肝癌细胞特异靶向双源性MR分子影像诊断的实验研究

The MRI imaging for hepatocellular carcinoma (HCC) diagnosis almost all used non-hepatoma cells targeted contrast agents, and there was still a great difficulty in the early diagnosis of HCC. In this project, the hepatoma cells targeting specific contrast agent was further developed on the basis of the previous studies of hepatocyte targeting. As follows: A54 peptide which indicated excellent binding ability to hepatoma cells was utilized to modify the solid lipid nanoparticles (SLN) after PEGylation, and the superparamagnetic iron oxide nanoparticles (USPIO) was used to mark targeted. Then to synthesize the molecular probe A54-PEG-SLN-USPIO with the capability of exogenous hepatoma cells targeted. Combining with the ferritin gene carrier promoted by AFP promoter, namely AFP-Fth, by ions interaction (endogenous hepatoma cells targeted) and further to synthesize the molecular probe (A54-PEG-SLN-USPIO/AFP-Fth) with capability of bidirectional hepatoma cells targeted. Take specific hepatoma cells and nude mice with orthotopic transplantation of liver cancer as models, to study the MR molecularly targeted imaging in vitro and in vivo, respectively, and further to validate the capacity of the probe targeting with hepatoma cells. The findings of this project is expected to increase the rate of early diagnosis of HCC. Meanwhile, the research aims to achieve early and specific diagnosis of HCC at the molecular level.

肝细胞癌（HCC）的磁共振成像（MRI）诊断几乎采用非肝癌细胞靶向对比剂，对肿瘤早期确诊仍存在较大困难。本项目设想在前期肝细胞靶向研究基础上，进一步研发针对肝癌细胞靶向特异对比剂。构想如下：利用对特定肝癌细胞具有明显特异靶向性的A54多肽修饰经聚乙二醇（PEG）化的固体脂质纳米粒(SLN)，并用超小超顺磁性氧化铁（USPIO）纳米粒进行靶向标记，构建对肝癌细胞具有外源性特异靶向作用的分子探针A54-PEG-SLN-USPIO。通过离子相互作用结合经AFP启动子启动的铁蛋白基因载体即AFP-Fth（内源性肝癌细胞靶向作用），构建肝癌细胞特异靶向双源性MR分子探针（A54-PEG-SLN-USPIO/AFP-Fth）。以肝癌细胞及肝癌裸鼠原位移植瘤为模型，进行体内外MRI分子靶向研究，验证该探针的靶向性。本项目的研究结果有望提升HCC的早期诊断率，实现HCC分子水平早期特异性诊断。

肝细胞癌（Hepatocellular carcinoma, HCC）是目前严重危害人类生命健康的疾病之一，肝细胞癌的磁共振成像（Magnetic resonance imaging, MRI）对比剂几乎采用非肝癌细胞靶向对比剂，对肿瘤早期确诊仍存在较大困难。人们已经认识到，构建肝肿瘤特异性靶向探针可以获得准确的成像信息，但仍面临挑战。本项目在前期肝细胞靶向研究基础上，进一步研发针对肝癌细胞具有特异靶向能力的双源性MRI对比剂。本研究首次制备了AGKGTPSLETTP肽(A54)修饰的超顺磁氧化铁(SPIO)负载的纳米结构脂质载体(A54-SNLC)，该载体可被肝癌细胞(Bel-7402)特异性摄取，在T2加权成像(T2WI)上显示出不同Fe浓度的超低成像信号强度，是一种有效的基因载体。在此基础上，设计了具有肿瘤特异性启动子(AFP-promoter)的内源性铁蛋白核磁共振报告基因(MRI)，该基因也可使T2WI信号强度降低。最后，以鱼精蛋白为阳离子介质，制备了A54-SNLC/鱼精蛋白/DNA (A54-SNPD)的新型三元纳米颗粒，作为肝肿瘤成像的活性双靶T2WI MRI造影剂。由于A54-SNLC与AFP启动子修饰的DNA靶向Bel-7402细胞的协同作用，通过三元复合纳米粒的T2增强效应，成功降低了肝脏肿瘤的T2成像强度值。值得强调的是，新型的A54-SNPD三元纳米颗粒作为活性双靶T2WI MRI造影剂，能够大大提高肝癌的诊断敏感性和特异性。