Tracers for Fluorescence-Guided Surgery: How Elongation of the Polymethine Chain in Cyanine Dyes Alters the Pharmacokinetics of a Dual-Modality c[RGDyK] Tracer

Abstract

The potential of receptor-mediated fluorescence-based image-guided surgery tracers is generally linked to the near-infrared emission profile and good-manufacturing-production availability of fluorescent dyes. Surprisingly, little is known about the critical interaction between the structural composition of the dyes and the pharmacokinetics of the tracers. In this study, a dual-modality tracer design was used to systematically and quantitatively evaluate the influence of elongation of the polymethine chain in a fluorescent cyanine dye on the imaging potential of a targeted tracer. As a model system, the integrin marker αβ was targeted using arginylglycylaspartisc acid [RGD]-based vectors functionalized with a In-diethylenetriaminepentaacetic acid (DTPA) chelate and a fluorescent dye: (Cy3-(SO)methyl-COOH [emission wavelength (λ), 580 nm], Cy5-(SO)methyl-COOH [λ, 680 nm], or Cy7-(SO)methyl-COOH [λ, 780 nm]). Tracers were analyzed for differences in photophysical properties, serum protein binding, chemical or optical stability, and signal penetration through tissue. Receptor affinities were evaluated using saturation and competition experiments. In vivo biodistribution (SPECT imaging and percentage injected dose per gram of tissue) was assessed in tumor-bearing mice and complemented with in vivo and ex vivo fluorescence images obtained using a clinical-grade multispectral fluorescence laparoscope. Two carbon-atom-step variations in the polymethine chain of the fluorescent cyanine dyes were shown to significantly influence the chemical and photophysical characteristics (e.g., stability, brightness, and tissue penetration) of the hybrid RGD tracers. DTPA-Cy5-(SO)methyl-COOH-c[RGDyK] structurally outperformed its Cy3 and Cy7 derivatives. Radioactivity-based evaluation of in vivo tracer pharmacokinetics yielded the lowest nonspecific uptake and highest tumor-to-background ratio for DTPA-Cy5-(SO)methyl-COOH-c[RGDyK] (13.2 ± 1.7), with the Cy3 and Cy7 analogs trailing at respective tumor-to-background ratios of 5.7 ± 0.7 and 4.7 ± 0.7. Fluorescence-based assessment of tumor visibility revealed a similar trend. These findings underline that variations in the polymethine chain lengths of cyanine dyes have a profound influence on the photophysical properties, stability, and in vivo targeting capabilities of fluorescent imaging tracers. In a direct comparison, the intermediate-length dye (Cy5) yielded a superior c[RGDyK] tracer, compared with the shorter (Cy3) and longer (Cy7) analogs.

Hybrid Tracers Based on Cyanine Backbones Targeting Prostate-Specific Membrane Antigen: Tuning Pharmacokinetic Properties and Exploring Dye–Protein Interaction

“…For fluorescence confocal imaging, cells were seeded on glass-bottom culture dishes (MatTek Corp.) and placed in the incubator overnight. Confocal imaging was performed on viable cells, using an SP8 WLL fluorescence confocal microscope (Leica) (42). The cells were incubated with 1 mM EuK-Cy5-mas 3 , EuK-(SO 3 )Cy5-mas 3 , EuK-Cy5(SO 3 )-mas 3 , EuK-(Ar) Cy5-mas 3 , or EuK-Cy5(Ar)-mas 3 for 1 h at 37°C and washed with phosphate-buffered saline 3 times.…”

“…At 2 h after injection, SPECT imaging was performed followed by ex vivo evaluation of the biodistribution (in total, 6 animals per tracer). Total-body imaging and data reconstruction for PC346C-tumor-bearing mice was performed on a U-SPECT scanner (MILabs) using previously described methods (42,47).…”

“…Ex vivo images of excised tissues were acquired at Cy5 (l ex , 640 nm; l em , 680 nm). For imaging with the fluorescence laparoscope, the tumor was placed on top of a heightadjustable lift and imaged with a setup identical to one described previously (42,48). Images were acquired using a clinical-grade Image 1 S camera system (Karl Storz Endoskope GmbH) equipped with a 0°l aparoscope.…”

“…Excitation was achieved using a Cy5-modified D-Light C light source (Karl Storz Endoskope GmbH). Cy5 was detected by placement of an additional standard eyepiece adaptor (catalog number 20100034; Karl Storz Endoskope GmbH) between the camera and the laparoscope (42,48).…”

New Developments in Dual-Labeled Molecular Imaging Agents

“…In another study, Buckle et al elucidated how the elongation of the polymethine chain of cyanine dyes alters the pharmacokinetic disposition of otherwise similar dual-labeled tracers (25). Cy3 (Ex/Em, 550/570 nm), Cy5, and Cy7 were conjugated to DTPA-labeled c[RGDyK], and their bioactivity was confirmed in vitro.…”