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.

Although F-5-fluoro-N-(2-[diethylamino]ethyl)picolinamide (F-5-FPN) is considered a promising radiopharmaceutical for PET imaging of melanoma, it accumulates at high concentrations in the liver. The aim in this research was to optimize the structure of F-5-FPN with triethylene glycol to reduce liver uptake as well as improve pharmacokinetics, and to evaluate its performance in detection of melanoma liver and lung metastases.F-PEG-FPN was successfully prepared with a high radiolabeling yield (44.68% ± 5.99%) and radiochemical purity (>99%). The uptake of F-PEG-FPN by pigmented B16F10 melanoma cells was significantly higher than that by amelanotic melanoma A375 cells. The binding to B16F10 cells could be blocked by excess F-PEG-FPN. On small animal PET images, B16F10 tumors, but not A375 tumors, were clearly delineated after F-PEG-FPN injection. More importantly, F-PEG-FPN uptake by liver (2.27 ± 0.45 and 1.74 ± 0.35% ID/g, at 1 and 2 h) was significantly lower than that of F-5-FPN, and the lesions in lung and liver could be clearly detected byF-PEG-FPN PET imaging in mouse models of pulmonary or hepatic metastases. Overall, we successfully synthesized F-PEG-FPN, which has higher labeling efficacy and better in vivo pharmacokinetics along with lower liver uptake compared to F-5-FPN. This suggestsF-PEG-FPN as a candidate for pigmented melanoma liver and lung metastasis detection.