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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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, PhD
National for Biotechnology Information, NLM, NIH, Bethesda, MD
Corresponding author.

Created: ; Last Update: February 2, 2012.

Chemical name:Cyclo(Cys-Arg-Gly-Asp-Cys)-Gly-Lys-Cy5.5
Abbreviated name:c(CRGDC)-GK-Cy5.5
Agent category:Peptide
Target:Integrin αvβ3
Target category:Receptor
Method of detection:Optical, near-infrared (NIR) fluorescence imaging
Source of signal:Cy5.5
  • Checkbox In vitro
  • Checkbox Rodents
No structure is available in PubChem.



Optical fluorescence imaging is increasingly being used to monitor biological functions of specific targets (1-3). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 nm) detection avoids the natural background fluorescence interference of biomolecules, providing a high contrast between target and background tissues in small animals. NIR fluorophores have a wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. NIR fluorophores also have high sensitivity, attributable to low background fluorescence, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is a non-invasive alternative to radionuclide imaging in small animals or with probes in close proximity of the target in humans (4, 5). Among the various optical imaging agents, only indocyanine green (ICG), with NIR fluorescence absorption at 780 nm and emission at 820 nm, is approved by the United States Food and Drug Administration for clinical applications in angiography, blood flow evaluation, and liver function assessment. It is also under evaluation in several clinical trials for other applications, such as optical imaging and mapping of both the lymphatic vessels and lymph nodes in cancer patients for surgical dissection of tumors and endoscopic imaging of the pancreas and colon.

Integrins are a family of cell-surface heterodimeric glycoproteins that mediate diverse biological events involving cell–cell and cell–matrix interactions (6). They consist of an α and a β subunit, and they are important for cell adhesion and signal transduction. The αvβ3 integrin is the most prominent receptor class affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (7-12). The αvβ3 integrin is strongly expressed on tumor cells and activated endothelial cells. In contrast, expression of αvβ3 integrin is weak on resting endothelial cells and most normal tissues. The αvβ3 antagonists are being studied as anti-tumor and anti-angiogenic agents (9, 13, 14), and the agonists are being studied as angiogenic agents for coronary angiogenesis (15, 16). A tripeptide sequence consisting of Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including αvβ3. Various radiolabeled antagonists have been introduced for imaging of tumors and tumor angiogenesis (17).

Cyclo(RGDyK) was conjugated with Cy5.5 to study in vivo biodistribution of the tracer in tumor-bearing mice. Cy5.5 is a NIR fluorescent dye with an absorbance maximum at 675 nm and an emission maximum at 694 nm, with a high extinction coefficient of 250,000 (mol/L)–1cm–1. c(RGDyK)-Cy5.5 was found to have high and long-lasting accumulation in αvβ3-positve U87MG human glioblastoma tumor cells in nude mice (18). The binding of c(RGDyK)-Cy5.5 to the integrin receptor was found to be specific both in vitro and in vivo. von Wallbrunn et al. (19) performed optical NIR fluorescence imaging studies using a two-dimensional planar fluorescence reflectance imaging system and three-dimensional fluorescence-mediated tomography (FMT) of cyclo(Cys-Arg-Gly-Asp-Cys)-Gly-Lys-Cy5.5 (c(CRGDC)-GK-Cy5.5) in tumor-bearing nude mice.



Cy5.5 monofunctional N-hydroxysuccinimide (NHS) ester was used to conjugate c(CRGDC)-GK to form c(CRGDC)-GK-Cy5.5, which was purified with high-performance liquid chromatography (19). Chemical yields of 75%–90% were obtained. The NHS ester of Cy5.5 was reacted with the ε-amino group of the lysine in c(CRGDC)-GK. The identity of c(CRGDC)-GK-Cy5.5 was confirmed with high-resolution electrospray mass spectrometry.

In Vitro Studies: Testing in Cells and Tissues


Flow cytometry analysis showed that ~98% of human melanoma M21 cells, ~16% of human adenocarcinoma MCF-7 cells, and ~1% of human fibrosarcoma HT-1008 cells were positive for the αvβ3 receptors (19). Binding of c(CRGDC)-GK-Cy5.5 to the three cell types was analyzed with fluorescence microscopy. M21cells exhibited a higher fluorescence intensity signal than MCF-7 cells, whereas little intensity signal was observed in the HT-1008 cells. The binding of c(CRGDC)-GK-Cy5.5 to M21 cells could be blocked with ten-fold excess of c(CRGDC)-GK.

Animal Studies



von Wallbrunn et al. (19) used FRI and FMT to perform biodistribution studies of c(CRGDC)-GK-Cy5.5 in nude mice (n = 5/group) bearing M21 tumors. Surface-weighted FRI studies were performed at 1, 3, and 24 h after injection of 2 nmol c(CRGDC)-GK-Cy5.5. The tumor accumulation of c(CRGDC)-GK-Cy5.5 could be clearly seen from 1 h to 24 h. The tumor/background ratios were 1.33 ± 0.14 and 1.46 ± 0.31 at 1 h and 24 h after injection. Pretreatment with 200 nmol c(CRGDC)-GK 10 min before injection of c(CRGDC)-GK-Cy5.5 reduced the tumor/background ratio to 1.13 ± 0.13 at 1 h after injection (P < 0.05).

FMT studies were performed at 1, 3, 5, and 24 h after injection of c(CRGDC)-GK-Cy5.5 in mice bearing M21 (n = 5), MCF-7 (n = 5), or HT-1008 (n = 6) tumors. Quantitative analysis of NIF optical images at 1 h after injection showed Cy5.5 concentrations of 417.6 ± 105.8 nM, 353.7 ± 54.0 nM, and 262.8 ± 155.4 nM in the M21, MCF-7, and HT-1008 tumors, respectively. A maximal accumulation was observed at 1 h in the tumors, which slowly washed out over time. A strong fluorescence signal was observed in the liver (269.1 ± 65.5 nM Cy5.5) at 1 h. The tumor/muscle ratios at 1 h were 6.4, 5.6, and 4.0 for M21, MCF-7, and HT-1008 tumors, respectively. The tracer accumulation could be blocked by 30% (P < 0.05) after pretreatment (10 min) with 100 nmol c(CRGDC)-GK in M21 tumors. Western blotting of M21, MCF-7, and HT-1080 cell and tumor tissue lysates using a murine β3-specific monoclonal antibody showed a similar expression of the murine β3 subunit on the tumor tissue but not on the human tumor cells. The higher binding of c(CRGDC)-GK-Cy5.5 on the M21 tumor tissue is probably due to binding of c(CRGDC)-GK-Cy5.5 to both the tumor vasculature and cells (expression of αvβ3 on M21 cells is higher than that on MCF-7 and HT-1080 cells). This proves to be a noninvasive method for imaging tumors in mice by using these two optical imaging techniques. FMT imaging allows three-dimensional reconstruction of NIR fluorescence distribution, which can be quantified.

Other Non-Primate Mammals


No publication is currently available

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.


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