Laboratory for In Vivo Cellular and Molecular Imaging, ICMI-BEFY, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
Leiden University Medical Center, Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University, 2311 Leiden, The Netherlands.
Biomolecules. 2021 Feb 26;11(3):360. doi: 10.3390/biom11030360.
Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a "multifunctional single attachment point" (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and In-labeling of DTPA, the tracer's characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer's ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.
术中使用靶向荧光示踪剂进行引导，有可能为外科医生提供关于切除边缘肿瘤组织存在的实时反馈。为了克服荧光光的有限穿透深度，已经提出了将荧光与 SPECT/CT 成像和/或伽马射线追踪相结合的方法。在这里，我们描述了一种新型双模态纳米体示踪剂的设计和临床前验证，该示踪剂使用“多功能单附着点”（MSAP）标签标记，将 Cy5 荧光团和二乙烯三胺五乙酸（DTPA）螯合剂整合到单个结构中。在将双模态 MSAP 缀合到抗 HER2 纳米体 2Rs15d 的伯胺上并对 DTPA 进行 In 标记后，在体外评估了示踪剂的特性。随后，通过 SPECT/CT 和荧光成像在 24 小时内评估了其生物分布和肿瘤靶向性。最后，在荷有 HER2 过表达 SKOV3.IP1 腹膜病变的小鼠中研究了示踪剂识别小的、弥散性肿瘤病变的能力。[In]In-MSAP.2Rs15d 在缀合后保留了其亲和力，并且在 24 小时内保持稳定。体内 SPECT/CT 和荧光图像显示，在 HER2 过表达的肿瘤中有特异性摄取，背景低。在 1 小时时获得了高的肿瘤与肌肉比，并且在 24 小时的 SPECT/CT 和荧光图像上分别保持 19 倍和 3 倍。在腹腔内弥散模型中，示踪剂通过核成像允许检测较大的病变，而荧光则能够准确切除亚毫米病变。因此，可以通过将携带荧光团和放射性标记螯合剂的单个分子 MSAP 试剂缀合来方便地设计双模态核/荧光纳米体示踪剂。这些示踪剂有望在临床应用中得到应用。
