Integrin αvβ3 and aminopeptidase N (APN, also known as CD13) are two important targets involved in the regulation of angiogenesis, tumor proliferation, invasion, and metastasis. In this study, we developed a heterodimeric tracer consisting of arginine-glycine-aspartic (RGD) and asparagine-glycine-arginine (NGR) peptides targeting αvβ3 and CD13, respectively, for PET imaging of breast cancer. The NGR peptide was first modified with N3-NOtB2 and then conjugated to BCN-PEG4-c(RGDyK) via copper-free click chemistry. The resulting precursor was purified and radiolabeled with gallium-68. Small-animal PET/CT imaging and post-imaging biodistribution studies were performed in mice bearing human breast cancer MCF-7, MDA-MB-231, MDA-MB-468, and MX-1 xenografts and pulmonary metastases models. The expression levels of αvβ3 and CD13 in tumors were checked via immunochemical staining. The heterodimeric tracer was successfully synthesized and radiolabeled with gallium-68 at a molar activity of 45-100 MBq/nmol at the end of synthesis. It demonstrated high in vitro and in vivo stability. In static PET/CT imaging studies, the MCF-7 tumor could be clearly visualized and exhibited higher uptake at 30 min post injection of 68Ga-NGR-RGD than that of either 68Ga-RGD or 68Ga-NGR alone. High specificity was shown in blocking studies using Arg-Gly-Asp (RGD) and Asp-Gly-Arg (NGR) peptides. The MCF-7 tumor exhibited the highest uptake of 68Ga-NGR-RGD followed by MDA-MB-231, MDA-MB-468, and MX-1 tumors. This was consistent with their expression levels of CD13 and αvβ3 as confirmed by western blot and immunohistochemical staining. Metastatic lesions in the lungs were clearly detectable on 68Ga-NGR-RGD PET/CT imaging in mouse models of pulmonary metastases. 68Ga-NGR-RGD, a CD13 and αvβ3 dual-receptor targeting tracer, showed higher binding avidities, targeting efficiency, and longer tumor retention time compared with monomeric 68Ga-NGR and 68Ga-RGD. Its promising in vivo performance makes it an ideal candidate for future clinical translation.

中文翻译：

---

用于乳腺癌成像的整合素 αvβ3 和氨肽酶 N 双受体靶向示踪剂的评估。

整合素αvβ3和氨肽酶N（APN，也称为CD13）是参与调节血管生成、肿瘤增殖、侵袭和转移的两个重要靶标。在这项研究中，我们开发了一种异二聚示踪剂，由精氨酸-甘氨酸-天冬氨酸（RGD）和天冬酰胺-甘氨酸-精氨酸（NGR）肽组成，分别靶向αvβ3和CD13，用于乳腺癌的PET成像。NGR 肽首先用 N3-NOtB2 进行修饰，然后通过无铜点击化学与 BCN-PEG4-c(RGDyK) 缀合。纯化所得前体并用镓68进行放射性标记。在携带人乳腺癌 MCF-7、MDA-MB-231、MDA-MB-468 和 MX-1 异种移植物和肺转移模型的小鼠中进行了小动物 PET/CT 成像和成像后生物分布研究。通过免疫化学染色检查肿瘤中αvβ3和CD13的表达水平。成功合成了异二聚体示踪剂，并在合成结束时用镓 68 以 45-100 MBq/nmol 的摩尔活度进行放射性标记。它表现出较高的体外和体内稳定性。在静态 PET/CT 成像研究中，MCF-7 肿瘤可以清晰可见，并且在注射 68Ga-NGR-RGD 30 分钟后表现出比单独 68Ga-RGD 或 68Ga-NGR 更高的摄取。使用 Arg-Gly-Asp (RGD) 和 Asp-Gly-Arg (NGR) 肽进行的阻断研究显示出高特异性。MCF-7 肿瘤对 68Ga-NGR-RGD 的摄取最高，其次是 MDA-MB-231、MDA-MB-468 和 MX-1 肿瘤。这与蛋白质印迹和免疫组织化学染色证实的 CD13 和 αvβ3 表达水平一致。在小鼠肺转移模型中，通过 68Ga-NGR-RGD PET/CT 成像可以清楚地检测到肺部转移病灶。68Ga-NGR-RGD是一种CD13和αvβ3双受体靶向示踪剂，与单体68Ga-NGR和68Ga-RGD相比，表现出更高的结合亲合力、靶向效率和更长的肿瘤保留时间。其良好的体内性能使其成为未来临床转化的理想候选者。