Mateusiak Łukasz, Chigoho Dora M, Floru Sam, Pollenus Sofie, Debie Pieterjan, van Willigen Danny M, van Leeuwen Fijs W B, Hernot Sophie
Laboratory for Molecular Imaging and Therapy, MITH, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium.
Interventional Molecular Imaging Laboratory, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333ZA Leiden, The Netherlands.
ACS Pharmacol Transl Sci. 2025 May 28;8(6):1659-1668. doi: 10.1021/acsptsci.5c00024. eCollection 2025 Jun 13.
Over the past two decades, it has become evident that fluorescence imaging holds substantial value in preclinical research and could also play a pivotal role in clinical applications such as intraoperative molecular imaging. The latter relies on applying targeted fluorescent agents designed to recognize specific biomarkers expressed in diseased tissues. Targeting moieties, such as camelid-derived nanobodies (Nbs), exhibit remarkable pharmacokinetics for molecular imaging owing to their robustness and compact size. However, the relatively small size of Nbs makes their pharmacokinetics sensitive to the chemical structure of attached fluorophores. In this study, we conducted a comparative analysis between Nbs labeled with three different sulfoCy5 derivatives (Cy5 (charge -2), Cy5 (charge -1), and Cy5° (charge 0)). Nb-Cy5 and Nb-Cy5° allowed specific visualization of subcutaneous tumors in mice within 1 h with minimal background. Conversely, Nb-Cy5 required at least 3 h to achieve sufficient contrast and exhibited nonspecific liver accumulation. Remarkably, Nb-Cy5 was able to overcome the renal retention typically observed for Nbs. Microscopy analyses of kidney sections revealed differential accumulation for Nb-Cy5 and Nb-Cy5° at the level of the proximal tubule cells, with only Nb-Cy5 showing internalization in lysosomes and endosomes and subsequent metabolization. In conclusion, this study underscores the significant influence of dye charges on the biodistribution profile and tumor-targeting capabilities of Nb tracers. Among the tested variants, the fluorescent dye Cy5 emerged as a promising choice to use in combination with Nbs for molecular imaging applications, in particular due to its low renal retention.
在过去二十年中，荧光成像在临床前研究中的重要价值已变得显而易见，并且在诸如术中分子成像等临床应用中也可能发挥关键作用。后者依赖于应用经过设计以识别病变组织中表达的特定生物标志物的靶向荧光剂。靶向部分，如骆驼科动物源纳米抗体（Nb），由于其稳定性和紧凑尺寸，在分子成像中表现出卓越的药代动力学特性。然而，Nb相对较小的尺寸使其药代动力学对连接的荧光团的化学结构敏感。在本研究中，我们对用三种不同的磺化Cy5衍生物（Cy5（电荷-2）、Cy5（电荷-1）和Cy5°（电荷0））标记的Nb进行了比较分析。Nb-Cy5和Nb-Cy5°能够在1小时内特异性地可视化小鼠皮下肿瘤，且背景最小。相反，Nb-Cy5至少需要3小时才能获得足够的对比度，并且表现出非特异性肝脏蓄积。值得注意的是，Nb-Cy5能够克服Nb通常观察到的肾脏潴留。肾脏切片的显微镜分析显示，Nb-Cy5和Nb-Cy5°在近端小管细胞水平存在差异蓄积，只有Nb-Cy5显示出在溶酶体和内体中的内化以及随后的代谢。总之，本研究强调了染料电荷对Nb示踪剂的生物分布特征和肿瘤靶向能力的重大影响。在测试的变体中，荧光染料Cy5成为与Nb联合用于分子成像应用的有前景的选择，特别是由于其低肾脏潴留。
