^(131)I-VEGF siRNA/SPIO在人肝细胞癌移植瘤裸鼠体内的血液清除动力学及生物分布

The kinetics of blood clearance and biodistribution of ^(131)I-VEGF siRNA/SPIO in nude mice bearing human hepatocellular carcinoma

目的研究在外加磁场作用下^(131)I-VEGF si RNA/SPIO在人肝细胞癌移植瘤裸鼠体内的血液清除动力学及生物分布特性。方法以Bolton-Hunter法使VEGF si RNA标记上^(131)I,以氧化铁超顺磁性纳米颗粒(superparamagneticiron oxide nanoparticles,SPIO)包裹^(131)I-VEGF si RNA。以人肝细胞癌细胞株Hep G2细胞悬液臀部皮下注射建立人肝细胞癌移植瘤裸鼠模型。45只人肝细胞癌移植瘤裸鼠随机分成外加磁场组(尾静脉注射^(131)I-VEGF si RNA/SPIO+肿瘤部位外加磁场)、非外加磁场组(尾静脉注射^(131)I-VEGF si RNA/SPIO+肿瘤部位无外加磁场)及对照组(尾静脉注射^(131)I-VEGF si RNA+肿瘤部位无外加磁场)。然后进行:(1)血液清除动力学研究:三组人肝细胞癌移植瘤裸鼠(每组5只)尾静脉给药后,分别于0.5 h、1.0 h、1.5 h、2.0 h、3.0 h、4.0 h、6.0 h、8.0 h、10.0 h、12.0 h时间点尾静脉采血20μL,测量血样每分钟放射性计数(counts per minute,cpm)值并绘制放射性-时间曲线,计算血液半衰期;(2)体内生物分布研究:三组人肝细胞癌移植瘤裸鼠尾静脉给药1 h后进行SPECT显像(每组5只)及MRI显像(每组5只),SPECT及MRI显像完毕,依次摘取移植瘤裸鼠肿瘤、皮肤、肌肉、骨、甲状腺、胃、小肠、大肠、肺、脾、性腺、肝、心、肾、膀胱等脏器称重并测量cpm值,然后计算各离体组织的%ID/g[即组织的放射性比活度(cpm/g)/注入标记物的放射性比活度(cpm/g)]。结果本研究分别以薄层层析硅胶板为载体、1∶1丙酮-生理盐水为展开剂和以新华一号滤纸为载体、1∶1甲醇-5%醋酸铵为展开剂测定^(131)I标记VEGF si RNA的放化纯分别为81.15%和84.05%。外加磁场组、非外加磁场组及对照组移植瘤裸鼠的血液半衰期分别约为(2.27±0.14)h、(2.93±0.20)h和(3.06±0.23)h,外加磁场组半衰期小于其它两组(差异有统计学意义,P<0.01)。SPECT显像显示外加磁场组肿瘤局部明显放射性增浓,非外加磁场组及对照组肿瘤局部未见明显放射性增浓;尾静脉给药前后MRI T1WI显示外加磁场组肿瘤局部信号明显强化,非外加磁场组及对照组肿瘤局部信号未见明显强化;外加磁场组肿瘤组织的%ID/g分布较非外加磁场组及对照组的均明显增高(P<0.01)。结论在外加磁场的作用下,以SPIO作为si RNA载体能够较成功地将^(131)I-VEGF si RNA转导至人肝细胞癌移植瘤裸鼠臀部皮下的肿瘤部位,对进一步研究肝细胞癌的VEGF靶向治疗、基因治疗以及示踪体内基因转导均有重要的意义。

Objective To explore the kinetics of blood clearance and biodistribution of ^131I-VEGF siRNA/SPIO in nude mice subcutaneously xenografted with human hepatocellular carcinoma （HCC） mediated by external magnetic field （EMF）. Methods VEGF siRNA was labeled with ^131I through Bohon-Hunter method, and a kind of superparamagnetic iron oxide nanoparticles （SPIO） named SilenceMag was employed as gene carrier. The BALB/c nude mouse xenograft model was established by transplanting HCC cell strain （Hep G2） subcutaneously in the right buttock, and 45 of them were randomly divided into EMF group （injection of ^131I-VEGF siRNA/SPIO through the tail vein ＋ EMF on the tumor ）, non-EMF group （injection of ^131I-VEGF siRNA/SPIO through the tail vein ＋ without EMF on the tumor ） and control group （injection of ^131I- VEGF siRNA through the tail vein ＋ without EMF on the tumor ）. （1）Kinetics of blood clearance was carried out by measuring the cpm （counts per minute） of 20 μL tail vein blood in different time points of 0.5 h, 1.0 h, 1.5 h, 2.0 h, 3.0 h, 4.0 h, 6.0 h, 8.0 h, 10.0 h and 12.0 h after ^131I-VEGF siRNA/SPIO administered through the tail vein, then the activity-time curve and the half-life in blood were achieved. （2）For the biodistibution research, three groups of nude mice were SPECT （each group was 5） and MRI （each group was 5） imaged after ^131I-VEGF siRNA/SPIO was administered the tail vein, and after that the tissues of tumor, skin, muscle, bone, thyroid, stomach, small intestine, large intestine, lung, spleen, gonad, liver, heart, kidney and bladder were successively taken and weighed, and then the cpm and %ID/g （percentage of injected doses per gram of tissue） of each tissue measured. Results Tow methods of thin layer chromatography （TLC） of the ^131I-VEGF siRNA/SPIO product indicated good radiochemical purity, 81.15% for Silica gel plate as the stationary phase and a mixture of freshly prepared acetone and normal saline （1： 1） as the mobile phase, 84.05% for Xinhua Filter Paper No.1 as the stationary phase and a mixture of freshly prepared methanol and 5% ammonium acetate （1 ： 1） as the mobile phase. ^131I-VEGF siRNA/SPIO remained in the blood for a short time in the tumor-bearing nude mice, and the half-life were （2.27±0.14） h for the EMF group, （2.93± 0.20） h for the non-EMF group and （3.06±0.23） h for the control group, respectively. The half-life in blood of the EMF group was even shorter than that of the other two groups （P〈 0.01）. In the SPECT and MRI study, ^131I-VEGF siRNA/SPIO accumulated more in the tumor in the EMF group nude mice, and the other two group showed no obvious accumulation in the tumor. The %ID/g of ^131I-VEGF siRNA/SPIO in tumors of the EMF group nude mice was much greater than that of the other two groups （P 〈 0.01）. Conclusion Mediated by EMF, SPIO can successfully deliver VEGF siRNA to the tumors in nude mice bearing HCC, and it will contribute a lot to the target therapy and gene therapy of VEGF against HCC, as well as to the real time monitoring of gene delivery.