三氧化二砷聚乙二醇-聚乳酸偶联人源抗血管内皮细胞生长因子受体2隐形纳米粒的抗肝癌机制探讨

摘要

目的：探讨以聚乙二醇-聚乳酸（ PEG-PLA）为载体材料包载的三氧化二砷（ As2 O3），同时偶联人源抗血管内皮细胞生长因子受体2（VEGFR-2）的隐形纳米粒的抗肝癌机制。方法以PEG-PLA为载体材料， W／O／W型超声乳化制备 As2 O3纳米粒，同时偶联具有肝癌靶向作用的VEGFR-2，得到人源抗VEGFR-2／As2 O3-PEG-PLA纳米粒，同时以相同方法制作香豆素6（ C6）-PEG-PLA及抗VEGFR-2／C6-PEG-PLA纳米粒。测定纳米粒粒径分布、 Zata电位； MTT法计算As2 O3、 As2 O3-PEG-PLA及抗VEGFR-2／As2 O3-PEG-PLA对Bel 7402肝癌细胞IC50值；采用流式细胞实验和激光共聚焦显微镜实验检测人肝癌 Bel 7402细胞摄取 C6-PEG-PLA及抗VEGFR-2／C6-PEG-PLA纳米粒的速度和强度；将18只肝癌裸鼠采用随机数字表法分为3组，每组分别经尾静脉注射As2 O3、 As2 O3-PEG-PLA、抗VEGFR-2／As2 O3-PEG-PLA溶液，给药后12 h处死小鼠，取肿瘤、脾、心、肺、肝、肾等组织，测试其中As2 O3浓度；再将另24只肝癌裸鼠采用随机数字表法分为4组，每组分别经尾静脉注射As2 O3、 As2 O3-PEG-PLA、抗VEGFR-2／As2 O3-PEG-PLA及0.9％氯化钠溶液，给药后第17 d，处死小鼠，计算肿瘤抑制率。结果抗VEGFR-2／As2O3-PEG-PLA平均粒径为（141.9±13.2） nm （PDI＝0.23），呈正态分布， Zata电位为（10.2±1.1） mV。 As2O3、 As2O3-PEG-PLA、抗VEGFR-2／As2O3-PEG-PLA的 IC50值分别为（1.53±0.22）μmol／L，（2.30±0.18）μmol／L和（1.80±0.22）μmol／L。不同剂型IC50值比较，差异有统计学意义（F＝4.503， P＝0.018）。不同时间点人肝癌Bel 7402细胞摄取抗VEGFR-2／C6-PEG-PLA的速度均高于C6-PEG-PLA，差异有统计学意义（t＝2.012， P＝0.045； t＝2.231， P＝0.035； t＝2.311， P＝0.022）。 As2O3组不同组织As2O3含量比较，差异有统计学意义（F＝5.568， P＝0.018）； As2O3-PEG-PLA组不同组织As2O3含量比较，差异有统计学意义（F＝4.659， P＝0.034）；抗VEGFR-2／As2O3-PEG-PLA组不同组织As2O3含量比较，差异有统计学意义（F＝5.001， P＝0.022）。对照组、 As2 O3组、 As2 O3-PEG-PLA组和抗VEGFR-2／As2 O3-PEG-PLA组肿瘤抑制率分别为32.6％、45.9％、66.2％和87.9％。4组肿瘤抑制率比较，差异有统计学意义（χ2＝4.189， P＝0.018）。结论以PEG-PLA为载体材料制备得到As2 O3纳米制剂，且偶联具有肝癌靶向作用的VEGFR-2，用于肝癌的治疗，可显著提高As2 O3的生物利用度，为构建As2 O3肝癌靶向纳米递送系统提供了理论依据。%Objective To investigate the anti -hepatoma mechanism of human anti -VEGFR-2/As2 O3 -PEG-PLA stealth nanoparticles . Methods PEG-PLA was used as the vector material to prepare As 2 O3 nanoparticles with W/O/W ultrasonic emulsification method , and VEGFR-2 that is targeted at liver cancer was coupled to obtain human anti -VEGFR-2/As2 O3 -PEG-PLA nanoparticles.C6 -PEG-PLA and anti -VEGFR-2/C6 -PEG-PLA stealth nanoparticles were also prepared using the same method .Size distribution and Zata potential of the nanoparticles were characterized using laser particle analyzer; IC50 values of As2 O3 , As2 O3 -PEG-PLA and anti -VEGFR-2/As2 O3 -PEG-PLA against liver cancer cell Bel 7402 were determined using MTT method; laser scanning confocal microscope and flow cytometry were used to measure the speed and intensity of the uptake of C 6 -PEG-PLA and anti -VEGFR-2/C6 -PEG-PLA nanoparticles by liver cancer cell Bel 7402; using random number table method , we divided 18 nude mice with liver cancer into three groups and assigned the three groups to receive the injection of As 2 O3 , As2 O3 -PEG-PLA and anti -VEGFR-2/As2 O3 -PEG-PLA solutions respectively through caudal veins , and we killed the mice 12 months after the injection and obtained their tumors , spleens, hearts, lungs, livers and kidneys to measure the As 2 O3 concentration within these tissues; using random number table method , we divided another 24 nude mice with lung cancer and assigned the three groups to receive the injection of As 2 O3 , As2 O3 -PEG-PLA, anti-VEGFR-2/As2 O3 -PEG-PLA and 0.9% sodium chloride solutions respectively through caudal veins , and we killed the mice 17 days after the injection and calculated the tumor inhibiting rates . Results The average particle size of anti -VEGFR-2/As2O3 -PEG-PLA was (141.9 ±13.2) nm (PDI=0.23) with a normal distribution.The Zata potential of anti-VEGFR-2/As2 O3 -PEG-PLA was ( 10.2 ±1.1 ) mV.The IC50 values of As2 O3 , As2 O3 -PEG-PLA and anti -VEGFR-2/As2 O3 -PEG-PLA were ( 1.53 ±0.22 ) μmol/L, ( 2.30 ±0.18 ) μmol/L and ( 1.80 ±0.22 ) μmol/L respectively, with the differences significant (F=4.503, P=0.018).At different time points, lung cancer Bel 7402 cells were faster in the uptake of anti -VEGFR-2/C6-PEG-PLA than in the uptake of C6-PEG-PLA ( t=2.012, P=0.045;t=2.231, P=0.035; t=2.311, P=0.022).In the As2O3 group, As2O3 content varied significantly with different tissues (F=5.568, P=0.018); in the As2O3 -PEG-PLA group, As2O3 content varied significantly with different tissues (F=4.659, P=0.034); in the anti -VEGFR-2/As2O3 -PEG-PLA group, As2O3 content varied significantly with different tissues (F=5.001, P=0.022).The cancer inhibition rates of control group , As2O3 group, As2O3 -PEG-PLA group and anti-VEGFR-2/As2 O3 -PEG-PLA group were 32.6%, 45.9%, 66.2% and 87.9% respectively , with the differences significant (χ2 =4.189, P=0.018).Conclusion The As2O3 nanoparticles prepared with PEG-PLA as vector and coupled with VEGFR-2 that is targeted at liver cancer can evidently improve the bioavailability of As 2 O3 and provide theoretical basis for the building of As2O3 liver cancer-targeted nanometer delivery system.