A biocompatible NIR squaraine dye and dye-antibody conjugates for versatile long-term in vivo fluorescence bioimaging

The demand for dependable near-infrared (NIR) probes, capable of sustained fluorescence within living systems and facile conjugation with biomolecules like antibodies and proteins, has been significantly on the rise, attributed to the substantial rise in the use of NIR imaging techniques and devices, with extensive integration into clinical diagnostics. Antibody conjugates are vital for targeted and selective bioimaging, enabling precise visualization of specific biomolecules within complex biological systems. Their multiplexing capability allows simultaneous detection of multiple targets, while their dynamic imaging capability enables real-time monitoring of cellular processes. Clinically, antibody conjugates have significant applications in disease prognosis, diagnosis, and monitoring. In this work, we report the synthesis of a new symmetrical NIR squaraine dye (SQ-58) with multiple carboxy anchoring groups for ease of coupling with antibodies. The dye showed decreased absorption and fluorescence intensity in phosphate buffer (PB) due to enhanced dye-aggregate formation. However, in the presence of bovine serum albumin (BSA) in PB, SQ-58 showed an enhanced fluorescence signal along concentrations of BSA. SQ-58 showed no cytotoxicity when tested in white laboratory mice while providing strong fluorescence when injected in vivo. Conjugation of SQ-58 through the carboxylic groups to the isotypic mouse IgG antibodies (IgG-SQ-58) resulted in uniform distribution of the targeted molecule in the whole cardiovascular system. The NIR signal of IgG-SQ-58 was stable for at least 7 days allowing the possibility of long-term imaging. Conjugation of SQ-58 to antibodies raised against NK-Ly lymphoma tumor cells allowed efficient discrimination of tumor cells grown in the abdomen of laboratory mice. Thus, to the best of our knowledge, we report for the first time a biocompatible NIR dye, SQ-58, that can be easily conjugatable to biomolecules, and its antibody conjugates for a wide range of bioimaging applications.