基于大环类DOTA-Gd的GPC3靶向PLA-PEG纳米探针的研制及其在肝癌微灶检测中的价值

It is difficult to locate and diagnose the micro hepatocellular carcinoma (mHCC)　using the standard imaging techniques, which has been becoming a bottleneck for the further diagnosis and therapy. Recently, the targeted molecular imaging technique shows a good potential to detect these micro tumors. However, the defects of many molecular probes using Gd-DTPA in current studies are as follows: Ⅰ.The toxic gadolinium may decompose and escape from the loading Gd-DTPA. Ⅱ. Heterogeneous monoclonal antibody represents large molecular weight and immunogenicity. Ⅲ.The expressed rate of the mHCC is not high. To solve the problem above, we synthesize the novel targeted molecular nanoprobes with the stable macrocyclic DOTA, small non-immunogenic humanized single-chain antibody, and the specifically high-expressed GPC3 in the mHCC. In preliminary study, we have designed a multifunctional polymeric nanoparticle contrast agent simultaneously modified with Gd-DTPA and anti- VEGF antibody to deliver the Gd-DTPA to the tumor and achieved the specific imaging of the HCC. Based on this nano-platform, in this study we try to synthesize the novel, stable, secure, specific and sensitive nanoprobes for the detection and specific diagnosis of the mHCC. Firstly, the nanocarrier will be made of the biocompatible materials, PLA-PEG or other materials, using self-assembly techniques. Secondly, the DOTA-Gd will be loaded to the nanoparticles with a high loading, relaxivity and security. Finally the targeted nanoprobe will be made by linking to the anti-GPC3 humanized single-chain antibody. The MR imaging experiments in vitro and in vivo will be performed to identify the clinical value of detection and specific diagnosis of the mHCC. The targeted molecular imaging using this novel nanoprobe will significantly improve the diagnostic accuracy and therapeutic effect and prove a novel tool for the evaluetion. Is is also consistent with the national strategic needs and planning about the accurate diagnosis of major diseases in the early stage.

传统影像学技术对肝癌微灶定位、定性困难，成为目前制约其诊治的瓶颈。靶向分子成像技术在解决此难题方面展示出良好潜力，但目前研究中含钆靶向探针的缺陷为：荷载的Gd-DTPA可分解游离有毒性的钆、异源性抗体分子量较大并有免疫源性、肝癌标记物往往表达效率不高。为解决此问题，我们利用生物相容性PLA-PEG等材料,并结合稳定的大环类DOTA、小分子量和无免疫原性的人源化单链抗体、及在小肝癌中高特异性和高表达的GPC3来制备靶向分子探针。在前期成功制备VEGF-含钆纳米探针并实现特异性成像的基础上，利用自组装技术制备PLA-PEG等纳米载体，结合单链抗体及DOTA-Gd后制备新的高载荷、高驰豫性和安全稳定的靶向分子探针，再利用动物模型进行肝癌微灶MR成像，突破成像盲区，在显示微灶的同时也实现了特异性诊断，促进诊断率、治疗效果及疗效评估水平的提高，也符合重大疾病早期精确诊断国家重大战略需求和规划。

目前传统影像学在肝癌诊断方面的难点是不典型病变定性困难及小病灶定位困难，已成为制约临床诊治的瓶颈。靶向分子成像为解决上述难题提供了可能。本研究利用生物相容性材料制备纳米粒，在探索合理的制备工艺的基础上制备纳米分子探针，探讨其在肝癌特异性成像及小病灶检测中的价值；也对诊断-治疗纳米粒的价值进行初步探索。研究分为三部分：纳米分子探针制备工艺的优化与筛选：采用生物相容性SLN、PLA-PEG、PLA-PEG-PLL，利用4种不同的合成等方法制备4种含钆载体纳米粒。结果：SLN、吸附型PLA-PEG、接枝型PLA-PEG及接枝型PLA-PEG-PLL纳米粒平均粒径为196.1、187.9、265.9、69.8nm左右，分散性和血浆稳定性好。其中接枝型PLA-PEG-PLL荷钆纳米粒粒径优良、性质稳定、活性化学基团多，成像效果最好。靶向荷钆PLA-PEG-PLL纳米探针在小肝癌特异性成像中的价值：利用化学合成法制备PLA-PEG-PLL纳米载体和靶向纳米探针，对种荷H22肝癌小鼠模型进行MR成像。结果：体外MR显影显示靶向探针的驰豫率与浓度成线性关系，当靶向探针浓度为8.0 μM/mL时R1值为18.394mM.mL-1.s-1。MR成像显示注射靶向探针后大小肿瘤均呈延迟期明显强化，峰值为2-3h。该结果证实该纳米探针可实现小肝癌的特异性靶向成像，并在在小肝癌的检测方面具有较好的应用前景。多功能聚合物纳米粒在肝癌诊治中的价值初步研究：将PLA-PEG-PLL-DTPA纳米粒与PLH-PEG-生物素结合，封装进索拉菲尼后连接Gd和VEGFR，构建成PH值敏感型多功能纳米复合物。电镜显示该复合物呈球形或椭圆形，粒径为181.4nm、包封率达95.02%、药物载荷率为2.38%。当pH 值降低至5时，索拉非尼释放率达到99.32%，对H22肝癌的疗效优于口服或注射。MRI显示T1弛豫率为17.300 mM-1s-1，是马根维显的3.63倍，H22小鼠模型肿瘤显示强化明显。本结果证实，研究制备的双载药（抗肿瘤药物和Gd）纳米复合物，可同时实现特异性成像和抗肿瘤治疗，显示出良好的临床应用潜力。经过1年的努力，本研究基本达到了实验预期设计的目标。目前发表文章3篇，包括Nanoscale（IF=6.23）、中华肝胆外科杂志及实用放射学杂志各1篇，并为下一步深入研究奠定了基础。