TfR受体介导的aptamer/七甲川菁复合物的肿瘤靶向显像及抗瘤效应

Theranostics has been considered as an important strategy to overcome challenges in tumor therapy. Selection of tumor marker is the initial step for tumor targeting imaging and therapy. In our previous research, we have identified that Transferrin receptor (TfR) is generally overexpressed on cancer cell membrane and could represent a suitable tumor target for molecular imaging and therapy. Aptamers are oligonucleotides with unique three-dimensional structures that can recognize and bind to specific tumor targets. With higher affinity and specificity, aptamers represent a class of a more attractive candidate than antibody and peptide do in molecular imaging applications. Heptamine cyanine dye has unique near infrared fluorescence emission properties and shows a significant accumulation in mitochondria of tumor cells. By conjugated these dye with drug, it can be used as carriers for the safe delivery of chemotherapies to tumor site. Moreover theses dyes can be utilized as effective agent for photodynamic therapy. Heptamine cyanine dye has attracted much attention in tumor therapy and imaging. If Heptamine cyanine dye was linked with transferrin receptor targeted aptamer to develop a new multimodal probe, it would have a higher specificity binding and uptake in the tumor cell membrane. Our objective was to develop a new molecular probe in vitro and in vivo. The method of study includes cell uptake experiments, biodistribution and dynamic kinetic experiments, microPET imaging and near infrared fluorescence imaging in tumor bearing mice. It may provide a new way for tumor early diagnosis and therapy.

肿瘤诊疗一体化是近来研究热点，靶点选择是其关键环节之一。课题组前期研究发现，转铁蛋白受体(TfR)在多种肿瘤细胞膜表面高表达，是肿瘤显像及治疗的理想靶点。与抗体或多肽相比，适配体(aptamer)能与特定肿瘤靶点高度选择性结合，具有更高的亲和力和特异性，是极具潜力的肿瘤分子探针。七甲川菁类化合物具有较强的近红外荧光显影特性，同时具有肿瘤细胞线粒体高效靶向蓄积、能进行光动力治疗、易载带小分子化学药物等优点，兼具肿瘤靶向诊治特性。. 由此设想，如将TfR靶向性aptamer与七甲川菁进行化学连接，能否通过TfR介导的特异性结合和内化作用进一步提高探针的肿瘤“主动”靶向性和膜穿透性，获得近红外荧光/PET多模态显像、多功能诊治一体化探针？本项目拟从整体-组织-细胞多层次明确此探针的选择性转运机制及成像效能，并初步分析细胞杀伤及动物抑瘤效应，从而为肿瘤诊疗一体化策略提供新思路。

项目背景: 转铁蛋白受体（TfR，transferrin receptor）在多种肿瘤细胞膜表面高表达，是肿瘤诊疗的理想靶点。核酸适配体（aptamer）能与特定肿瘤靶点高度选择性结合，与抗体相比具有更高的亲和力和特异性，是有转化潜力的肿瘤分子探针。. 主要研究内容:针对TfR 为靶点筛选出高度特异性的aptamer，选择合适的化学修饰方法及核素标记条件，探讨探针的体外细胞结合特性、亚细胞定位、荷瘤裸鼠的体内 显像及生物学分布；同时合成针对TfR 为靶点的小分子多肽及diabody，分析各种探针的显像效能及转化潜能。. 重要结果:三种探针均与阳性表达TfR的肿瘤特异、高亲和性结合，而阴性表达TfR的肿瘤相对摄取较低。但三者在生物学分布有显著差异，小分子多肽和aptamer主要从肾脏排泄，靶/非靶比值较高，而Diabody在肝脏排泌较多，影响上腹部图像质量。. 关键数据: 筛选出TfR特异性aptamer，并可有效被99mTc标记，放化纯达90%以上，稳定性较好；TfR-aptamer对于TfR阳性细胞HepG2、CT11具有较强的亲和力，弱表达TfR的MX-1未见明显结合；荷PANC-1、MCF-7肿瘤裸鼠显像证实，核素标记TfR-aptamer展现肿瘤高摄取，且主要从肾脏排泄，30min即可获得较高的靶/非靶比值；本项目尚进行了TfR-小分子多肽T7及双链抗体diabody显像，对比研究发现小分子多肽具有aptamer类似特性，而diabody相对分子量较大，肝脏本底较高，靶/非靶比值低于aptamer和小分子多肽。. 科学意义: 通过对比aptamer、小分子多肽及diabody三种探针体内外特性，初步建立靶向TfR阳性肿瘤显像的新方法。