去唾液酸糖蛋白受体介导的新型肝细胞靶向磁共振对比剂的体外实验研究

In vitro experimental study of asialoglycoprotein receptor-mediated superparamagnetic nanoparticles as a novel hepatocyte-targeted magnetic resonance contrast agent

目的 :探讨去唾液酸糖蛋白受体(Asialoglycoprotein receptor,ASGPR)介导的新型肝细胞靶向磁共振对比剂(Fe-HSA-LA)对大鼠正常肝细胞(BRL 3A)的靶向性及利用MR检测的可行性。方法:制备Fe-HSA-LA,透射电镜(TEM)观测其表征,MR检测其T2弛豫率。采用CCK-8法检测Fe-HSA-LA的细胞毒性。以10μg Fe/mL和50μg Fe/mL浓度的Fe-HSA-LA和PEG-USPIO分别孵育BRL 3A细胞及Colon26细胞后普鲁士蓝染色,对细胞悬液进行MR T2加权成像及T2map成像,利用原子吸收光谱法(AAS)测定各组标记细胞的结合铁量。结果:Fe-HSA-LA和PEG-USPIO的Z-平均水合粒径分别为(53.38±17.07)nm和(16.57±4.18)nm,Zeta电位分别为(-23.3±9.10)mV和(2.57±0.83)mV,T2弛豫率分别为168.4 mM-1s-1和416.3 mM-1s-1。细胞毒性实验表明,Fe-HSA-LA浓度≤50μg Fe/mL时,BRL 3A细胞存活率达到95%以上。普鲁士蓝染色显示BRL 3A细胞结合的Fe-HSA-LA含量最高(P<0.05)。磁共振检测显示,当Fe-HSA-LA浓度≥3.125μg Fe/mL时,Fe-HSA-LA标记的BRL 3A细胞的信号降低最明显,细胞内的铁含量最高(P<0.05)。标记细胞R2值和细胞内铁含量之间具有良好的相关性。结论:Fe-HSA-LA对BRL 3A细胞具有靶向效应,可采用3.0T MR对其进行监测。

Objective: To explore the specificity of novel hepatocyte-targeted magnetic resonance contrast agent(Fe-HSA-LA) mediated by asialoglycoprotein receptor(ASGPR) for BRL 3A cells and the feasibility of MR. Methods: After synthesizing the asialoglycoprotein receptor-mediated hepatocyte-targeted magnetic resonance contrast agents(Fe-HSA-LA). It’s characterization was observed by transmission electron microscope(TEM), and the T2 relaxation rate was measured by MR. The cytotoxicity of Fe-HSA-LA was measured by CCK-8 method. BRL 3 A cells and Colon26 cells were incubated with Fe-HSA-LA and PEG-USPIO at concentrations of 10 μg Fe/mL and 50 μg Fe/mL, respectively, and stained with Prussian blue. MR T2 weighted imaging and T2 map imaging were performed on the cell suspension, and the bound iron content of each group of labeled cells was determined by atomic absorption spectroscopy(AAS). Results: The Z-average hydrated particle sizes of Fe-HSA-LA and PEG-USPIO were(53.38±17.07) nm and(16.57±4.18) nm, the Zeta potentials were(-23.3±9.10) mV and(2.57±0.83) m V,and the T2 relaxation rates were 168.4 mM-1s-1 and 416.3 mM-1s-1, respectively. The cytotoxicity experiment showed that the cell viability of BRL3A cells was over 95% when the concentration of Fe-HSA-LA was ≤50 μg Fe/mL. The MR T2 and the AAS results showed that when the iron incubation concentration was equal to or greater than 3.125 μg Fe/mL, the signal intensity of the Fe-HSA-LA labeled BRL 3 A cells decreased obviously, and the iron content of the labeled cells was the highest(P<0.05). There was a good correlation between the MR R2 value and the intracellular iron content. Conclusion: Fe-HSA-LA nanoparticles had good targeting specificity for BRL 3A cells and could be monitored with 3.0 T MR.