到加強腫瘤毒殺效果，故命名此新型微脂體為雙效型微脂體 Dual effect liposome (Lipo-Dox-Ce6)。本研究首先確定雙效型微脂體Lipo-Dox-Ce6能被腫瘤細胞吸收，並且證實 Lipo-Dox-Ce6於光激發後，確實能加速其中所包覆之 Dox釋放至細胞核。在細胞實驗中，相較於 Lipo-Ce6、Lipo-Dox，雙效型微脂體 Lipo-Dox-Ce6有更好之細胞毒殺效果。活體老鼠實驗中也發現，Lipo-Dox-Ce6確實比Lipo-Ce6、Lipo-Dox具有更好之抑制腫瘤生長效應，並延長老鼠之存活時間。Photodynamic therapy (PDT), which is based on the uptake of a photosensitizer followed by the selective irradiation of light, is a therapeutic modality for cancer treatment. Due to the limited light penetration, PDT is generally considered as a local treatment. To enhance the efficacy of cancer treatment, we prepared a polyethylene glycol (PEG) modified liposome simultaneously encapsulated with Chlorin e6 (Ce6) and Doxorubicin (Dox). Here we named as dual-effect liposome, which has Ce6 encapsulated in the lipid bilayer and Dox in the aqueous interior. There is no significant difference in the cellular uptake between the dual-effect liposome and liposomal Ce6. The cellular localization of this dual-effect liposome was in the cytoplasmic area only. Under light irradiation, significant nuclear localization of Dox was found under confocal fluorescence microscope. To address the cytotoxicity of these dual-effect liposomes, colony formation assay was performed after light irradiation. At the light dose of 20 J/cm2, 5 ng/ml of liposome Ce6 can produce 40% cytotoxicity; however, dual-effect liposome can produce over 80% cytotoxicity. Finally, we examined the anti-tumor efficacy of dual-effect liposome with C-26 syngeneic tumor model in BALB/cByJ mice. Compared liposomal Ce6 or liposomal Dox, this dual-effect liposome achieved greater therapeutic effect.中文摘要-------------------------------------------------------------------------------------------- i 文摘要------------------------------------------------------------------------------------------- ii錄------------------------------------------------------------------------------------------------- iii寫表-----------------------------------------------------------------------------------------------iv 一章 緒論.1 癌症及其治療方式--------------------------------------------------------------- P.1.2 Doxorubicin ----------------------------------------------------------------------- P.5.3 Liposomal-Doxorubicin --------------------------------------------------------- P.6.4 光動力治療 ----------------------------------------------------------------------- P.8 .5 光感物質 ------------------------------------------------------------------------ P.11.6 研究動機與目的 --------------------------------------------------------------- P.12二章 材料與方法.1 藥物與儀器 --------------------------------------------------------------------- P.14.2 細胞株 --------------------------------------------------------------------------- P.16.3 細胞計數 ------------------------------------------------------------------------ P.17.4 微脂體製備-----------------------------------------------------------------------P. 18.5 光動力治療 --------------------------------------------------------------------- P.18.6 Ce6 uptake ----------------------------------------------------------------------- P.19.7 共軛焦顯微鏡 ------------------------------------------------------------------ P.19.8 細胞存活率檢測 --------------------------------------------------------------- P.20.9 細胞死亡型態檢測 ------------------------------------------------------------ P.22.10 活體動物實驗 ------------------------------------------------------------------ P.23計分析 ---------------------------------------------------------------------------------P.24三章 結果.1 雙效型微脂體光動力作用在細胞分子層次的研究 --------------------- P.25.2 雙效型微脂體光動力作用在活體動物實驗的應用 --------------------- P.32四章 討論 ---------------------------------------------------------------------------------- P.34五章 結論 ---------------------------------------------------------------------------------- P.39igure 1~12 ------------------------------------------------------------------------------------- P.41圖一至六------------------------------------------------------------------------------------- P.62考文獻 --------------------------------------------------------------------------------------- P.6