人源抗VEGFR-2／As2O3-PEG-PLA隐形纳米粒的构建及质量控制研究

目的：探讨三氧化二砷（As2O3）纳米新剂型的制备和质量控制。方法以聚乙醇化聚乳酸（PEG‐PLA）为载体材料，W／O／W型超声乳化制备As2O3纳米粒，同时偶联具有肝癌靶向作用的VEGFR‐2，最终得到人源抗VEGFR‐2／As2O3‐PEG‐PLA纳米粒。对其粒径分布、Zata电位、载药量和包封率进行测定，通过透射电镜（TEM）观察其表观形态，同时考察其体外释药和稳定性。选择24只肝癌裸鼠，随机分为VEGFR‐2／As2O3‐PEG‐PLA组及As2O3‐PEG‐PLA组，通过尾静脉注射纳米粒，高效液相色谱法测定As2O3在两组裸鼠体内的分布；免疫组化及蛋白质印迹法（Westernblot）检测血管内皮细胞生长因子（VEGF）表达量。结果本实验制得的As2O3纳米制剂VEGFR‐2／As2O3‐PEG‐PLA粒径为（141．9±13．2）nm，Zata电位为（10．2±1．1）mV；经TEM观察呈圆形或椭圆形颗，粒状、大小较一致，分散性较好；载药量为（5．51±1．83）％，包封率为（62．12±5．98）％。体外释放发现其具有缓释效果，半数释放时间t1／2分别为10h；初步稳定性考察结果发现该制剂稳定性良好。与As2O3‐PEG‐PLA组比较，VEGFR‐2／As2O3‐PEG‐PLA组中肿瘤、肝组织的As2O3浓度较高，心、血液、肾组织的As2O3浓度较低（P＜0．05），且肿瘤组织中VEGFR‐2阳性率及蛋白表达较低。结论以PEG‐PLA为载体材料制备得到As2O3纳米制剂，且偶联具有肝癌靶向作用的VEGFR‐2，最终得到粒径分布均匀，包封率和载药量高，稳定性良好的纳米制剂，且初步证实在肝癌裸鼠体内具有良好的靶向作用。

Preparation and quality control of human anti-VEGFR-2/As2 O3-PEG-PLA nanoparticle

Objective To explore the preparation and quality control of As2 O3 nanoparticle .Methods PEG‐PLA was used as the vector material to prepare As2 O3 nanoparticle with ultrasonic emulsification method ,and the VEGFR‐2 was coupled to obtain VEGFR‐2/As2 O3‐PEG‐PLA nanoparticle .The particle size distribution ,Zata potential ,loading efficiency (LE) ,encapsulation effi‐ciency(EE) ,drug release in vitro and stability was determined ,and morphological characteristics was observed by transmission elec‐tron microscope(TEM) .Tweety‐four hepatocellular carcinoma nude mices were randomly divided into VEGFR‐2/As2O3‐PEG‐PLA nanoparticles group and As2 O3‐PEG‐PLA nanoparticles group ,by tail vein injection of nanoparticles .High performance liquid chro‐matography was used to determine content of As2 O3 .After 21 d ,six nude mices in each group were killed ,and the immunohisto‐chemistry and western blot method was used to detect the expression of VEGFR‐2 .Results The particle size of VEGFR‐2/As2 O3‐PEG‐PLA was determined to be (141 .9 ± 13 .2)nm ,Zata potential was (10 .2 ± 1 .1)mV .It was found to spherical or oval shape , with uniform size and dispersibility under TEM .LE and EE was (5 .51 ± 1 .83)% and (62 .12 ± 5 .98)% ,respectively .Drug release in vitro showed that VEGFR‐2/As2 O3‐PEG‐PLA exhibited controlled release effect ,with half of the release time as 10 h .Besides , VEGFR‐2/As2 O3‐PEG‐PLA showed a good stability in 3 days .Compared with As2 O3‐PEG‐PLA nanoparticles group ,the concen‐tration of As2 O3 in tumor and liver tissue was high ,the concentration of As2 O3 in blood ,heart ,kidney tissue was low ,the expression of VEGFR‐2 in tumor tissue was low in VEGFR‐2/As2O3‐PEG‐PLA nanoparticles group(P< 0 .05) .Conclusion The prepared As2 O3 nanoparticle using PEG‐PLA as vector and VEGFR‐2 as target showed uniform size ,high EE and LE ,good stability .And it preliminarily proved that VEGFR‐2 could be targeted in nude mice .