利用ATRP聚合製備陽離子高分子作為基因載體之評估

基因治療，在現今許多遺傳、心血管疾病與癌症的治癒上展現了新的契機，因此基因載體在基因治療中，扮演著不可或缺的角色，而理想的基因載體必須具備低毒性及維持高效率的轉染能力，近幾十年來，陽離子高分子系統逐漸受到重視，然而此系統雖具有高度的基因表現，但也伴隨著高細胞毒性的缺失。本研究利用陽離子高分子Poly(2-dimethyl amino)ethyl methacrylate (PDMAEMA)與三嵌段共聚物PluronicF127 (PF127)為基礎，並藉由原子轉移自由基聚合方式(atom transfer radical polymerization, ATRP)設計出五嵌段共聚物PF127-pDMAEMA，在體外測試(in vitro)中，我們發現此嵌段共聚物隨著PDMAEMA鏈段的增加而提升轉染效能，但亦具有高度的細胞毒殺能力。隨著研究的發展，為克服初步研究中PDMAEMA鏈段所引發的細胞毒性問題，在第二階段研究中，我們將五嵌段共聚物進行改質，由於我們發現PDMAEMA鏈段因具有大量正電荷，是造成細胞毒殺的主要原因，因此，我們設計出含有陰離子結構(acrylic acid, AA)的嵌段共聚物PF127-p(DMAEMA-AA)，此共聚物相較於PF127-pDMAEMA，同樣具有良好的緩衝效能與DNA結合能力，複合體的粒徑能控制在80 – 180 nm且表面電位維持正電荷，在體外測試(in vitro)中，PF127-p(DMAEMA-AA)具有優越的基因表現，且大幅改善細胞毒殺性的缺失，並由共軛焦顯微鏡觀察，PF127-p(DMAEMA-AA)複合體能有效地傳遞DNA進入細胞內，另一方面，我們可藉由化學修飾接枝上螢光分子，探討複合體於細胞胞飲作用(endocytosis)，發現以clathrin-mediated 及 caveolae-mediated endocytosis為細胞主要攝取路徑。近年來超順磁氧化鐵奈米粒子已廣泛應用於生物醫學領域，而為了提高基因傳遞效率與兼具多功能性質，我們開發出新穎的磁性基因載體系統；此系統藉由熱溶劑法(solvothermal)進行製備超順磁氧化鐵奈米粒子做為高分子起始劑 (IO-Br)，並利用ATRP方式進行表面聚合反應，設計出陽離子磁性奈米粒子(IO-PDMAEMA)，在MRI影像測試上，此奈米粒子展現出優越的核磁共振影像對比性，在轉染效能評估上，藉由外加磁場的導引下，即使於血清環境中，亦仍有優異的基因表現，且具備良好的細胞存活率，甚至優於市售磁性轉染試劑- PolyMag，此結果說明此奈米粒子具備高度潛力，可作為多功能基因傳遞系統。

關鍵字

基因治療；共聚物；原子轉移自由基聚合

並列摘要

Gene therapy shows much promise in tackling various genetic diseases and cancers, viral infection, and cardiovascular disorders. The most challenging task in gene therapy is the design of gene delivery vectors with low cytotoxicity and high transfection efficiency. Cationic polymers are the major type of non-viral gene delivery vectors investigated in the past decade. Because of the highly toxic concern using cationic polymers as a gene delivery vector, the aim of the present work is to synthesize pentablock copolymers of PluronicF127 (PF127) and Poly(2-dimethyl amino)ethyl methacrylate (PDMAEMA), with different PDMAEMA block lengths using atom transfer radical polymerization (ATRP). The PF127-pDMAEMA/pDNA showed excellent gene transfection efficiency in 293T cells. The higher block length of PDMAEMA indeed showed the higher transfection efficiency but resulted in the higher cytotoxicity as well. To overcome the cytotoxicity caused by a high chain length of PDMAEMA >100 repeating units, we tried to modify the internal structure of PF127-pDMAEMA by copolymerizing a negatively charged acrylic acid (AA) to produce PF127-p(DMAEMA-AA). The copolymers showed high buffering capacity and efficiently complexed with plasmid DNA (pDNA) to form nanoparticles with 80 – 180 nm in diameter and with positive zeta potentials. In the absence of 10% FBS, PF127-p(DMAEMA-AA) showed the highest gene expression and the lowest cytotoxicity in 293T cells. After AA groups had been linked with a fluorescent dye, the confocal laser scanning microscopic image showed that PF127-p(DMAEMA-AA)/pDNA could efficiently entered the cells. Both clathrin-mediated and caveolae-mediated endocytosis mechanisms were involved. In the second part of studies, superparamagnetic iron oxide nanoparticles (SPION) have been widely used for multiple biomedical applications. In this work, poly(2-dimethyl amino)ethyl methacrylate-bound iron oxide nanoparticles (IO-PDMAEMA) were generated using a graft-from approach via ATRP as a gene vector. We designed a simple method to produce an IO-initiator containing bromide groups (IO-Br). The IO-Br was synthesized by reacting iron oxide nanoparticles with 2-bromoisobutyric acid using a one-pot solvothermal method at a high temperature. We optimized IO-PDMAEMA with a controllable PDMAEMA molecular weight that allows higher gene expression with lower cytotoxicity. The transversal relaxivity studies suggested that IO-PDMAEMA can be a contrast agent for magnetic resonance imaging. The magnetofection efficacy of IO-PDMAEMA/pDNA was measured in 293T cells with or without fetal bovine serum (FBS). The IO-PDMAEMA/pDNA magnetoplexes exhibited remarkably improved gene expression in the presence of a magnetic field and 10% FBS as compared with a commercial product, PolyMag/pDNA. No significant cytotoxicity of IO-PDMAEMA/pDNA was observed with different incubation time periods with or without the magnetic field. Our results showed that IO-PDMAEMA has great potential to be a gene delivery vector.