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Patient survival depends on the completeness of resection of peritoneal ovarian cancer metastases (POCM), and therefore, it is important to develop methods to enhance detection. Previous probe designs based on activatable galactosyl human serum albumin (hGSA)-fluorophore pairs, which target lectin receptors expressed on POCM, have used only visible range dyes conjugated to hGSA. However, imaging probes emitting fluorescence in the NIR range are advantageous because NIR photons have deeper in vivo tissue penetration and result in lower background autofluorescence than those emitting in the visible range. A NIR-activatable hGSA fluorophore was synthesized using a bacteriochlorin-based dye, NMP1. NMP1 has two unique absorption peaks, one in the green range and the other in the NIR range, but emits at a NIR peak of 780 nm. NMP1, thus, has two different Stokes shifts that have the potential to allow imaging of POCM both at the peritoneal surface and just below it. hGSA was conjugated with 2 NMP1 molecules to create a self-quenching complex (hGSA-NMP1). The activation ratio of hGSA-NMP1 was measured by the fluorescence intensity before and after exposure to 10% SDS. The activation ratio of hGSA-NMP1 was ~100-fold in vitro. Flow cytometry, fluorescence microscopy, and in vivo spectral fluorescence imaging were carried out to compare hGSA-NMP1 with hGSA-IR800 and hGSA-ICG (two always-on control agents with similar emission to NMP1) in terms of comparative fluorescence signal and the ability to detect POCM in mice models. The sensitivity and specificity of hGSA-NMP1 for POCM implant detection were determined by colocalizing NMP1 emission spectra with red fluorescent protein (RFP) expressed constitutively in SHIN3 tumor implants at different depths below the peritoneal surface. In vitro, SHIN3 cells were easily detectable after 3 h of incubation with hGSA-NMP1. In vivo submillimeter POCM foci were clearly detectable with spectral fluorescence imaging using hGSA-NMP1. Among 555 peritoneal lesions, hGSA-NMP, using NIR and green excitation light, respectively, detect 75% of all lesions and 91% of lesions ~0.8 mm or greater in diameter. Few false positives were encountered. Nodules located at a depth below the small bowel surface were only depicted with hGSA-NMP1. We conclude that hGSA-NMP1 is useful in imaging peritoneal ovarian cancer metastases, located both superficially and deep in the abdominal cavity.

bioconjugate chemistry

Galactosyl Human Serum Albumin-NMP1 Conjugate: A Near Infrared (NIR)-Activatable Fluorescence Imaging Agent to Detect Peritoneal Ovarian Cancer Metastases