基于开环反应构建新型还原响应型支化聚赖氨酸基因载体
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摘要
通过"一锅法"开环反应构建了一种含有双硫键的还原响应型可降解支化聚赖氨酸基因载体(SS-HP).同时通过相似的方法合成了不含双硫键的支化聚赖氨酸基因载体(CC-HP)作为对照.聚阳离子/pDNA复合物的水合粒径和电位通过动态光散射仪测定,其在还原性环境中的降解情况通过凝胶阻滞电泳、原子力显微镜测试评价. SS-HP和CC-HP的体外转染能力及细胞毒性通过荧光素酶报告基因和MTT法在脑胶质瘤C6和肝癌HepG2细胞中测定.癌细胞中过量表达的谷胱甘肽能够使SS-HP在细胞内降解,加速pDNA的释放,使该载体有良好的基因转染能力.同时,载体的可降解性以及由开环反应给载体引入的大量羟基,使载体有较低的细胞毒性. KillerRed蛋白是一种在可见光照射下能产生单线态氧的红色荧光蛋白.使用SS-HP携载pKillerRed (pKR)质粒进行体外光动力抗癌评价. SS-HP/pKR转染C6细胞后能成功表达出KillerRed蛋白,光照后可以促进肿瘤细胞的凋亡.
Gene therapy is widely concerned as an excellent treatment for cancer. One of the most important things in gene therapy is to construct gene delivery systems with good biodegradability, biocompatibility, and gene delivery capability. Biodegradable non-viral gene vectors based on different microenvironments between cancer cells and normal cells have been paid more attention. In this work, the reduction-responsive branched polylysine(SS-HP) with disulfide bonds was synthesized via a one-pot ring-opening reaction. To make a comparison, the branched polylysine without disulfide bonds(CC-HP) was synthesized by the same method. All the SS-HP/pDNA and CC-HP/pDNA complexes with various weight ratios were prepared by mixing polycationbased solution and pDNA solution completely, and stood for 30 min. The particle size and zeta potential of SSHP/pDNA and CC-HP/pDNA were measured by dynamic light scattering(DLS). The degradability of polyplexes in reductive environment was visualized by agarose gel electrophoresis and atomic force microscopy(AFM). The in vitro transfection efficiencies and cell viability of SS-HP and CC-HP were evaluated in C6 and HepG2 cell lines using luciferase reporter gene, green fluorescence protein gene, and MTT assay. The concentration of reductive glutathione(GSH) is higher in some cancer cells than that in normal cells. SS-HP showed high gene transfection efficiency in vitro due to the breakdown of reduction-responsive disulfide bonds. Moreover, SS-HP exhibited low cytotoxicity due to the good biodegradability of SS-HP and plenty of hydroxy groups induced by ring-opening reactions. KillerRed protein is a red fluorescence protein which could produce reactive oxygen species(ROS) upon the induction of visible light. From in vitro antitumor assays, the plasmid pKillerRed(pKR)delivered by SS-HP was expressed in C6 cells. KillerRed protein expressed in C6 cells could contribute to the cell apoptosis via photodynamic therapy(PDT). This study provides a novel approach for designing the nextgeneration gene delivery systems.
Gene therapy is widely concerned as an excellent treatment for cancer. One of the most important things in gene therapy is to construct gene delivery systems with good biodegradability, biocompatibility, and gene delivery capability. Biodegradable non-viral gene vectors based on different microenvironments between cancer cells and normal cells have been paid more attention. In this work, the reduction-responsive branched polylysine(SS-HP) with disulfide bonds was synthesized via a one-pot ring-opening reaction. To make a comparison, the branched polylysine without disulfide bonds(CC-HP) was synthesized by the same method. All the SS-HP/pDNA and CC-HP/pDNA complexes with various weight ratios were prepared by mixing polycationbased solution and pDNA solution completely, and stood for 30 min. The particle size and zeta potential of SSHP/pDNA and CC-HP/pDNA were measured by dynamic light scattering(DLS). The degradability of polyplexes in reductive environment was visualized by agarose gel electrophoresis and atomic force microscopy(AFM). The in vitro transfection efficiencies and cell viability of SS-HP and CC-HP were evaluated in C6 and HepG2 cell lines using luciferase reporter gene, green fluorescence protein gene, and MTT assay. The concentration of reductive glutathione(GSH) is higher in some cancer cells than that in normal cells. SS-HP showed high gene transfection efficiency in vitro due to the breakdown of reduction-responsive disulfide bonds. Moreover, SS-HP exhibited low cytotoxicity due to the good biodegradability of SS-HP and plenty of hydroxy groups induced by ring-opening reactions. KillerRed protein is a red fluorescence protein which could produce reactive oxygen species(ROS) upon the induction of visible light. From in vitro antitumor assays, the plasmid pKillerRed(pKR)delivered by SS-HP was expressed in C6 cells. KillerRed protein expressed in C6 cells could contribute to the cell apoptosis via photodynamic therapy(PDT). This study provides a novel approach for designing the nextgeneration gene delivery systems.
引文
1 Fan W,Yung B,Huang P,Chen X.Chem Rev,2017,117(22):13566-13638
2 Mahalingam S M,Kularatne S A,Myers C H,Gagare P,Norshi M,Liu X,Singhal S,Low P S.J Med Chem,2018,61(21):9637-9646
3 Chen J,Tian H,Dong X,Guo Z,Jiao Z,Li F,Kano A,Maruyama A,Chen X.Macromol Biosci,2013,13(10):1438-1446
4 Guan X,Li Y,Jiao Z,Lin L,Chen J,Guo Z,Tian H,Chen X.ACS Appl Mater Interfaces,2015,7(5):3207-3215
5 Lin Lin(林琳),Guo Zhaopei(郭兆培),Chen Jie(陈杰),Tian Huayu(田华雨),Chen Xuesi(陈学思).Acta PolymericaSinica(高分子学报),2017,(2):321-328
6 Wang Y,Xiao H,Fang J,Yu X,Su Z,Cheng D,Shuai X.Chem Commun,2016,52(6):1194-1197
7 Liu X,Xiang J J,Zhu D C,Jiang L M,Zhou Z X,Tang J B,Liu X R,Huang Y Z,Shen Y Q.Adv Mater,2016,28(9):1743-1752
8 Putnam D.Nat Mater,2006,5(6):439-451
9 Fu Huili(付慧莉),Cheng Sixue(程巳雪),Zhuo Renxi(卓仁禧).Acta Polymerica Sinica(高分子学报),2009,(2):97-103
10 Kim H,Kim W J.Small,2014,10(1):117-126
11 Yamano S,Dai J,Hanatani S,Haku K,Yamanaka T,Ishioka M,Takayama T,Yuvienco C,Khapli S,Moursi A M,Montclare J K.Biomaterials,2014,35(5):1705-1715
12 Qian X,Long L,Shi Z,Liu C,Qiu M,Sheng J,Pu P,Yuan X,Ren Y,Kang C.Biomaterials,2014,35(7):2322-2335
13 Li Y,Bing X,Tao B,Liu W.Biomaterials,2015,55:12-23
14 Ma D,Lin Q M,Zhang L M,Liang Y Y,Xue W.Biomaterials,2014,35(14):4357-4367
15 Zhang Qian(张倩),Yuan Zhi(袁直).Acta Polymerica Sinica(高分子学报),2018,(7):776-785
16 Wang D,Zhao T,Zhu X,Yan D,Wang W.Chem Soc Rev,2015,44(12):4023-4071
17 Jin H,Huang W,Zhu X,Zhou Y,Yan D.Chem Soc Rev,2012,41(18):5986-5997
18 Huang Y,Wang D,Zhu X,Yan D,Chen R.Polym Chem,2015,6(15):2794-2812
19 Wang K,Peng H,Thurecht K J,Puttick S,Whittaker A K.Polym Chem,2016,7(5):1059-1069
20 Huang Y,Ding X,Qi Y,Yu B,Xu F J.Biomaterials,2016,106:134-143
21 Fletcher N L,Houston Z H,Simpson J D,Veedu R N,Thurecht K J.Chem Commun,2018,54(82):11538-11541
22 Zhang C,Moonshi S S,Wang W,Ta H T,Han Y,Han F Y,Peng H,Kral P,Rolfe B E,Gooding J J,Gaus K,Whittaker A K.ACS Nano,2018,12(9):9162-9176
23 Zhang S,Liu Y,Derakhshanfar S,He W,Huang Q,Dong S,Rao J,Luo G X,Zhong W,Liao W,Shi M,Xing M.AdvHealthc Mater,2018:1800118
24 Xu H,Cao W,Zhang X.Acc Chem Res,2013,46(7):1647-1658
25 Li Yunkun(李芸焜),Li Yachao(李亚超),Xu Xianghui(徐翔晖),Gu Zhongwei(顾忠伟).Acta Polymerica Sinica(高分子学报),2017,(2):375-385
26 Liu H,Wang H,Yang W,Cheng Y.J Am Chem Soc,2012,134:17680-17687
27 Duan S,Yu B,Gao C,Yuan W,Ma J,Xu F J.ACS Appl Mater Interfaces,2016,8(43):29334-29342
28 Qi Y,Song H,Xiao H,Cheng G,Yu B,Xu F J.Small,2018,14(42):1803061
29 Liao Z,Li Y,Lu H,Sung H.Biomaterials,2014,35(1):500-508
30 Tseng S,Liao Z,Kao S,Zeng Y,Huang K,Li H,Yang C,Deng Y,Huang C,Yang S,Yang P,Kempson I.NatCommun,2015,6:6456
31 Ping Y,Liu C D,Tang G P,Li J S,Li J,Yang W T,Xu F J.Adv Funct Mater,2010,20(18):3106-3116
2 Mahalingam S M,Kularatne S A,Myers C H,Gagare P,Norshi M,Liu X,Singhal S,Low P S.J Med Chem,2018,61(21):9637-9646
3 Chen J,Tian H,Dong X,Guo Z,Jiao Z,Li F,Kano A,Maruyama A,Chen X.Macromol Biosci,2013,13(10):1438-1446
4 Guan X,Li Y,Jiao Z,Lin L,Chen J,Guo Z,Tian H,Chen X.ACS Appl Mater Interfaces,2015,7(5):3207-3215
5 Lin Lin(林琳),Guo Zhaopei(郭兆培),Chen Jie(陈杰),Tian Huayu(田华雨),Chen Xuesi(陈学思).Acta PolymericaSinica(高分子学报),2017,(2):321-328
6 Wang Y,Xiao H,Fang J,Yu X,Su Z,Cheng D,Shuai X.Chem Commun,2016,52(6):1194-1197
7 Liu X,Xiang J J,Zhu D C,Jiang L M,Zhou Z X,Tang J B,Liu X R,Huang Y Z,Shen Y Q.Adv Mater,2016,28(9):1743-1752
8 Putnam D.Nat Mater,2006,5(6):439-451
9 Fu Huili(付慧莉),Cheng Sixue(程巳雪),Zhuo Renxi(卓仁禧).Acta Polymerica Sinica(高分子学报),2009,(2):97-103
10 Kim H,Kim W J.Small,2014,10(1):117-126
11 Yamano S,Dai J,Hanatani S,Haku K,Yamanaka T,Ishioka M,Takayama T,Yuvienco C,Khapli S,Moursi A M,Montclare J K.Biomaterials,2014,35(5):1705-1715
12 Qian X,Long L,Shi Z,Liu C,Qiu M,Sheng J,Pu P,Yuan X,Ren Y,Kang C.Biomaterials,2014,35(7):2322-2335
13 Li Y,Bing X,Tao B,Liu W.Biomaterials,2015,55:12-23
14 Ma D,Lin Q M,Zhang L M,Liang Y Y,Xue W.Biomaterials,2014,35(14):4357-4367
15 Zhang Qian(张倩),Yuan Zhi(袁直).Acta Polymerica Sinica(高分子学报),2018,(7):776-785
16 Wang D,Zhao T,Zhu X,Yan D,Wang W.Chem Soc Rev,2015,44(12):4023-4071
17 Jin H,Huang W,Zhu X,Zhou Y,Yan D.Chem Soc Rev,2012,41(18):5986-5997
18 Huang Y,Wang D,Zhu X,Yan D,Chen R.Polym Chem,2015,6(15):2794-2812
19 Wang K,Peng H,Thurecht K J,Puttick S,Whittaker A K.Polym Chem,2016,7(5):1059-1069
20 Huang Y,Ding X,Qi Y,Yu B,Xu F J.Biomaterials,2016,106:134-143
21 Fletcher N L,Houston Z H,Simpson J D,Veedu R N,Thurecht K J.Chem Commun,2018,54(82):11538-11541
22 Zhang C,Moonshi S S,Wang W,Ta H T,Han Y,Han F Y,Peng H,Kral P,Rolfe B E,Gooding J J,Gaus K,Whittaker A K.ACS Nano,2018,12(9):9162-9176
23 Zhang S,Liu Y,Derakhshanfar S,He W,Huang Q,Dong S,Rao J,Luo G X,Zhong W,Liao W,Shi M,Xing M.AdvHealthc Mater,2018:1800118
24 Xu H,Cao W,Zhang X.Acc Chem Res,2013,46(7):1647-1658
25 Li Yunkun(李芸焜),Li Yachao(李亚超),Xu Xianghui(徐翔晖),Gu Zhongwei(顾忠伟).Acta Polymerica Sinica(高分子学报),2017,(2):375-385
26 Liu H,Wang H,Yang W,Cheng Y.J Am Chem Soc,2012,134:17680-17687
27 Duan S,Yu B,Gao C,Yuan W,Ma J,Xu F J.ACS Appl Mater Interfaces,2016,8(43):29334-29342
28 Qi Y,Song H,Xiao H,Cheng G,Yu B,Xu F J.Small,2018,14(42):1803061
29 Liao Z,Li Y,Lu H,Sung H.Biomaterials,2014,35(1):500-508
30 Tseng S,Liao Z,Kao S,Zeng Y,Huang K,Li H,Yang C,Deng Y,Huang C,Yang S,Yang P,Kempson I.NatCommun,2015,6:6456
31 Ping Y,Liu C D,Tang G P,Li J S,Li J,Yang W T,Xu F J.Adv Funct Mater,2010,20(18):3106-3116