複合式接枝型幾丁聚醣對於人類肝癌細胞轉染效率之研究

Abstract

本實驗中以幾丁聚醣為遞送載體的骨架來進行化修飾，目的是將以甲氧基聚乙二醇(methoxy poly(ethylene glycol), mPEG)或聚乙二醇二羧酸 (poly(ethylene glycol) diacid, PEG diacid)以共價鍵(covalent bond)的方式接枝在幾丁聚醣的第二位碳上，並將一具有肝標把作用的醣基以醚鏈鍵結(ether linkage)的方式接枝於幾丁聚醣的第六位碳上，而本實驗採用的醣基為半乳糖(galactose)，其含有可與去唾液酸胎醣蛋白(asialoglycoprotein, ASGP-R)進行辨認。經由化學修飾後的幾丁聚醣變成具有親水基團的標靶式遞送載體後，接著以帶有負電荷的質體DNA與幾丁聚醣以聚電解質複合反應(polyelectrolyte complexation)形成奈米複合體，並對此奈米複合體進行物性探討，接著利用奈米複合體進行細胞存活率測試及細胞轉染試驗，以比較不同修飾基對於基因轉染效率的影響。 研究中所用來進行一系列化學修飾之幾丁聚醣是先經由去乙醯化反應得到去乙醯化幾丁聚醣(DA-CS)後，再以去聚合作用的方式控制去乙醯化幾丁聚醣的數目平均分子量介於5000~6000之間，稱之為去聚合作用之去乙醯化幾丁聚醣(DADP-CS)，並以膠體滲透系統作分子量評估。經由化學修飾之幾丁聚醣分別為接枝型半乳糖基-去聚合作用之去乙醯化幾丁聚醣(DADP-CS-C6-Gal)、接枝型甲氧基聚乙二醇-去聚合作用之去乙醯化幾丁聚醣(DADP-CS-C2-mPEG)、接枝型聚乙二醇二羧酸-去聚合作用之去乙醯化幾丁聚醣(DADP-CS-C2-PEG diacid)、接枝型(半乳糖基/甲氧基聚乙二醇)-去聚合作用之去乙醯化幾丁聚醣(DADP-CS-C6-Gal-C2-mPEG)以及接枝型(半乳糖基/聚乙二醇二羧酸)-去聚合作用之去乙醯化幾丁聚醣(DADP-CS-C6-Gal-C2-PEG diacid)等五種高分子，將以紅外線光譜儀、核磁共振儀確認分子結構，以核磁共振儀用積分方式推估甲氧基聚乙二醇和聚乙二醇二羧酸於幾丁聚醣之二位碳的接枝比例，及以蒽酮-硫酸方法定量半乳糖的接枝率。將去聚合作用之去乙醯化幾丁聚醣與化學修飾之幾丁聚醣以聚電解質複合反應包覆帶有負電荷之質體DNA形成奈米複合體，以圓二色光譜儀確認物性結構，並以奈米粒徑分析儀測定粒徑大小與表面電荷，另以穿透式電子顯微鏡檢視型態。細胞試驗則是分析奈米複合體材料毒性以及細胞轉染效率，同時利用共軛焦顯微鏡分析影像的螢光強度。 根據每個化學修飾的幾丁聚醣之半乳糖、甲氧基聚乙二醇和聚乙二醇二羧酸定量結果，DADP-CS-C6-Gal的半乳糖取代度(DS%)約13.2~44.8%，DADP-CS -C2-mPEG的甲氧基聚乙二醇取代度(DS%)約2.3~5.3%，DADP-CS-C2-PEG diacid則有4~29%的聚乙二醇二羧酸取代度(DS%)，其接枝比例結果皆隨反應莫耳比的增加而上升。DADP-CS-C6-Gal-C2-mPEG的Gal和mPEG取代度(DS%)分別為27.3%、3.67%，而DADP-CS-C6-Gal-C2-PEG diacid 的Gal和PEG diacid取代度(DS%)則為 37.9% 和43.5%。經由三種重量比例(2：1、10：1、20：1)形成的奈米複合體，其粒徑與表面電荷結果顯示隨著高分子的重量比例增加，粒徑大小也隨之增大，整體平均粒徑大小約上升35%，而粒徑分散度(polydispersity index, PdI)也皆從0.2上升至0.4左右，表面電荷，皆維持正電荷，重量比例(2：1)約為17~45 mV、重量比例(10：1)約為25~65 mV及重量比例(20：1)約為29~72 mV。圓二色光譜儀結果顯示被包覆在內的質體DNA並無明顯的改變其CD的訊號，亦證明接枝型幾丁聚醣-質體DNA形成奈米複合體後，並不會對DNA造成結構上的影響。 接枝型幾丁聚醣的細胞毒性試驗結果，隨著給予的幾丁聚醣溶液濃度增加，其細胞存活率仍有60~70%。細胞轉染效率的部分，重量比例(20：1) 的DADP-CS其轉染效率最高，其次為DADP-CS-C6-Gal，DADP-CS-C6-Gal-C2-mPEG與DADP-CS-C6-Gal-C2-PEG diacid可能因為聚乙二醇立體障礙關係，阻擋了半乳糖進行標把作用，而因降低其轉染效率。根據共軛焦影像分析，DADP-CS同樣也是表達最高綠螢光蛋白強度。

In our studies, we used chitosan as the backbone to condout a series of chemical modification. The grafted chitosan composing hydrophilic group of methoxy poly(ethylene glycol) (mPEG) or poly(ethylene glycol) diacid (PEG diacid) via covalent bonding at C2-NH2 of chitosan. The target sugar molecule of galactose, which can recognize aisaloplycoprotein (ASGP-R) was futher introduced into the chitosan structure. An polyelectrolyte complexation method was used to prepare nanocomplex containing plasmid DNA as a gene carrier. Invesgating the nanocomplex physicochemical properties, then undergoing cell viability test and cell transfection efficiency experiment, to compare the diffetent modification chitosan transfection efficiency. The original material of chitosan was deacetylated and depolymerized first, then analysed the molecular weight by the gel permeation chromatography (GPC). Synthesis a series of modification chitosan were DADP-CS-C6-Gal、DADP-CS-C2-mPEG 、DADP-CS-C2-PEG diacid、DADP-CS-C6-Gal-C2-mPEG、DADP-CS-C6-Gal-C2-PEG diacid and identified by FT-IR, H1-NMR, GPC. Using H1-NMR to calculated mPEG and PEG diacid the degree of substitution (DS%) of chitosan and measured the glactose amount which linkage at C6-OH of chitosan by the anthrone-sulfuric acid colorimetric assay. The comformation copolymer-plasmid DNA nanocomplex were comfirmed the physical structure by circular dichroism (CD), detected the size and zeta potential and examined the properties of nanocomplex by TEM. The cell experiments were to analysis the materials cytotoxity, transfection efficiency and the EGFP fluorescence intensity by confocal. The result of galactose DS(%) on DADP-CS-C6-Gal were 13.2%~44.8%. The pegylation of DADP-CS-C2-mPEG、DADP-CS-C2-PEG diacid were 2.3%~5.3% and 4%~29%, separately. These results indicated that more feed molar ratio to the DADP-CS, the higher of degree of subsititution to the DADP-CS. DADP-CS-C6-Gal-C2-mPEG galactose DS(%) and PEG DS(%) were 27.3%, 3.67% and DADP-CS-C6-Gal-C2-PEG diacid were 37.9%, 43.5%. The nanocomplex was formed by copolymer and plasmid DNA in three weight ratio (2：1, 10：1, 20：1). The complex size was average rise to 33%, the polydispersity (PdI) were also from 0.2 rise to 0.4. The zeta potential results showed that the three weight ratio maintain positive charge, weight ratio 2：1 were 17~45 mV, weight ratio 10：1 were 25~65 mV and weight ratio 20：1 were 29~72 mV. The signal of circular dichroism form three weight ratio obstructnanocomplex indicated that there was no significant change for the plasmid DNA which packaged in the nanocomplex. In other words, the nanocomplex wouldn’t effect the plasmid DNA structure. The cell viability assay was examine the series of modification chitosan cytotoxicity. According to the result, the cells still had 60%~70% viability in the highest copolymer concentration, which means those copolymer were harmless to the cells. In the transfeciton experiment, DADP-CS showed the best transfeciton efficiency and the second was DADP-CS-C6-Gal. It was proved that the galactose has the targeting ability to ASGP-R. However, DADP-CS-C6-Gal-C2-mPEG and DADP-CS-C6-Gal-C2-PEG diacid were not in our expected results, we assumed the PEG was obstruct the galactose target on the ASGP-R causing the low transfection efficiency. The results of confocal, also indicated that DADP-CS had the highest the EGFP fluorescence intensity.