Dual Functionality of [⁶⁴Cu]Cu-NOTA-San A-Cy7 for Diagnostic Imaging and Surgical Guidance in Hsp90α-Positive Tumors

Intraoperative fluorescence navigation in esophageal cancer enables the clinical translation of fluorescence imaging. Heat shock protein 90 alpha (Hsp90α) plays a vital role in the progression of malignant disease, and elevated Hsp90α expression has been reported in esophageal cancer. The aim of this study was to develop a dual-modality probe, [⁶⁴Cu]Cu-NOTA-San A-Cy7, for imaging Hsp90α expression in vivo via both positron emission tomography (PET) and fluorescence imaging in esophageal cancer. In this study, the Hsp90α-targeting cyclopeptide Sansalvamide A (San A) was chemically modified with a Cy7 dye and NOTA chelator simultaneously. Experimental assays confirmed that NOTA-San A-Cy7 has a favorable affinity for Hsp90α-positive EC109 cells, with a dissociation constant (K_d) of 1.08 ± 0.19 μM. The probe [⁶⁴Cu]Cu-NOTA-San A-Cy7 was successfully synthesized with ⁶⁴CuCl₂, achieving a high radiochemical purity of over 95%. Furthermore, the probe demonstrated excellent stability in both saline and serum solutions. The probe was subsequently evaluated in a Hsp90α-positive EC109 tumor-bearing model via PET imaging, which confirmed that Hsp90α-specific uptake was significantly reduced by the co-administration of an excess blocking agent. Biodistribution studies revealed that at 24 hours post-injection, the tumor uptake of the probe was 1.35 ± 0.29%ID/g in the nonblocking group and significantly decreased to 0.73 ± 0.15%ID/g in the blocking group (p < 0.05). Concurrent with the PET experiment, fluorescence imaging was conducted, revealing substantial tumor uptake in the EC109 model. As a proof of concept, imaging-guided surgery utilizing the fluorescent component of this probe was performed. This approach demonstrated the potential for providing surgical guidance in mice positive for Hsp90α, highlighting the dual functionality of the probe for both diagnostic imaging and intraoperative navigation. In summary, our findings unequivocally demonstrate that the dual-modality probe [⁶⁴Cu]Cu-NOTA-San A-Cy7 holds significant promise as an agent for imaging Hsp90α-positive tumors in vivo, offering a valuable tool for the detection and potential management of such tumors.