精氨酸-甘氨酸-天冬氨酸修饰的星型高分子负载雷珠单抗对脉络膜新生血管靶向治疗作用

Targeted therapy effect of arginine-glycine-aspartate modified star polymer loaded ranibizumab on choroidal neovascularization

目的探讨精氨酸-甘氨酸-天冬氨酸(RGD)修饰的聚乙二醇星型高分子(S-PEG)负载雷珠单抗(RBZ)通过静脉注射方式对激光诱导小鼠脉络膜新生血管(CNV)的靶向治疗作用。方法分别采用Nano-ZS型纳米粒度分析仪及Zeta电位分析仪对合成的材料S-PEG-ICG-RGD-RBZ NPs(SPIRR NPs)进行表征测试,应用SPIRR NPs分别对人视网膜色素上皮细胞系ARPE-19以及经血管内皮生长因子(VEGF)诱导后的人脐静脉内皮细胞(HUVEC)进行干预,CCK-8法检测细胞毒性,管腔形成实验检测管腔形成情况。建立C57BL/6小鼠CNV模型,尾静脉注射SPIRR NPs后行眼底照相、眼底血管造影检查,并对CNV面积进行测量,检测小鼠肝、肾功能及电解质水平变化以观察SPIRR NPs在体内的生物安全性。结果成功制备材料SPIRR NPs并对其进行表征,在所研究的浓度范围内,SPIRR NPs具有良好的体外安全性。在HUVEC管腔形成实验中,正常对照组、VEGF干预组、低浓度治疗组、高浓度治疗组管腔节数分别为55.00±16.00、110.00±13.00、44.67±14.36及25.33±17.00,管腔节点数分别为16.00±4.58、31.67±5.51、12.67±5.03及8.00±5.20,管腔节段数分别为20.67±7.37、45.33±8.08、14.33±6.03及7.67±6.66,相对管腔长度分别为1.00±0.00、1.51±0.08、0.97±0.18及0.75±0.15。与正常对照组相比,VEGF干预组的管腔节数、节点数、节段数及相对管腔长度均有大幅度增加,两治疗组各指标均较VEGF干预组显著降低(均为P<0.05)。静脉注射后,脉络膜ICGA可见SPIRR NPs靶向至CNV病灶部位并长期留存,FFA可见荧光渗漏信号减弱,RPE-脉络膜-巩膜铺片可见CNV病灶面积明显缩小。结论RGD修饰的S-PEG作为一种强有力的载体,可以将抗VEGF药物运送到CNV区域,并且其具有良好的细胞相容性,在CNV治疗中具有很大的应用潜力。

Objective To investigate the targeted therapeutic effect of arginine-glycine-aspartate(RGD)-modified star shaped polymer(S-PEG)loaded with ranibizumab(RBZ)on laser-induced choroidal neovascularization(CNV)in mice.Methods Nano-ZS nanoparticle size analyzer and Zeta potential analyzer were used to characterize S-PEG-ICG-RGD-RBA NPs(SPIRR NPs).SPIRR NPs was applied to intervene ARPE-19 cells and human umbilical vein epithelial cells(HUVEC)induced by vascular endothelial growth factor(VEGF).We used CCK-8 method to detect cytotoxicity and cell tubule formation assay to detect lumen formation.After the CNV model of C57BL/6 mice was established and intravenous injection SPIRR NPs,fundus photography and fundus angiography were performed,and CNV area was also measured.Besides,the liver and kidney functions and electrolyte levels were detected to observe the biological safety of SPIRR NPs in vivo.Results SPIRR NPs was successfully prepared and characterized.Within the concentration range studied,SPIRR NPs had shown safety in vitro and could inhibit VEGF induced catheter formation.In HUVEC tubule formation assay,the lumen section numbers of the normal control group,the VEGF-intervention group,the low-concentration therapy group and the high-concentration therapy group were 55.00±16.00,110.00±13.00,44.67±14.36 and 25.33±17.00.Besides,the number of lumen nodes were 16.00±4.58,31.67±5.51,12.67±5.03 and 8.00±5.20,the number of lumen segments were 20.67±7.37,45.33±8.08,14.33±6.03 and 7.67±6.66 and the relative length of lumens were 1.00±0.00,1.51±0.08,0.97±0.18 and 0.75±0.15.Compared with the normal control group,the number of lumen sections,lumen nodes,lumen segments and the relative length of lumens of the VEGF-intervention group had shown an obviously increase.Notably,these parameters of the two therapy groups was much lower than those of the VEGF-intervention group(all P<0.05).After intravenous injection,SPIRR NPs were observed to target to the lesion site and remain for a long time by choroidal indocyanine green angiography.Fluorescence leakage signal was decreased in fundus fluorescein angiography,and CNV lesion area was significantly reduced by RPE-choroidal-sclera lamination.Conclusion As a powerful carrier,RGD modified S-PEG can deliver anti-VEGF drugs to CNV region and shows good cytocompatibility,which has great potential in the treatment of CNV.