摘要
第一部分MR分子探针Tf-SPION的合成与表征
目的：合成MR成像分子探针转铁蛋白-超顺磁性氧化铁纳米粒螯合物Tf-SPION,并对其理化性质进行表征和评价。
材料与方法：按酰胺化反应合成用Tf-SPION。将超顺磁性氧化铁纳米粒用EDCA/NHS活化,15分钟后磁板分离,用PBS将沉淀重新分散。将转铁蛋白加入上述SPION溶液中,混匀后放置过夜,次日吸附沉淀、去除上清,加入缓冲液将Tf-SPION溶液浓度调整为5mg/ml。EDAC/NHS活化转铁蛋白,加入超顺磁性氧化铁纳米粒,过夜反应10-24小时,磁板分离,加入缓冲液将Tf-SPION溶液浓度调整为5mg/ml,4℃储存备用。通过红外光谱、透射电镜、动态激光散射仪对Tf-SPION的结构和粒径进行观察,测定螯合物Tf-SPION的T2弛豫率。
结果：成功合成MR分子探针Tf-SPION。经过红外光谱检测,证实转铁蛋白Tf与超顺磁性氧化铁纳米粒SPION已经偶联为Tf-SPION。透射电镜检测Tf-SPION微粒呈球形,颗粒均匀,分散良好,粒子核心平均粒径为18.3±1.1nm；SPION平均粒径为7.9±0.5nm；动态激光散射仪检测Tf-SPION粒径为143.5±49.36nm。Tf-SPION弛豫率为94.618Mm/s,单纯SPION弛豫率173.39Mm/s。
结论：螯合物Tf-SPION满足MR分子探针所需的条件,能够用于MR分子成像。
第二部分移植性肝肿瘤转铁蛋白受体MR分子成像的在体实验研究
目的：观察分别经静脉、动脉途径引入Tf-SPION对肝肿瘤的靶向性成像效果。
材料与方法：40只肝左外侧叶种植Walker-256肿瘤SD大鼠被随机分为A、B、C、D、E五组(每组各8只大鼠)。A、B、C两组分别为经尾静脉注射0.2ml生理盐水,0.2ml SPION和0.2ml Tf-SPION。D、E两组在手术显微镜下将聚乙烯微导管经胃十二指肠动脉逆行插入肝动脉,分别经导管肝动脉注射0.2mlTf-SPION和0.2ml SPION。以上各组SD大鼠在干预后1小时内、4小时、24小时、96小时分别行腹部磁共振T2横断位扫描。扫描完成后,选取肿瘤最大径层面,分别测量肿瘤组织及同层面背部肌肉组织的T2信号强度,计算肿瘤的T2相对信号强度比RR。每组各4只大鼠在干预后1小时和96小时MR扫描结束后处死,通过普鲁士蓝染色观察肝脏组织中铁粒子含量及分布。
结果：SD大鼠移植性肝肿瘤模型造模成功。MR扫描显示,瘤块多呈类圆形,最大直径平均值1.06±0.35cm。统计学分析表明,A、B组间RR在干预后各时间点均无显著性差异(P>0.05)；C、B组间,以及D、C组间RR值在干预后各时间点均有显著性差异(P<0.01)；D、E组间RR值在干预后1小时存在显著性差异(P<0.01)。普鲁士蓝染色显示,与其他组相比,D组肝肿瘤摄取铁粒子效率最高。
结论：SPION在偶联转铁蛋白后,能够增加对肿瘤细胞转铁蛋白受体的亲和力,促进肿瘤细胞的摄取；经动脉途径引入Tf-SPION,能够更好地靶向性显示移植性肝肿瘤。
Part Ⅰ:Preparation and characterization of the magnetic resonance molecular imaging probes:transferrin (Tf)-superparamagnetic iron oxide nanoparticles (SPION)
Objective:To synthesis the MR molecular imaging probes Tf-SPION and characterize the physicochemical properties of the prepared probes.
Materials and Methods:Tf-SPION is synthesized according to the principle of the amidation reaction. The carboxyl groups in the surface of SPION are actived by the activating agent1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) and protective agent N-hydroxy succinimide (NHS). The actived SPION particles are separated as the form of precipitation with a magnetic plate. The precipitation is washed with phosphate buffer solution (PBS) to remove the unreacted EDC and NHS. Then the precipitation is dispersed in the PBS. Transferrin is added into the actived SPION solution and reactived overnight at room temperature. Adsorb the precipitation with a magnetic plate, and remove the supernatant. The concentration of Tf-SPION is adjusted to5mg/ml with PBS.
The infrared spectroscopy is used to identify the compounds of the synthesized precipitation which is a single component of Tf-SPION but not a mixture. The surface morphology is detected by the transmission electron microscopy (TEM) and dynamic light scattering (DLS). Then the T2relaxation rate is tested respectively by MR.
Results:The MR molecular imaging probes Tf-SPION is synthesized successfully with the amidation reaction method.
The precipitation is conformed to be the single compound of Tf-SPION but not the mixture of the Tf and SPION by infrared spectroscopy. Tf-SPION particles are spherical and uniform in the image of TEM and DLS, with a particle mean size is18.3±1.1nm,143.5±49.36nm respectively, and the SPION is7.9±0.5nm,71.45±29.80nm respectively. The Tf-SPION relaxation rate is94.618mM/s and the SPION relaxation rate is173.39mM/s.
Conclusion:The chelate Tf-SPION we have synthesized shows the conditions for MR molecular image probes and can be used for MR molecular imaging.
Part Ⅱ:In vivo study of Tf-SPION for MR molecular imaging in a rat liver cancer model
Objective:To investigate the characteristic of Tf-SPION for molecular imaging in a rat hepatic tumor model via artery or vein route. Materials and methods:Fourty Spraque-Dawley(SD) rats implanted with Walker-256tumors in the hepatic left lateral lobes were randomly divided into five groups (A, B, C, D and E) with8rats each. Retrograde gastroduodenal arterial catheterization with polyethylene microcatheter was performed under the surgical microscope in the group D and E.0.2ml Tf-SPION and0.2ml SPION was injected into hepatic artery in the group D and group E respectively.0.2ml saline,0.2ml SPION and0.2ml Tf-SPION was injected into tail vein in the group A, B and group C respectively. Abdominal MRI was performed at1hour,4hours,1day and4days after the injection of Tf-SPION or SPIO in each group. T2signal intensity of the tumor and the back muscle tissue was measured in the maximum tumor diameter section, and the relative signal intensity ratio(RR) was calculated. Four rats were sacrified at1hour and4days after MRI in each group, iron particles in the liver were tested by Prussian blue staining.
Results:The rat liver cancer model was established successfully. There was no significant difference of RR between group A and group B (P<0.01). And there was significant difference of RR between group C and group B (P<0.01) at each timepoint after intervention, and the same difference between group D and group C(P<0.01). The statistics difference was found between group D and group E at the first timepoint (P<0.01). Group D showed the most efficient hepatic tumor uptake of iron particles compared with other groups by Prussian blue staining. Conclusion:The results suggested Tf-SPION as a potential MR imaging probe for cell tracking. Introduction of Tf-SPION via artery may be used as a new novel method for the diagnosis evaluation of hepatic tumor.
引文
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