Chia Nan University of Pharmacy & Science Institutional Repository:Item 310902800/24950
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    標題: 陽離子型PLGA-PEI奈米微粒之製備及做為基因傳輸材料之研究
    Preparation of Cationic Plga-Pei Nanoparticles and Studies on Gene Transfection
    作者: 蕭明達
    貢獻者: 生物科技系(所)
    關鍵字: 基因治療
    細胞毒性
    胞飲作用
    轉染效率
    日期: 2011
    上傳時間: 2012-01-03 13:57:29 (UTC+8)
    摘要: 基因治療是指將一段正常基因送進病人細胞中,使細胞能夠合成正常生理所需的蛋白質,進而治療疾病。基因透過傳遞系統有效率地傳送至細胞,需要一安全且轉染率高的基因載體。在陽離子型高分子的基因傳遞系統中,高分子與DNA 自組裝地形成一奈米尺度的複合體,並藉由細胞胞飲作用進入細胞質,且能夠保護DNA 免於受到核酸酵素分解。然而,陽離子型高分子/DNA 複合體經由胞飲作用進入細胞之後,主要仍留在內質體及溶小體中,限制了它們繼續在細胞質中運輸,因此,DNA 自內質體釋出將是基因傳輸系統中達成轉染效果的限制步驟。除此之外,PLL、PDMAEMA、及PEI 都是非生物可分解的材料,且具有明顯得細胞毒性,為了改善上述高分子的缺點,本研究室將以下五項特性作為合成新的陽離子型高分子基因傳遞系統結構設計的準則:(1)聚集 DNA 的能力(2)水溶蝕性(3)生物相容性(低細胞毒性)(4)pKa 值介於5.0-7.4(5)容易合成。以目前而言,質體DNA 被廣泛用於治療藥物及疫苗接種,然而,深入的研究發現,DNA 接種疫苗成功關鍵在於傳輸系統。高分子材料亦被常用於疫苗接種的傳遞系統,而此類的高分子乃以物理包陷的方式包覆DNA 疫苗且成功地傳遞至細胞。在本研究計畫中,我們將合成含三種不同分子量PEI 之PLGA-PEI 高分子材料,並利用乳化-擴散-揮發法將其製備成陽離子性奈米微粒。利用原子力顯微鏡及動態光散射儀器測試其粒徑大小,以表面電位及膠體電泳來了解其表面性質及與帶負電DNA 複合之能力,陽離子性 PLGA-PEI 與DNA 複合微粒之細胞毒性則以XTT 方法來評估,至於轉染效率我們擬以綠螢光及ONPG 來測試。
    Gene therapy is defined as delivery of therapeutic gene expression into patient’s host cells to enable production of proteins to correct or moderate disease. To achieve gene delivery, safe vectors with selective and high transfection efficiency are required. The polycation/DNA complexes not only can protect the DNA from nuclease degradation, but also has a nanoscale size small enough to enter the cell via endocytosis. However, the delivery vehicle is localized in the endosomes and will ultimately be trafficked to the lysosome. Release of the delivery vehicle from these compartments into cytoplasm is believed to be the limiting step in transfection mediated by many cationic polymers. In addition, these polycations such PLL 、PDMAEMA 、and HMWPEI are nonbiodegradable materials and associated with a considerable degree of cytotoxicity. In point of improving their drawback, we outlined five criteria to drive the rational synthesis to formulate new cationic polymer gene delivery systems. (1) DNA condensation capability(size of complex below 150 nm to allow efficient endocytosis) (2)hydrolytic degradation (3)biocompatibility(to minimize potential toxic effect) (4)pKa between lysosome pH(~5.0)and pH(~7.4)(to provide buffer capacity) (5) facile synthesis. In this study, PLGA-PEIs with different molecular weight PEI were synthesized and relative nanoparticles were obtained by an emulsion-diffusion evaporation technique using PVA as a stabilizer. The size of modified particles was characterized by atomic force microscopy (AFM) and dynamic light scattering (DLS). Also Zeta potential and gel electrophoresis studies were performed to understand the surface properties of cationic nanoparticles and their ability to bind negatively-charged DNA. The cytotoxicity of the cationic nanoparticle/DNA complexes was detected by XTT assay. The DNA delivering of PLGA-PEI nanoparticles was evaluated by measuring pEGFP and pCMV-βgalexpressions with fluorescence and ONPG assay, respectively.
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