簡易合成硫酸化軟骨素接枝聚乙烯亞胺-氧化鐵奈米粒子於磁導引式基因轉染之應用研究

Chondroitin Sulfate -Polyethylenimine Copolymer-Coated Superparamagnetic Iron Oxide Nanoparticles as an Efficient Magneto-Gene Carrier

羅于倫(Yu-Lun Lo)

指導教授 ： 王麗芳

高雄醫學大學/生命科學院/醫藥暨應用化學系博士班/博士(2015年)

摘要

聚乙烯亞胺(Polyethylenimine, PEI)是極具有潛力的基因載體，但應用上受限於它嚴重的細胞毒性。因此於本研究中藉由結合天然高分子硫酸化軟骨素(Chondroitin Sulfate, CS)，成為新的共聚物(Chondroitin Sulfate grafted Polyethylenimine, CP)，減低原本的毒性問題，並維持良好的基因傳遞能力。在體外測試(in vitro)中，我們發現掛接PEI比例最低的CP(L)在包覆DNA後擁有最佳的轉染效率及最低的毒性；另一方面，探討細胞胞飲作用(endocytosis)機制，發現CP/DNA複合體是以Clathrin-mediated, Caveolae-mediated endocytosis和CD44 receptor-mediated為細胞主要攝取路徑。在先前的研究中，PEI可與聚丙烯酸修飾的高順磁氧化鐵奈米粒子(Polyacrylic acid-bound iron oxide, PAAIO)結合而成具磁性的奈米載體，可以藉磁導引方式來增加轉染效果。因此，我們利用毒性較高的CP(H)，以製備PEI-PAAIO相同的方式來製備超順磁氧化鐵奈米粒子 (CS-PEI-PAAIO, CPIO)。在體外測試(in vitro)中，當施加外加磁場後，以CPIO做為基因載體時，呈現出優於市售磁性轉染試劑- PolyMag的基因表現，並具備更良好的細胞存活率。使用CPIO來搭載對於神經膠質瘤細胞有抑制生長作用的pMIRNA-128基因片段，並證實其基因表現。在小鼠生物分布性實驗中，我們也驗證CPIO於磁場導引下，能夠有效率地將要遞送的pDNA聚集在腫瘤組織。

關鍵字

基因傳遞 ； 硫酸化軟骨素 ； 聚乙烯亞胺 ； 轉染 ； 氧化鐵奈米粒子

並列摘要

Polyethylenimine (PEI) has been demonstrated as a gold satndard for nonviral gene delivery vectors. However, the severe cytotoxicity of PEI is a serious issue. To solve these problems, an anionic polysaccharide- chondroitin sulfate (CS) was conjugated with PEI to minimize the cytotoxicity of PEI and enhance the transfection efficiency. In vitro test, CP(L) showed the least cytotoxic and the best gene expression as compared to Lipofectamine. For endocytosis test, the fluorescent probe, FITC was conjugated onto CP(L) to observe internalization pathways. The good cellular uptake of CP(L)/pDNA into U87 cells was primarily based on clatherin-dependent and CD44-mediated endocytosis. In our previous study, PEI was associated with polyacrylic acid-bound superparamagnetic iron oxide (PAAIO) through electrostatic interactions (PEI-PAAIO). Similarly, CP(H) with a high PEI content was used to complex with PAAIO named as CPIO through electrostatic interactions. In vitro test, CPIO exhibited superior gene expression with an assisted magnetic field to commercial-available magnetofection reagent, PolyMag. For delivering miRNA, we constructed a pDNA containing glioblastoma supressor-microRNA-128 sequence and trasfected it successfully into U87 cells. From an animal study, we demonstrated that CPIO could accumulate in the tumor tissue with an assisted magnetic field as compared to without.

並列關鍵字

gene delivery ； chondroitin sulfate ； polyethylenimine ； transfection ； iron oxide nanoparticles

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