Fluorescence-guided surgery is an emerging imaging technique that can enhance the ability of surgeons to detect tumors when compared with visual observation. To facilitate characterization, fluorescently labeled probes have been dual-labeled with a radionuclide to enable cross-validation with nuclear imaging. In this study, we selected the somatostatin receptor imaging agent DOTATOC as the foundation for developing a dual-labeled analog. We hypothesized that a customized dual-labeling approach with a multimodality chelation (MMC) scaffold would minimize steric effects of dye conjugation and retain agonist properties. Methods: An MMC conjugate (MMC-TOC) was synthesized on solid-phase and compared with an analog prepared using conventional methods (DA-TOC). Both analogs were conjugated to IRDye 800 using copper-free click chemistry. The resulting compounds, MMC(IR800)-TOC and DA(IR800)-TOC, were labeled with Cu and ⁶⁴Cu and tested in vitro in somatostatin receptor subtype 2–overexpressing HEK-293 cells to assess agonist properties, and in AR42J rat pancreatic cancer cells to determine receptor binding characteristics. Multimodality imaging was performed in AR42J xenografts. Results: Cu-MMC(IR800)-TOC demonstrated higher potency for cyclic adenosine monophosphate inhibition (half maximal effective concentration [EC₅₀]: 0.21 ± 0.18 vs. 1.38 ± 0.54 nM) and receptor internalization (EC₅₀: 41.9 ± 29.8 vs. 455 ± 299 nM) than Cu-DA(IR800)-TOC. Radioactive uptake studies showed that blocking with octreotide caused a dose-dependent reduction in ⁶⁴Cu-MMC(IR800)-TOC uptake whereas ⁶⁴Cu-DA(IR800)-TOC was not affected. In vivo studies revealed higher tumor uptake for ⁶⁴Cu-MMC(IR800)-TOC than ⁶⁴Cu-DA(IR800)-TOC (5.2 ± 0.2 vs. 3.6 ± 0.4 percentage injected dose per gram). In vivo blocking studies with octreotide reduced tumor uptake of ⁶⁴Cu-MMC(IR800)-TOC by 66%. Excretion of ⁶⁴Cu-MMC(IR800)-TOC was primarily through the liver and spleen whereas ⁶⁴Cu-DA(IR800)-TOC was cleared through the kidneys. Ex vivo analysis at 24 h confirmed PET/CT data by showing near-infrared fluorescence signal in tumors and a tumor-to-muscle ratio of 5.3 ± 0.8 as determined by γ-counting. Conclusion: The findings demonstrate that drug design affected receptor pharmacology and suggest that the MMC scaffold is a useful tool for the development of dual-labeled imaging agents.

荧光引导手术是一种新兴的成像技术，与目测观察相比，可以增强外科医生检测肿瘤的能力。为便于表征，已将荧光标记的探针用放射性核素进行了双重标记，以实现与核成像的交叉验证。在这项研究中，我们选择了生长抑素受体显像剂DOTATOC作为开发双标记类似物的基础。我们假设具有多模态螯合（MMC）支架的定制双标记方法将使染料偶联的空间效应最小化，并保留激动剂特性。方法：MMC共轭物（MMC-TOC）在固相上合成，并与使用常规方法制备的类似物（DA-TOC）进行比较。使用无铜咔嗒化学将两种类似物偶联到IRDye 800上。将生成的化合物MMC（IR800）-TOC和DA（IR800）-TOC标记为Cu和⁶⁴ Cu，并在生长抑素受体亚型2过表达的HEK-293细胞中进行体外测试，以评估激动剂特性，并在AR42J大鼠胰腺中进行测试确定癌细胞与受体的结合特性。在AR42J异种移植物中进行了多模态成像。结果： Cu-MMC（IR800）-TOC表现出更高的抑制环磷酸腺苷的能力（半数最大有效浓度[EC ₅₀ ]：0.21±0.18 vs. 1.38±0.54 nM）和受体内在化（EC₅₀：41.9±29.8与455±299 nM）相比，Cu-DA（IR800）-TOC。放射性吸收研究表明，用奥曲肽阻断可导致⁶⁴ Cu-MMC（IR800）-TOC吸收剂量依赖性降低，而⁶⁴ Cu-DA（IR800）-TOC则不受影响。体内研究显示，与⁶⁴ Cu-DA（IR800）-TOC相比，⁶⁴ Cu-MMC（IR800）-TOC的肿瘤吸收更高（5.2±0.2 vs. 3.6±0.4％注射剂量/克）。奥曲肽的体内阻断研究将⁶⁴ Cu-MMC（IR800）-TOC的肿瘤吸收降低了66％。⁶⁴ Cu-MMC（IR800）-TOC的排泄主要是通过肝脏和脾脏，而⁶⁴Cu-DA（IR800）-TOC通过肾脏清除。在24小时的离体分析通过显示肿瘤中的近红外荧光信号和通过γ计数确定的肿瘤与肌肉的比为5.3±0.8证实了PET / CT数据。结论：研究结果表明药物设计影响受体药理学，并表明MMC支架是开发双标记成像剂的有用工具。