Construction of a Novel Reactive Oxygen Species-responsive Cationic Copolymer and Its Performance in Gene Delivery

doi:10.6023/A21030090

Acta Chimica Sinica ›› 2021, Vol. 79 ›› Issue (6): 794-802.DOI: 10.6023/A21030090 Previous Articles Next Articles

Article

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

韩旭, 张留伟, 张强, 睢晞航, 钱明, 陈麒先^*(), 王静云^*()

大连理工大学生物工程学院大连 116024

投稿日期:2021-03-11 发布日期:2021-04-30
通讯作者: 陈麒先, 王静云
基金资助:
国家自然科学基金(21878041); 国家自然科学基金(22078050); 中央高校基本科研业务费专项资金(DUT17RC(3)059); 中央高校基本科研业务费专项资金(DUT20YG126); 大连市科技创新基金(2020JJ26SN050); 大连市科技创新基金(2020JJ26GX025)

Construction of a Novel Reactive Oxygen Species-responsive Cationic Copolymer and Its Performance in Gene Delivery

Xu Han, Liuwei Zhang, Qiang Zhang, Xihang Sui, Ming Qian, Qixian Chen(), Jingyun Wang()

School of Bioengineering, Dalian University of Technology, Dalian 116024, China

Received:2021-03-11 Published:2021-04-30
Contact: Qixian Chen, Jingyun Wang
Supported by:
National Natural Science Foundation of China(21878041); National Natural Science Foundation of China(22078050); Fundamental Research Funds for the Central Universities(DUT17RC(3)059); Fundamental Research Funds for the Central Universities(DUT20YG126); Dalian Science & Technology Innovation Fund(2020JJ26SN050); Dalian Science & Technology Innovation Fund(2020JJ26GX025)

1. Supporting Information-A21030090.pdf(609KB)

Abstract

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

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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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Fig. & Tab. 6

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

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

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

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

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

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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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

Construction of a Novel Reactive Oxygen Species-responsive Cationic Copolymer and Its Performance in Gene Delivery

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