载三氧化二砷脑胶质瘤靶向纳米递药系统i RGD/TGN-PEG-PAMAM/ATO的构建及体外研究
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摘要
目的以树状大分子为载体材料并修饰血脑屏障(blood brain barrier,BBB)靶向短肽TGN和肿瘤靶向短肽i RGD构建脑胶质瘤靶向递药系统(i RGD/TGN-PEG-PAMAM/ATO),旨在解决三氧化二砷(arsenictrioxide,As2O3,ATO)在治疗脑胶质瘤过程中分布缺乏特异性、透BBB难等问题,使其具有更好抗脑胶质瘤作用。方法核磁共振图谱(1H-NMR)、透射电子显微镜(TEM)等考察载体的理化性质;电感耦合等离子发射光谱(ICP)、透析袋法分析其包封率及体外释放情况;通过激光共聚焦及流式细胞仪分析i RGD、TGN对细胞摄取的影响;MTT法考察纳米载体对脑微血管内皮细胞(HBMEC)和脑胶质瘤U87细胞的毒性及BBB模型中递药系统抑制U87细胞生长的情况。结果成功合成了i RGD/TGN-PEG-PAMAM载体,其形态规整,大小均匀,测得其粒径(24.87±0.84)nm,电位(17.26±1.64)m V;该载体对HBMEC和U87细胞均具有较小的毒性;递药系统i RGD/TGN-PEG-PAMAM/ATO的包封率为(71.92±1.17)%,体外释放表明ATO经载体包载后呈现一种缓慢释放趋势,且在酸性条件下更有利于ATO的释放;细胞摄取结果提示iRGD/TGN的修饰有利于U87细胞对递药系统的摄取;体外跨BBB抑制U87细胞生长实验结果表明,i RGD/TGN-PEG-PAMAM/ATO组具有更好的跨BBB抑制U87细胞生长效果。结论 i RGD/TGN-PEG-PAMAM/ATO脑胶质瘤靶向递药系统具有较好的体外跨BBB抑制U87细胞生长的效果,为脑胶质瘤治疗提供了新的策略。
Objective To construct a glioma targeting delivery system, PAMAM G5 were modified with the oligopeptide of blood brain barrier(BBB) targeting TGN and tumor targeting oligopeptide iRGD to solve the problem of non-specificity in distribution and difficulty in permeating BBB of ATO, in order to have better anti-glioma effect. Methods The physical and chemical properties of nanocarriers were investigated by 1 H-NMR and transmission electron microscopy(TEM); The encapsulation efficiency and in vitro release were analyzed by inductively coupled plasma emission spectrum(ICP) and dialysis bag method; The effects of iRGD and TGN on cellular uptake of the carriers were analyzed by laser confocal and flow cytometry. The cytotoxicity of nanocarriers on brain microvascular endothelial cells(HBMEC) and glioma cells(U87), the inhibition effect on U87 cells of drug delivery systems after acrossing the BBB model in vitro were investigated by MTT method. Results The iRGD/TGN-PEG-PAMAM was synthesized successfully. The TEM results showed that iRGD/TGN-PEG-PAMAM was regular in shape and uniform in size. The particle size of i RGD/TGN-PEG-PAMAM/ATO was(24.87 ± 0.84) nm and the potential was(17.26 ± 1.64) mV. The synthesized carrier had less toxicity to HBMEC and U87 cells. The encapsulation efficiency of iRGD/TGN-PEG-PAMAM/ATO delivery system was(71.92 ±1.17)%. The in vitro release showed that ATO had a slow release trend after entrapment, and it was more favorable for ATO release under acidic conditions. The cell uptake indicated that iRGD/TGN modification was more beneficial for U87 cell to uptake the drug delivery system. The in vitro inhibition effect on U87 cells after acrossing the BBB model showed iRGD/TGN-PEG-PAMAM/ATO had better inhibition effect on U87 cells. Conclusion The iRGD/TGN-PEG-PAMAM/ATO targeting drug delivery system has good inhibition effect on U87 cells effect after acrossing the BBB model in vitro, which provides a new strategy for the treatment of glioma.
Objective To construct a glioma targeting delivery system, PAMAM G5 were modified with the oligopeptide of blood brain barrier(BBB) targeting TGN and tumor targeting oligopeptide iRGD to solve the problem of non-specificity in distribution and difficulty in permeating BBB of ATO, in order to have better anti-glioma effect. Methods The physical and chemical properties of nanocarriers were investigated by 1 H-NMR and transmission electron microscopy(TEM); The encapsulation efficiency and in vitro release were analyzed by inductively coupled plasma emission spectrum(ICP) and dialysis bag method; The effects of iRGD and TGN on cellular uptake of the carriers were analyzed by laser confocal and flow cytometry. The cytotoxicity of nanocarriers on brain microvascular endothelial cells(HBMEC) and glioma cells(U87), the inhibition effect on U87 cells of drug delivery systems after acrossing the BBB model in vitro were investigated by MTT method. Results The iRGD/TGN-PEG-PAMAM was synthesized successfully. The TEM results showed that iRGD/TGN-PEG-PAMAM was regular in shape and uniform in size. The particle size of i RGD/TGN-PEG-PAMAM/ATO was(24.87 ± 0.84) nm and the potential was(17.26 ± 1.64) mV. The synthesized carrier had less toxicity to HBMEC and U87 cells. The encapsulation efficiency of iRGD/TGN-PEG-PAMAM/ATO delivery system was(71.92 ±1.17)%. The in vitro release showed that ATO had a slow release trend after entrapment, and it was more favorable for ATO release under acidic conditions. The cell uptake indicated that iRGD/TGN modification was more beneficial for U87 cell to uptake the drug delivery system. The in vitro inhibition effect on U87 cells after acrossing the BBB model showed iRGD/TGN-PEG-PAMAM/ATO had better inhibition effect on U87 cells. Conclusion The iRGD/TGN-PEG-PAMAM/ATO targeting drug delivery system has good inhibition effect on U87 cells effect after acrossing the BBB model in vitro, which provides a new strategy for the treatment of glioma.
引文
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[14]蔡宝,魏为添,刘岸,等.三氧化二砷对胰腺癌BXPC-细胞移植瘤的体内抑制作用[J].中草药,2010,41(1):90-93.
[15]Sun L Y,Wang H,Zhou J.Clinical implementation of arsenic trioxide[J].Chin Herb Med,2016,8(4):308-313.
[16]Wang K,Zhang X,Liu Y,et al.Tumor penetrability and anti-angiogenesis using iRGD-mediated delivery of doxorubicin-polymer conjugates[J].Biomaterials,2014,35(30):8735-8747.
[17]Qian Y,Zha Y,Feng B,et al.PEGylated poly(2-(dimethylamino)ethyl methacrylate)/DNA polyplex micelles decorated with phage-displayed TGN peptide for brain-targeted gene delivery[J].Biomaterials,2013,34(8):2117-2129.
[18]Du J,Lu W L,Ying X,et al.Dual-targeting topotecan liposomes modified with Tamoxifen and wheat germ agglutinin significantly improve drug transport across the blood-brain barrier and survival of brain tumor-bearing animals[J].Mol Pharm,2009,6(3):905-917.
[19]Xiong Z,Shen M,Shi X.Dendrimer-based strategies for cancer therapy:Recent advances and future perspectives[J].Sci China Mater,2018,61(11):1387-1403.
[2]Gladson C L,Prayson R A,Liu W M.The pathobiology of glioma tumors[J].Annu Rev Pathol-Mech,2010,5(1):33-50.
[3]Jiang Y,Lv L,Shi H,et al.PEGylated polyamidoamine dendrimer conjugated with tumor homing peptide as a potential targeted delivery system for glioma[J].Colloid Surface B,2016,147:242-249.
[4]高会乐.基于不同靶向策略的脑肿瘤靶向递药系统设计及评价[D].上海:复旦大学,2013.
[5]张洪兵,张铁军,李亚卓,等.中药新型给药系统局部药动学研究概况[J].中草药,2015,46(21):3258-3263.
[6]Maryam G,Gholamreza D,Fereydoon A G,et al.Surface functionalized dendrimers as controlled-release delivery nanosystems for tumor targeting[J].Eur J Pharm Sci,2018,122:311-330.
[7]Puig-Saus C,Rojas L A,Laborda E,et al.i RGDtumor-penetrating peptide-modified oncolytic adenovirus shows enhanced tumor transduction,intratumoral dissemination and antitumor efficacy[J].Gene Ther,2014,21(8):767-774.
[8]Liu C,Yao S,Li X,et al.iRGD-mediated core-shell nanoparticles loading carmustine and O6-benzylguanine for glioma therapy[J].J Drug Target,2017,25(3):12.
[9]许婷,李秀妍,李琳,等.纳米有机金属框架与精氨酸-甘氨酸-天冬氨酸序列短肽偶联最佳工艺[J].中草药,2015,46(10):1460-1463.
[10]Zhang C,Wan X,Zheng X,et al.Dual-functional nanoparticles targeting amyloid plaques in the brains of Alzheimer’s disease mice[J].Biomaterials,2014,35(1):456-465.
[11]李婧炜.噬菌体展示技术筛选脑靶向功能肽及其修饰纳米粒的脑内递药研究[D].上海:复旦大学,2012.
[12]张燕,费伟东,陶姣阳,等.Angiopep-2修饰的载三氧化二砷介孔二氧化硅脂质囊纳米递药系统的构建及体外评价[J].中草药,2018,49(6):1289-1297.
[13]郭曼曼,王国伟,徐骏军,等.载三氧化二砷pH值响应介孔二氧化硅纳米粒的制备及体内外评价[J].中草药,2015,46(7):982-989.
[14]蔡宝,魏为添,刘岸,等.三氧化二砷对胰腺癌BXPC-细胞移植瘤的体内抑制作用[J].中草药,2010,41(1):90-93.
[15]Sun L Y,Wang H,Zhou J.Clinical implementation of arsenic trioxide[J].Chin Herb Med,2016,8(4):308-313.
[16]Wang K,Zhang X,Liu Y,et al.Tumor penetrability and anti-angiogenesis using iRGD-mediated delivery of doxorubicin-polymer conjugates[J].Biomaterials,2014,35(30):8735-8747.
[17]Qian Y,Zha Y,Feng B,et al.PEGylated poly(2-(dimethylamino)ethyl methacrylate)/DNA polyplex micelles decorated with phage-displayed TGN peptide for brain-targeted gene delivery[J].Biomaterials,2013,34(8):2117-2129.
[18]Du J,Lu W L,Ying X,et al.Dual-targeting topotecan liposomes modified with Tamoxifen and wheat germ agglutinin significantly improve drug transport across the blood-brain barrier and survival of brain tumor-bearing animals[J].Mol Pharm,2009,6(3):905-917.
[19]Xiong Z,Shen M,Shi X.Dendrimer-based strategies for cancer therapy:Recent advances and future perspectives[J].Sci China Mater,2018,61(11):1387-1403.