Jiang Lei, Kimura Richard H, Ma Xiaowei, Tu Yingfeng, Miao Zheng, Shen Bin, Chin Frederick T, Shi Hongcheng, Gambhir Sanjiv Sam, Cheng Zhen
Department of Nuclear Medicine, Zhongshan Hospital, Fudan University , 180 Fenglin Road, Shanghai, China 200032.
Mol Pharm. 2014 Nov 3;11(11):3885-92. doi: 10.1021/mp500018s. Epub 2014 Apr 28.
A divalent knottin containing two separate integrin binding epitopes (RGD) in the adjacent loops, 3-4A, was recently developed and reported in our previous publication. In the current study, 3-4A was radiofluorinated with a 4-nitrophenyl 2-(18)F-fluoropropinate ((18)F-NFP) group and the resulting divalent positron emission tomography (PET) probe, (18)F-FP-3-4A, was evaluated as a novel imaging probe to detect integrin αvβ3 positive tumors in living animals. Knottin 3-4A was synthesized by solid phase peptide synthesis, folded, and site-specifically conjugated with (18/19)F-NFP to produce the fluorinated peptide (18/19)F-fluoropropinate-3-4A ((18/19)F-FP-3-4A). The stability of (18)F-FP-3-4A was tested in both phosphate buffered saline (PBS) buffer and mouse serum. Cell uptake assays of the radiolabeled peptides were performed using U87MG cells. In addition, small animal PET imaging and biodistribution studies of (18)F-FP-3-4A were performed in U87MG tumor-bearing mice. The receptor targeting specificity of the radiolabeled peptide was also verified by coinjecting the probe with a blocking peptide cyclo(RGDyK). Our study showed that (18)F-FP-3-4A exhibited excellent stability in PBS buffer (pH 7.4) and mouse serum. Small animal PET imaging and biodistribution data revealed that (18)F-FP-3-4A exhibited rapid and good tumor uptake (3.76 ± 0.59% ID/g and 2.22 ± 0.62% ID/g at 0.5 and 1 h, respectively). (18)F-FP-3-4A was rapidly cleared from the normal tissues, resulting in excellent tumor-to-normal tissue contrasts. For example, liver uptake was only 0.39 ± 0.07% ID/g and the tumor to liver ratio was 5.69 at 1 h p.i. Furthermore, coinjection of cyclo(RGDyK) with (18)F-FP-3-4A significantly inhibited tumor uptake (0.41 ± 0.12 vs 1.02 ± 0.19% ID/g at 2.5 h) in U87MG xenograft models, demonstrating specific accumulation of the probe in the tumor. In summary, the divalent probe (18)F-FP-3-4A is characterized by rapid and high tumor uptake and excellent tumor-to-normal tissue ratios. (18)F-FP-3-4A is a highly promising knottin based PET probe for translating into clinical imaging of tumor angiogenesis.
一种在相邻环3-4A中含有两个独立整合素结合表位（RGD）的二价结蛋白，最近已被开发并在我们之前的出版物中报道。在本研究中，用4-硝基苯基2-(18)F-氟丙酸酯((18)F-NFP)基团对3-4A进行放射性氟化，所得的二价正电子发射断层扫描（PET）探针(18)F-FP-3-4A，作为一种新型成像探针用于检测活体动物中整合素αvβ3阳性肿瘤进行了评估。结蛋白3-4A通过固相肽合成法合成、折叠，并与(18/19)F-NFP进行位点特异性缀合，以产生氟化肽(18/19)F-氟丙酸酯-3-4A((18/19)F-FP-3-4A)。在磷酸盐缓冲盐水（PBS）缓冲液和小鼠血清中测试了(18)F-FP-3-4A的稳定性。使用U87MG细胞进行了放射性标记肽的细胞摄取试验。此外，在U87MG荷瘤小鼠中进行了(18)F-FP-3-4A的小动物PET成像和生物分布研究。通过将探针与封闭肽环(RGDyK)共同注射，也验证了放射性标记肽的受体靶向特异性。我们的研究表明，(18)F-FP-3-4A在PBS缓冲液（pH 7.4）和小鼠血清中表现出优异的稳定性。小动物PET成像和生物分布数据显示，(18)F-FP-3-4A表现出快速且良好的肿瘤摄取（分别在0.5小时和1小时时为3.76±0.59% ID/g和2.22±0.62% ID/g）。(18)F-FP-3-4A从正常组织中迅速清除，从而产生优异的肿瘤与正常组织对比度。例如，在注射后1小时，肝脏摄取仅为0.39±0.07% ID/g，肿瘤与肝脏的比值为5.69。此外，在U87MG异种移植模型中，将环(RGDyK)与(18)F-FP-3-4A共同注射显著抑制了肿瘤摄取（在2.5小时时为0.41±0.12% ID/g对1.02±0.19% ID/g），证明了探针在肿瘤中的特异性积累。总之，二价探针(18)F-FP-3-4A的特点是肿瘤摄取快速且高，以及肿瘤与正常组织的比值优异。(18)F-FP-3-4A是一种非常有前途的基于结蛋白的PET探针，有望转化为肿瘤血管生成的临床成像。
