Photophysical Characterization and Biointeractions of NIR Squaraine Dyes for in Vitro and in Vivo Bioimaging

Abstract

The increasing demand for reliable near-infrared (NIR) probes exhibiting enduring fluorescence in living systems and facile compatibility with biomolecules such as peptides, antibodies or proteins is driven by the increasing use of NIR imaging in clinical diagnostics. To address this demand, a series of carboxy-functionalized unsymmetrical squaraine dyes (SQ-27, SQ-212, and SQ-215) along with non-carboxy-functionalized SQ-218 absorbing and emitting in the NIR wavelength range were designed and synthesized followed by photophysical characterization. This study focused on the impact of structural variations in the alkyl chain length, carboxy functionality positioning, and spacer chain length on dye aggregation and interaction with bovine serum albumin (BSA) as a model protein. In phosphate buffer (PB), the absorption intensity of the dyes markedly decreased accompanied by pronounced shoulders indicative of dye aggregation, and complete fluorescence quenching was seen in contrast to organic solvents. However, in the presence of BSA in PB, there was a enhancement in absorption intensity while regaining the fluorescence coupled with a remarkable increase in the intensity with increasing BSA concentrations, signifying the impact of dye−BSA interactions on preventing aggregation. Further analysis of Job's plot unveiled a 2:1 interaction ratio between BSA and all dyes, while the binding studies revealed a robust binding affinity (K a ) in the order of 10 7 /mol. SQ-212 and SQ-215 were further tested for their in vitro and in vivo imaging capabilities. Notably, SQ-212 demonstrated nonpermeability to cells, while SQ-215 exhibited easy penetration and prominent cytoplasmic localization in in vitro studies. Injection of the dyes into laboratory mice showcased their efficacy in visualization, displaying stable and intense fluorescence in tissues without toxicity, organ damage, or behavioral changes. Thus, SQ-212 and SQ-215 are promising candidates for imaging applications, holding potential for noninvasive cellular and diagnostic imaging as well as biomarker detection when coupled with specific vectors in living systems.

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

“…For quantum yield, rhodamine 6G whose Q R is 0.95 48 was used as the reference, and the calculated fluorescence quantum ( φ ) yield of SQ-58 was 0.16 in methanol, which is typical of squaraine dyes. 49,50…”

Design and Synthesis of Novel Squaraine-Based Fluorescent Probe for Far-Red Detection of Chymotrypsin Enzyme

“…The squaraine dye’s chain length plays a pivotal role in its behavior in aqueous environments, influencing aggregate formation. Specifically, shorter chain lengths are prone to forming both H-aggregates and J-aggregates, whereas longer alkyl chain substitutions, such as octyl and dodecyl, are observed to mitigate this aggregation through steric hindrance [ 33 ]. The lipophilicity of squaraine dye is increased by longer alkyl chains, which also have a hydrophobic effect that slightly lowers the chance of aggregate formation [ 34 ].…”