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標題: | 具CD44標的潛力之奈米微胞基因遞送系統的研究 Study of Self-Assembled Micelles with CD44 Targeting Potential for Gene Delivery |
作者: | Wei-Chi Lee 李瑋綺 |
指導教授: | 林文貞(Wen-Jen Lin) |
關鍵字: | 玻尿酸,硫酸軟骨素,聚乳酸-甘醇酸,基因遞送,CD44, hyaluronic acid,chondroitin sulfate,poly(lactide-co-glycolide),gene delivery,CD44, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 玻尿酸(Hyaluronic acid,HA)和硫酸軟骨素(Chondroitin sulfate,CD)為在人體內細胞外基質中的內生性多醣分子。而此二多醣分子不但擁有相似的化學結構,近年來更因為二者皆為會過度表現在癌細胞外之CD44受器的專一性配體,進而有大量的研究將玻尿酸或硫酸軟骨素用作抗癌藥物或基因遞送的載體。
此研究以帶正電的1,2-二油酰基-3-三甲基銨丙烷(DOTAP)吸附帶負電質體DNA形成帶正電的奈米複合體(D/P),雖其轉染效果佳,但許多研究指出,帶正電的載體進入體循環後,容易引起血液中紅血球凝集,故本研究進一步將聚乙二醇二胺化的聚乳酸-甘醇酸(PLGA-PEG-NH2)接枝在低分子量玻尿酸(LPP)、高分子量玻尿酸(HPP)或硫酸軟骨素(CPP)的末端(直鏈)或側鏈,作為基因遞送的載體,包覆帶正電的奈米複合體分別形成LPP-D/P、HPP-D/P、CPP-D/P、LPPg-D/P、HPPg-D/P、CPPg-D/P,以增加其安全性及標靶遞送。 首先,合成的聚乙二醇二胺化的聚乳酸-甘醇酸接枝玻尿酸或硫酸軟骨素先以核磁共振(1H-NMR)、傅立葉轉換紅外線光譜儀(FT-IR)確認其化學結構及接枝率的定量。以透析方法形成微胞並包覆奈米複合體後,以Zetasizer測所形成的粒徑大小以及表面電位、以凝膠阻滯實驗確認質體DNA的被包覆性,並以PicoGreen reagent進行質體DNA包覆率的定量,最後也進行質體DNA在pH4.0的醋酸緩衝溶液與pH7.4磷酸緩衝溶液的體外釋放試驗,以確認質體DNA的釋放模式。以流式細胞儀篩選出過度表現CD44的細胞株(U87、MDA-MB-231、L929、MCF-7)進行奈米微胞對細胞之轉染試驗,並針對效果較佳的玻尿酸或硫酸軟骨素修飾奈米微胞(LPP-D/P、HPP-D/P、CPP-D/P)進行細胞胞飲途徑的探討,最後進行細胞存活率試驗,以確定奈米微胞對於正常細胞的安全性。 結果顯示,不同比例的LPP、HPP、CPP、LPPg、HPPg、CPPg形成的奈米微胞的粒徑分別在184.4-360.8 nm之間,帶正電的奈米複合體被聚乙二醇二胺化的聚乳酸-甘醇酸接枝玻尿酸或硫酸軟骨素包覆形成奈米微胞後,表面電位皆變為帶負電,因而不會造成紅血球凝集。凝膠阻滯實驗的結果確認直鏈合成聚合物包覆的pDNA不會因電場的影響而釋放出來,而支鏈合成聚合物會受到電場的影響而有pDNA釋放。合成之直鏈聚合物的細胞轉染效果,在MDA-MB-231、MCF-7細胞株中,HPP包覆D/P的轉染效果最佳,而在U87這株細胞則為CPP包覆D/P的轉染效果最佳,而LPP包覆D/P的綠色螢光蛋白轉染效率無論在MDA-MB-231、U87或MCF-7的效果都不太明顯,但是在L929細胞的轉染率卻相當高。合成之枝鏈聚合物的細胞轉染效果,在U87、MDA-MB-231、MCF-7、L929細胞株中,轉染效率皆是以LPPg-D/P最佳,甚至CD44 negative的HepG2對於LPPg-D/P都能有31.25 %之細胞轉染率,間接說明雖然LPPg-D/P轉染率高,但可能不是藉由CD44胞攝入細胞的專一性途徑。 在藉由不同機轉之胞飲抑制劑探討LPP、HPP、CPP進入細胞之途徑實驗中,說明了LPP、HPP在U87細胞中皆為clathrin-mediated endocytosis及macropinocytosis,而CPP在U87僅有clathrin-mediated endocytosis。LPP、HPP、CPP在MDA-MB-231皆為macropinocytosis,而HPP在MDA-MB-231細胞中還有Caveolin-mediated endocytosis一途徑。 細胞存活率試驗,LPP-D/P、HPP-D/P、CPP-D/P對L929的細胞存活率皆大於64.8%,對U87的細胞存活率大於64.7%,而對MDA-MB-231則為大於76.0%,其安全性皆優於商品化的Lipofectamine 2000。 Hyaluronic acid (HA) and chondroitin sulfate (CD) are endogenous polysaccharides existed in the extracellular matrix. In recent years, these two endogenous polysaccharides have aroused much interest of scientists because of their specificity of binding to CD44 receptor which is overexpressed in several types of tumors. As a result, a lot of studies are dedicated to develop the HA or CD based drug and gene delivery systems. Several polymers are synthesized including LHA-b-PEG-PLGA(LPP)、LHA-g-PEG-PLGA(LPPg)、HHA-b-PEG-PLGA(HPP)、HHA-g-PEG-PLGA(HPPg)、CD-b-PEG-PLGA(CPP)、CD-g-PEG-PLGA (CPPg). The structure and conjugation ratio of synthesized polymers were comfirmed by FTIR and 1H-NMR. The positively charged DOTAP was used to complex with the negatively charged plasmid DNA (pDNA) to form lipoplex. The dialysis method was used to encapsulate the D/P into the synthesized polymers to form micelles. The size and zeta potential of the micelles were measured by Zetasizer and the pDNA encapsulation efficiency was quantified by PicoGreen reagent. The in vitro release of the pDNA was conducted in the PBS buffer (pH 7.4) and acetate buffer (pH 4.0) separately. The CD44 overexpressed U87, MDA-MB-231, L929, MCF-7 were screened out by flow cytometry and used for cellular transfection experiment. The endocytosis mechanism of the CD44 tareting micelles was investigated. Finally, the MTT assay was conducted to comfirm the safety of the micelles. The size of the micelles were around 184.4-360.8 nm and the zeta potential was converted to negative after encapsulating D/P into the synthesized polymers. The positively charged D/P led to erythrocytes agglutination but the micelles didn’t because of the negatively charged surface of the micelles. The resuls of the electrophoresis retardation assay comfirmed the micelles formed by LPP, HPP and CPP could retain the pDNA inside the micelles well but LPPg, HPPg and CPPg could not. The transfection efficiencies of the micelles formed by LPP, HPP, and CPP were evaluated by flow cytometry. The HPP micelle showed the best transfection efficiency in MDA-MB-231 cell and MCF-7 cell. The CPP micelle showed the best efficiency in U87 cell. The LPP micelle showed the poorest efficiency in three CD44 overexpressed cancer cell lines U87, MDA-MB-231 and MCF-7 but the best efficiency in mouse fibroblast L929.The transfection efficiency of the micelles formed by LPPg, HPPg, and CPPg were different from LPP, HPP, and CPP micelles. In U87, MDA-MB-231, MCF-7 and L929 cell, the LPPg micelle showed the best transfection efficiency. Although the CD44 expression level of the HepG2 was low, the transfection efficiency of the LPPg was high. This result indirectly indicated that the cellular uptake pathway of the LPPg micelle was not the CD44-meiated pathway. The endocytosis study comfirmed the pathways of LPP, HPP, and CPP micelles. LPP and HPP were uptaken by the pathways of clathrin-mediated endocytosis and macropinocytosis in U87. However, the CPP micelle was uptaken by clathrin-mediated endocytosis. LPP, HPP, and CPP micelles were uptaken by the pathway of macropinocytosis in MDA-MB-231. Besides the macropinocytosis, HPP micelle was also uptaken by caveolin-mediated endocytosis in MDA-MB-231.The MTT assay comfirmed the synthesized polymer formed micelles were safer than the commercial product Lipofectamine 2000. In conclusion, the HPP and CPP micelles could provide a safer and more specific way to deliver gene to cancer cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/55737 |
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