荧光引导手术（FGS）可以通过协助外科医生在术中充分观察恶性组织来提高根治切除率。设计的蛋白结构重复蛋白（DARPins）具有理想的药物动力学和其他性质，适合于体内成像。本研究旨在评估上皮细胞粘附分子（EpCAM）结合DARPins作为近红外荧光（NIRF）和光声（PA）肿瘤成像靶向载体的临床前潜力。EpCAM结合的DARPins Ac2、Ec4.1和非结合对照DARPin Off7被结合到IRDye 800CW，并在EpCAM阳性的HT-29和EpCAM阴性的COLO-320人类结肠癌细胞系上评估它们的结合效力。然后，在HT-29_luc2肿瘤携带小鼠中进行了所有三种结合物的NIRF和PA成像。注射后24小时，切除肿瘤和器官，分析示踪物的生物分布。Ac2-800CW和Ec4.1-800CW专门结合于HT-29细胞，而不结合COLO-320细胞。然后，建立了6 nmol和24小时作为两种DARPin示踪物的最佳体内剂量和成像时间点。注射后24小时，Ac2-800CW和Ec4.1-800CW的平均肿瘤背景比分别为2.60 ± 0.3和3.1 ± 0.3，使用临床Artemis NIRF成像仪可以清晰地描绘肿瘤。非肿瘤组织的生物分布分析只在肝脏和肾脏中显示高荧光信号，这反映了DARPin示踪物的清除。我们令人鼓舞的结果表明，EpCAM结合DARPins是一类有前途的靶向载体用于泛癌症靶向，注射后24小时可以提供清晰的肿瘤描绘。所述工作为基于DARPin的双模式NIRF/PA肿瘤成像提供了临床前基础。© 2023.作者。

Fluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer.EpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed.Ac2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers.Our encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer.© 2023. The Author(s).