构建和鉴定靶向穿膜的多功能分子探针iCREKA

The molecular probe is playing the extremely vital role in targeting diagnosis and treatment of malignant tumors, however the single function molecular probes apparently have been unable to fully meet the needs of basic and clinical research, and the development of the more sensitive, more specific, multifunctional molecular probe is just unfolding. In this study, we will design, synthesize and evaluate a multifunctional molecular probe iCREKA, which could initiatively target to broad-spectrum solid tumors, intelligently been released, efficiently transmembrane based on preliminary studies. The probe provides two binding sites for conjugates(such as the imaging signal, anticancer drug, gene vector,etc.)，which can be specifically imported and accumulated in the tumor cell. Through different combinations of the two conjugates, the iCREKA can achieve a variety of functions. The iCREKA is combined with a positron nuclide and a fluorescein, it can be used for PET diagnosis of cancer and for intraoperative guidance; The iCREKA is combined with an imaging signal and a gene vector, it can be used for monitoring of gene transduction in vivo; The iCREKA is combined with two anticancer drugs, it can be used for combination therapy. In short，the iCREKA can be used for diagnosis, therapy, drug development, gene diagnosis, gene therapy and other aspects in targeting tumor. If the iCREKA is synthesized successfully, it would have broad application prospects and provide reference for the development of new multifunctional molecular probes.

分子探针在肿瘤靶向诊断及靶向治疗中起着十分重要的作用，单一功能分子探针显然已无法完全满足基础及临床研究的需要。目前研发高特异性、高灵敏性的多功能分子探针方兴未艾，本课题在前期研究工作基础上结合现有的理论，拟构建一个广谱的肿瘤主动靶向、智能释放、高效穿膜的多功能分子探针（iCREKA）。该探针具有两个结合位点，可同时将两个结合物(如显像信号、抗肿瘤药物、基因载体等)特异性导入肿瘤细胞内，并在细胞内聚积。通过对探针两个结合物的不同组合，可实现多种功能：两个位点分别结合正电子核素和荧光素，既可用于PET肿瘤诊断，也可用于术中荧光成像导航；分别结合显像信号和基因载体，可以在活体监测基因转导；分别结合两种抗肿瘤药物，可以进行肿瘤联合治疗。如果该探针制备成功，可以在肿瘤的靶向诊断、靶向治疗、药物研发、基因诊断、基因治疗等多个方面具有广阔的应用前景；同时也可为新分子探针的研发提供可借鉴的思路。

目的：本研究旨在构建构建一个广谱的肿瘤主动靶向、智能释放、高效穿膜的多功能分子探针（iCREKA），并验证其用于荧光和PET成像的可行性。.方法：制备多功能分子探针（iCREKA），采用荧光素FITC标记iCREKA、CREKA。制备荧光探针FITC-iCREKA和FITC-CREKA。体外实验检测FITC-iCREKA在MMP-2作用下的细胞穿膜能力。通过荧光成像方法测定FITC-iCREKA和对照肽FITC-CREKA 经静脉注射后在肿瘤组织内的分布差异，以验证FITC-iCREKA与肿瘤间质的结合及继发的细胞穿膜能力。通过动物荧光成像方法分析FITC-iCREKA用于肿瘤显像的可行性及体内生物学分布。利用氨基酰化反应合成PET分子探针18F-iCREKA。用microPET/CT显像研究18F-iCREKA用于肿瘤显像的可行性及其体内放射性生物学分布。.结果：经质谱分析鉴定，iCREKA及FITC-iCREKA合成并标记成功。当FITC-iCREKA与胶质瘤细胞孵育时培养液中加入活化的MMP-2后，肿瘤细胞内出现大量绿色荧光聚集。对照组中未加入活化的MMP-2，则细胞内仅见少量绿色荧光。流式细胞仪检测显示加入和未加入活化的MMP-2后肿瘤细胞荧光摄取强度存在显著性差异。高表达MMP2的肿瘤组织荧光显像结果显示，静脉注射FITC-iCREKA 1小时后，大量荧光分布于肿瘤血管壁及周围间质内，肿瘤细胞内未见明显荧光分布；3小时以后，肿瘤细胞内可见明显荧光分布。对照组注射FITC-CREKA后荧光始终分布于肿瘤间质而未进入肿瘤细胞内。荷脑胶质瘤裸鼠体内荧光生物学分布研究显示，肿瘤病灶处FITC-iCREKA高摄取，而正常脑组织仅见少量荧光分布，肿瘤/脑比值明显高于注射FITC-CREKA的对照组。PET分子探针18F-iCREKA制备成功，放化纯度为97.0%。MicroPET/CT显像显示，静脉注射18F-iCREKA 30分钟后，肿瘤显影并随时间延长其影像更加清晰，而正常脑内未见放射性浓聚。.结论：体内及体外实验证明iCREKA构建成功，该探针能够靶向肿瘤间质纤维蛋白并穿膜进入肿瘤细胞内，可用于肿瘤的荧光及MicroPET/CT活体显像。