整合素αvβ3和氨肽酶N（APN/CD13）在肿瘤血管生成过程中发挥着重要作用。它们在各种肿瘤细胞和新生血管内皮细胞中高表达，在肿瘤成像中被视为极具潜力的靶点。精氨酸-甘氨酸-天冬酰胺（RGD）和天冬酰胺-甘氨酸-精氨酸（NGR）是两种与整合素αvβ3和CD13特异性结合的肽。在本研究中，我们优化了先前开发的探针，并在10种不同的皮下和原位肿瘤模型中对新的整合素αvβ3和CD13双靶向探针NOTA-RGD-NGR（简称HX01）进行了临床前评价，其以68Ga进行标记。我们确定了[68Ga]Ga-HX01的特异活性和放射化学纯度。通过一系列的阻断实验和局部淬灭的示踪剂使用BxPC-3异种移植模型，确认了双受体靶向能力。在BxPC-3异种移植模型中，探索了动态成像研究和剂量逐步增加研究，以确定最佳成像时间点和剂量。接下来，我们建立了多种皮下和原位肿瘤模型，包括胰腺（BxPC-3）、乳腺（MCF-7）、胆囊（NOZ）、肺（HCC827）、卵巢（SK-OV-3）、结直肠（HCT-8）、肝（HuH-7）、胃（NUGC-4）和脑胶质瘤（U87）。所有模型在接种后约2周进行了[68Ga]Ga-HX01 PET/CT成像，其中部分模型同时进行了[18F]FDG PET/CT扫描，并进行了比较。此外，还进行了离体生物分布研究，以验证非侵入性PET图像的半定量结果。在BxPC-3异种移植模型中，[68Ga]Ga-HX01明显优于单靶探针。所有阻断和单靶组均显著降低了肿瘤摄取量。在BxPC-3、MCF-7和NOZ皮下肿瘤中发现高的肿瘤摄取量（%ID/g > 1.1），而在HCC827、SK-OV-3、HCT-8和HuH-7皮下肿瘤中观察到中等摄取量（%ID/g 0.7-1.0）。由于低背景，[68Ga]Ga-HX01的肿瘤/肌肉和肿瘤/血液比值高于[18F]FDG。作为双靶成像剂，[68Ga]Ga-HX01在不同皮下和原位肿瘤模型中表现出优越的体内性能，而[18F]FDG及其相应的单受体靶向剂则没有。这为未来的肿瘤成像的临床应用提供了保证。© 2023. 该作者，其独家许可给Springer-Verlag GmbH Germany，属于Springer Nature的部分人士。

The integrin αvβ3 and aminopeptidase N (APN/CD13) play vital roles in the tumor angiogenesis process. They are highly expressed in a variety of tumor cells and proliferating endothelial cells during angiogenesis, which have been considered as highly promising targets for tumor imaging. Arginine-glycine-aspartic (RGD) and asparagine-glycine-arginine (NGR) are two peptides specifically binding to the integrin αvβ3 and CD13, respectively. In this study, we optimized our previously developed probe and preclinically evaluated the new integrin αvβ3 and CD13 dual-targeted probe, NOTA-RGD-NGR (denoted as HX01) radiolabeled with 68Ga, in 10 different subcutaneous and orthotopic tumor models.The specific activity and radiochemical purity of [68Ga]Ga-HX01 were identified. The dual-receptor targeting ability was confirmed by a series of blocking studies and partly muted tracers using BxPC-3 xenograft model. The dynamic imaging study and dose escalation study were explored to determine the optimal imaging time point and dosage in the BxPC-3 xenograft model. Next, we established a variety of subcutaneous and orthotopic tumor models including pancreas (BxPC-3), breast (MCF-7), gallbladder (NOZ), lung (HCC827), ovary (SK-OV-3), colorectal (HCT-8), liver (HuH-7), stomach (NUGC-4), and glioma (U87) cancers. All models underwent [68Ga]Ga-HX01 PET/CT imaging about 2 weeks post-inoculation, with a subset of them undergoing [18F]FDG PET/CT scan performed concurrently, and their results were compared. In addition, ex vivo biodistribution studies were also performed for verifying the semi-quantitative results of the non-invasive PET images.[68Ga]Ga-HX01 significantly outperformed single target probes in the BxPC-3 xenograft model. All blocking and single target groups exhibited significantly descending tumor uptake. The high tumor uptakes were found in BxPC-3, MCF-7, and NOZ subcutaneous tumors (%ID/g > 1.1), while middle uptakes were observed in HCC827, SK-OV-3, HCT-8, and HuH-7 subcutaneous tumor (%ID/g 0.7-1.0). Due to the low background, the tumor-to-muscle and tumor-to-blood ratios of [68Ga]Ga-HX01 were higher than that of [18F]FDG.[68Ga]Ga-HX01, as a dual target imaging agent, exhibited superior in vivo performance in different subcutaneous and orthotopic mice models of human tumors over [18F]FDG and its respectively mono-receptor targeted agents, which warrants the future clinical translation for tumor imaging.© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.