Aiming at the design of highly brilliant NIR emissive optical probes, e.g., for in vivo near-infrared fluorescence imaging (NIRF), we studied the absorption and fluorescence properties of the asymmetric cyanines Dy678, Dy681, Dy682, and Dy676 conjugated to the model antibody IgG. The ultimate goal was here to derive general structure-property relationships for suitable NIR fluorescent labels. These Dy dyes that spectrally match Cy5 and Cy5.5, respectively, were chosen to differ in chromophore structure, i.e., in the substitution pattern of the benzopyrylium end group and in the number of sulfonic acid groups. Spectroscopic studies of the free and IgG-bound fluorophores revealed a dependence of the obtained dye-to-protein ratios on dye hydrophilicity and control of the fluorescence quantum yields (Φ(f)) of the IgG conjugates by the interplay of different fluorescence reduction pathways like dye aggregation and fluorescence resonance energy transfer (FRET). Based upon aggregation studies with these dyes, the amount of dye dimers in the IgG conjugates was determined pointing to dye hydrophilicity as major parameter controlling aggregation. To gain further insight into the exact mechanism of dye dimerization at the protein, labeling experiments at different reaction conditions but constant dye-to-protein ratios in the reaction solution were performed. With Dy682 that displays a Φ(f) of 0.20 in PBS and 0.10 for moderate dye-to-protein ratio of 2.5, a low aggregation tendency, and a superior reactivity in IgG labeling, we identified a promising diagnostic tool for the design of NIR fluorescent probes and protein conjugates.

No abstract

Aiming at the identification of new fluorescent reporters for targeted optical probes, we assessed the application-relevant features of a novel asymmetric cyanine, DY-681, in comparison to the only clinically approved dye indocyanine green (ICG), the golden imaging standard Cy5.5, and the asymmetric cyanine DY-676 successfully exploited by us for the design of different contrast agents. This comparison included the analysis of the spectroscopic properties of the free fluorophores and their thermal stability in aqueous solution as well as their cytotoxic potential. In addition, the absorption and emission features of IgG-conjugated DY-681 were examined. The trimethine DY-681 exhibited spectral features closely resembling that of the pentamethine Cy5.5. Its high thermal stability in phosphate buffer saline (PBS) solution in conjunction with its low cytotoxicity, reaching similar values as determined for Cy5.5 and DY-676, renders this dye more attractive as ICG and, due to its improved fluorescence quantum yield in PBS, also superior to DY-676. Although in PBS, Cy5.5 was still more fluorescent, the fluorescence quantum yields (Phi(f)) of DY-681 and Cy5.5 in PBS containing 5 mass-% bovine serum albumin (BSA) were comparable. Labeling experiments with DY-681 and the model antibody IgG revealed promisingly high Phi(f) values of the bioconjugated dye.

To assess the suitability of asymmetric cyanine dyes for in vivo fluoro-optical molecular imaging, a comprehensive study on the influence of the number of negatively charged sulfonate groups governing the hydrophilicity of the DY-67x family of asymmetric cyanines was performed. Special attention was devoted to the plasma protein binding capacity and related pharmacokinetic properties. Four members of the DY-67x cyanine family composed of the same main chromophore, but substituted with a sequentially increasing number of sulfonate groups (n  =  1-4; DY-675, DY-676, DY-677, DY-678, respectively), were incubated with plasma proteins dissolved in phosphate-buffered saline. Protein binding was assessed by absorption spectroscopy, gel electrophoresis, ultrafiltration, and dialysis. Distribution of dye in organs was studied by intraveneous injection of 62 nmol dye/kg body weight into mice (n  =  12; up to 180 minutes postinjection) using whole-body near-infrared fluorescence imaging. Spectroscopic studies, gel electrophoresis, and dialysis demonstrated reduced protein binding with increasing number of sulfonate groups. The bovine serum albumin binding constant of the most hydrophobic dye, DY-675, is 18 times higher than that of the most hydrophilic fluorophore, DY-678. In vivo biodistribution analysis underlined a considerable influence of dye hydrophilicity on biodistribution and excretion pathways, with the more hydrophobic dyes, DY-675 and DY-676, accumulating in the liver, followed by strong fluorescence signals in bile and gut owing to accumulation in feces and comparatively hydrophilic DY-678-COOH accumulating in the bladder. Our results demonstrate the possibility of selectively controlling dye-protein interactions and, thus, biodistribution and excretion pathways via proper choice of the fluorophore's substitution pattern. This underlines the importance of structure-property relationships for fluorescent labels. Moreover, our data could provide the basis for the rationalization of future contrast agent developments.

Dye-biomolecule conjugation is frequently accompanied by considerable spectral changes of the dye's absorption spectrum that limit the use of the common photometrical method for the determination of labeling densities. Here, we describe an improvement of this method using the integral absorbance of the dye instead of its absorbance at the long wavelength maximum to determine the concentration of the biomolecule-coupled dye. This approach is illustrated for three different cyanine dyes conjugated to the antibody IgG.