靶向Neuropilin和GPC-3受体的双靶点PET分子探针的构建和鉴定

There is not a PET molecular probe which can detect hepatocellular carcinoma (HCC) sensitively and specifically. It has been identified that Neuropilin receptor （NRP）highly expressed on the tumor cells and the tumor neovascular surface,especially on the later. tLyP-1 is a peptide which can robustly and selectively binds to NRP with about 120 times more than control peptides and then produces intense extravasation and cell internalization activity.It has also been identified that Glypican-3（GPC-3）is over-expressed on HCC cells and has potential as a promising target for diagnosis and treatment. L5-2 is a peptide which can specifically target to GPC-3 receptor. The PET probe targeting to these two receptors may be able to detect the HCC sensitively and specifically. After the fluorescence labeling tLyP-1 was proved to be able to target specifically to the lesion of HCC by our research, we have conjugated this peptide with L5-2 and synthesized a novel heterodimeric peptide. The cell binding examination in vitro showed the heterodimeric peptide can be high sensitively uptaked by the HepG2 HCC cells.Based on these studys, in the present study, we will investigate how to radiolabel this novel heterodimeric peptide with positron emitter of 18F-floride and develop it to become a new molecule probe of PET (named 18F-tLyP-1-L5-2). By utilizing the highly efficient tumor neovascular targeting and extravasation potency of tLyP-1 and the dual-targeting binding capacity of the heterodimeric peptide probe, the present study may find a new road to realize the dream of specific diagnosis of HCC and in a meanwhile increase significantly the detective sensitivity.We suggest it would significantly promote the development of tumor targeted imaging in the future.

目前尚无一种PET分子探针能实现肝细胞癌高灵敏度、高特异性诊断。研究表明肝细胞癌及其新生血管高表达Neuropilin 受体，多肽tLyP-1能与其高效结合（其结合能力较对照肽高120倍）并能产生跨血管渗透作用。研究也表明肝细胞癌特异性高表达GPC-3受体，多肽L5-2能与其高效结合。靶向这2种受体的双靶点PET探针有望能实现肝细胞癌高灵敏度、高特异性诊断。我们在用荧光显像证实tLyP-1在体内具有优良的肝细胞癌靶向性后，已将该肽和L5-2相结合创建了一种新型的双靶向肽并经实验证实能为肝癌细胞高灵敏度摄取，在此基础上，本课题拟探索将其用18F标记发展为新型的PET分子探针（18F-tLyP-1-L5-2），拟利用tLyP-1的高效肿瘤血管靶向和跨血管渗透能力以及该探针与肝癌细胞的双靶点结合作用，在实现肝细胞癌靶向显像的同时明显提高探测灵敏度。此研究对肿瘤靶向显像将产生深远的促进作用。

PET/CT在肝细胞癌的诊断方面目前仍存在明显不足，本研究立足于探索、发展一种靶向Neuropilin受体和GPC3受体的新型双靶向杂和探针以解决此难题。经过研究，本项目成功地合成了靶向Neuropilin 受体、GPC3受体和双受体靶向多肽tLyP-1、L5和L5-tLyP-1,并分别用荧光素FAM标记成功为荧光分子探针，经细胞学和肿瘤模型荧光生物学分布研究证实以上3种荧光探针能为肝细胞癌HepG2、HEL7402特异性摄取，并与相应的受体分布相吻合。我们采用了18F-SFB成功标记了tLyp-1制备了分子探针18F-tLyP-1，该探针经荷瘤鼠microPET/CT显像证实具有体内肿瘤靶向性，肿瘤显像清楚，肿瘤靶/非靶比值达2.69 ± 0.52（60 min ）和3.11±0.25 （120 min）。我们又采用了NODA修饰L5并用18F-AlF标记成功了靶向GPC3受体的PET分子探针18F-AlF-NODA-MP-6-Aoc-L5，经荷瘤鼠体内放射性分布及microPET/CT显像证实该探针能特异性靶向高表达GPC3的肝细胞癌，为了减少正常肝脏对该探针的摄取，我们还采用亲水基团GGGRDN修饰L5制备了亲水性靶向GPC3多肽L-L5，再经NOTA修饰，也已成功地用18F-AlF标记成功一种新型的PET探针18F-AlF-NOTA-L-L5，经microPET/CT显像证实它能更好地用于肝细胞癌显像。我们虽然经细胞学证实了双靶向探针可促进肿瘤内摄，提高了探针在肿瘤部位的聚集量，也成功地研制成功了双靶向杂合PET探针18F-AlF-NOTA-L5-tLyP-1,但HPLC检测结果发现产物中存在多种放射性杂质，肿瘤显像效果不理解，目前正在研究其原因并想方法改进。