Oral Administration of a Bivalent Carbonic Anhydrase IX Near-Infrared Imaging Agent Detects Hypoxic Tumors in a Mouse Model

Early detection of breast cancer can significantly increase survival rates, and annual screening increases the chance of early detection. However, anatomical imaging methods, such as mammography, underperform in women with dense breast tissue, and alternatives such as ultrasound and magnetic resonance imaging can be more time- and cost-intensive. Molecular imaging methods have the potential to provide both spatial and molecular information, yielding advantages over traditional imaging. However, molecular imaging has previously been impractical for annual screening due to its high cost, risk of ionizing radiation, long acquisition time, and IV injection of contrast agents. Therefore, a cost-effective and lower-risk method is needed. This study reports the design of a bivalent near-infrared fluorophore sulfo-Cy5.5 carbonic anhydrase 9 (CAIX/CA9) targeting molecule (biAAZ-Cy5.5) for oral administration and fluorescent breast cancer screening. A challenge in developing orally delivered contrast agents is balancing sufficient oral bioavailability with efficient targeting and background clearance. Previous work from our group developed an integrin-targeting agent for oral delivery (α_Vβ₃-IRdye800CW) that binds α_Vβ₃ integrin with sufficient oral absorption to successfully detect breast tumors in a mouse model. However, similar to current screening methods, the potential for false positives caused by benign tumors limits its application. To increase the diagnostic potential of this approach, we developed a second molecular targeting agent for dual-channel imaging. By selecting the malignant tumor-associated marker CA9 and using acetazolamide (AAZ) as the targeting molecule, the bivalent biAAZ-Cy5.5 shows high specificity, high affinity, and low off-target binding. With ∼7.6% oral bioavailability in mice, biAAZ-Cy5.5 uptake was sufficient for <i>in vivo</i> imaging. Oral coadministration of α_Vβ₃-IRdye800CW and biAAZ-Cy5.5 in HT29 (CA9⁺, α_Vβ₃⁺) and HCT116 (CA9^-, α_Vβ₃⁺) tumor-bearing mice demonstrated that biAAZ-Cy5.5 selectively targets the CA9-expressing tumors. The combination of an 800 nm integrin-targeting agent for high sensitivity with the 680 nm CA9-targeting agent for improved specificity highlights the utility of dual-channel imaging.