活性氧响应的新型阳离子共聚物构建及其基因递送性能研究

doi:10.6023/A21030090

摘要/Abstract

引入快速、主动释放基因的机制是提升非病毒基因递送效率的关键. 本研究以2-甲基丙烯酰氧乙基磷酰胆碱(MPC)与(2-丙烯酰基)乙基(硼酸苄基)二乙基溴化铵(BD)为单体, 基于可逆加成-断裂链转移聚合(RAFT)反应制备具有活性氧响应性质的聚阳离子嵌段共聚物pM-pBD. 通过静电作用, 阳离子共聚物pM-pBD能够与带负电荷的DNA以分子自组装的方式形成纳米复合物. 其中, 阳离子pBD片段具有活性氧触发电荷反转的特性, 因此, 有助于获得活性氧触发的静电组装复合体结构解离, 从而实现可控基因释放的功能. 理化表征结果表明, pM-pBD与质粒DNA静电复合后能够形成粒径约为99.1 nm, ζ电位约为+13.8 mV, 显微形貌近似球形的纳米复合物. 在加入促过氧化氢产生的抗坏血酸后, 上述pM-pBD基因递送系统的转染效率得到了显著的提升. 因此, 本研究创制的pM-pBD为基因递送系统的可控释放提供了新的解决方案.

关键词: 聚阳离子, 电荷反转, 基因递送, 活性氧响应, 抗坏血酸

The development of rapid and active gene release function is the key to improve the efficiency of non-viral gene delivery system. Herein, a novel reactive oxygen-responsive cationic block copolymer non-viral gene delivery vector (termed as pM-pBD) consisting of biocompatible poly(2-methacryloyloxyethyl phosphorylcholine) (pMPC) segment and charge reversible poly[(2-acryloyl)-ethyl-(boronic acid benzyl)-diethylammonium bromide] segment (pBD) was synthesized via reversible addition-fragmentation chain transfer polymerization (RAFT). Dynamic light scattering (DLS) assay and ζ potential measurements, transmission electron microscope (TEM) and gel electrophoresis were performed to characterize pM-pBD/(plasmid DNA, pDNA) complexes. Gel electrophoresis retardation assay displayed that pM-pBD can firmly bind pDNA through electrostatic interaction even in the presence of high concentration of heparin, but capable of releasing pDNA in response to reactive oxygen (such as H₂O₂). The pM-pBD/pDNA complexes show low cytotoxicity against HeLa cells, even at high weight ratio of pM-pBD and pDNA (N/P) (up to 16) demonstrated by MTT (methylthiazolyldiphenyl- tetrazolium bromide) assay. The pM-pBD/pDNA complexes demonstrated appreciable colloidal stability in the presence of 10% fetal bovine serum. The pM-pBD/pDNA complexes at N/P ratio of 3 displayed spherical morphologies with average diameter of approximate 99.1 nm and ζpotential of approximate +13.8 mV. However, once upon incubation in presence of 1 mmol/L H₂O₂, the diameter of pM-pBD/pDNA complexes at N/P ratio of 3 was enlarged to 330 nm and ζ potential was reversed to –3.91 mV due to charge reversal of BD responsive to reactive oxygen. Flow cytometry revealed uptake efficiency (88.9%) and the highest transfection efficiency (31.2%) for pM-pBD at N/P ratio of 3 and 16, respectively. The transfection efficiency of pM-pBD/pDNA against HeLa cells was observed to be significantly augmented (1.5-fold) after the addition of ascorbic acid, which could stimulate the production of hydrogen peroxide. Therefore, pM-pBD represented intriguing utilities in fabrication of non-viral gene delivery systems, which enable spatiotemporal control of gene release and thereby facilitated the subsequent transcription machinery.

Key words: polycation, charge reversal, gene delivery, ROS-responsive, ascorbic acid

引用此文

韩旭, 张留伟, 张强, 睢晞航, 钱明, 陈麒先, 王静云. 活性氧响应的新型阳离子共聚物构建及其基因递送性能研究[J]. 化学学报, 2021, 79(6): 794-802.

Xu Han, Liuwei Zhang, Qiang Zhang, Xihang Sui, Ming Qian, Qixian Chen, Jingyun Wang. Construction of a Novel Reactive Oxygen Species-responsive Cationic Copolymer and Its Performance in Gene Delivery[J]. Acta Chimica Sinica, 2021, 79(6): 794-802.

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图/表 6

图式1. 载体与基因自组装形成纳米复合物及细胞内响应活性氧实现电荷反转释放DNA过程示意图

Scheme 1. Schematic representation of non-viral gene delivery system from reaction oxygen species (ROS)-responsive charge-reversal copolymers

图1. 活性氧响应的新型阳离子共聚物pM-pBD的合成路线

Figure 1. Synthetic route of ROS-responsive cationic copolymeric pM-pBD

图2. (a)不同N/P纳米复合物的粒径和表面电位. (b)动态光散射法测定不同N/P时, pM-pBD/pDNA在含有10%胎牛血清的缓冲液中粒径变化. (c)在N/P=3时pM-pBD/pDNA复合物的粒径分布和TEM显微图片(插图). (d)不同N/P时, pM-pBD/pDNA的凝胶阻滞电泳图

Figure 2. (a) The particle size and ζ potential of pM-pBD/pDNA at varied N/P ratios. (b) The particle size of pM-pBD/pDNA in buffer containing 10% fetal bovine serum was measured by dynamic light scattering. (c) The particle size distribution and morphologies of pM-pBD/pDNA complex at N/P=3 by TEM (inset). (d) Gel electrophoresis image of pM-pBD/pDNA at varied N/P ratios

图3. (a) pM-pBD共聚物响应ROS实现电荷反转的分子机理. (b)在1 mmol/L H2O2孵育不同时间(0、2、5、10、30、60、120 min)后释放出对羟基苯甲醇(4-HBA)的HPLC色谱图(内参为色氨酸). (c)动态光散射检测在1 mmol/L H2O2孵育60 min后粒径大小及电位变化. (d) 1 mmol/L H2O2孵育后pM-pBD/pDNA (N/P=3)的凝胶阻滞电泳图

Figure 3. (a) The schematic diagram of pM-pBD copolymer producing polyacrylic acid by charge reversal in response to ROS. (b) HPLC chromatogram of 4-hydroxybenzyl alcohol (4-HBA) released after incubation in presence of 1 mmol/L H2O2 for varied time (0, 2, 5, 10, 30, 60, 120 min) (internal reference: tryptophan). (c) The changes of particle size and ζ potential after incubation in presence of 1 mmol/L H2O2 for 60 min was measured by dynamic light scattering. (d) Gel electrophoresis image of pM-pBD/pDNA (N/P=3) under incubation in presence of 1 mmol/L H2O2

图4. (a) HeLa细胞在不同N/P的pM-pBD/pDNA纳米复合物中孵育24 h后的细胞存活率. (b)不同抗坏血酸浓度产H2O2的能力(插图: 抗坏血酸产生活性氧的机理). (c)激光共聚焦显微镜观察HeLa细胞对N/P=3的pM-pBD/Cy5-pDNA纳米复合物在孵育4 h及8 h后的摄取. (d)流式细胞术测定的HeLa细胞对pDNA和pM-pBD/Cy5-pDNA纳米复合物(N/P=3)的摄取效率

Figure 4. (a) The cell viability of pM-pBD/pDNA nanocomplex at varied N/P ratios for 24 h in HeLa cell. (b) Production of H2O2 at varied concentrations of ascorbic acid (illustration: the mechanism of ascorbic acid producing reactive oxygen species). (c) Confocal laser scanning microscopy of HeLa cells incubated with pM-pBD/Cy5-pDNA (N/P=3) nanocomplex for 4 h and 8 h. (d) the uptake efficiency of pDNA and pM-pBD/Cy5-pDNA complexes (N/P=3) in HeLa cells

图5. (a) pDNA、不同N/P的pM-pBD/pDNA和PEI/pDNA (N/P=6.25)在HeLa细胞中转染后的激光共聚焦显微镜图. (b) pDNA、不同N/P的pM-pBD/pDNA和PEI/pDNA (N/P=6.25)在HeLa细胞中的转染效率. (c)有/无抗坏血酸存在时pM-pBD/pDNA复合物(N/P=3)在HeLa细胞中的转染效率. (d)有/无抗坏血酸存在时pM-pBD/pDNA复合物(N/P=3)在HeLa细胞中转染后的激光共聚焦显微镜图

Figure 5. (a) Confocal laser scanning microscopy of HeLa cells against pDNA, pM-pBD/pDNA with varied N/P ratios and PEI/pDNA (N/P=6.25). (b) The transfection efficiency of pDNA, pM-pBD/pDNA with varied N/P ratios and PEI/pDNA (N/P=6.25) in HeLa cells. (c) The transfection efficiency of pM-pBD/pDNA complex (N/P=3) with or without ascorbic acid (AA) in HeLa cells. (d) Confocal laser scanning microscopy of the transfection of pM-pBD/pDNA complex (N/P=3) with or without ascorbic acid in HeLa cells

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