Leung K.

Publication Details



In vitro Rodents



Integrins are a family of cell surface heterodimeric glycoproteins that mediate diverse biological events involving cell-cell and cell-matrix interactions (1). They consist of an α and a β subunit. 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 (2-7). 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 (8, 9), and the agonists are being studied as angiogenic agents for coronary angiogenesis (10, 11). A tripeptide sequence consisting of Arg-Gly-Asp (RGD) is 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 and peptides were introduced for imaging of tumors and tumor angiogenesis (12).

Optical fluorescence imaging is increasingly used to obtain biological functions of specific targets (13-15). However, the intrinsic fluorescence of biomolecules poses a problem when visible light (350-700 nm) absorbing fluorophores are used. Near-infrared (NIR) fluorescence (700-1000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low infrared background, 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 becoming a noninvasive complement to radionuclide imaging.

Cypate is a reactive carbocyanine dye, which is derived from indocyanine green (ICG) (16). Cypate was previously conjugated to octreotate (Cyp-OC). Cyp-OC was not toxic to rats up to 10 μmol/kg (17). From the results of investigating a small library of RGD peptides for their binding activity to the αvβ3 integrin, a linear hexapeptide, Gly-Arg-Asp-Ser-Pro-Lys (GRDSPK), lacking the RGD sequence was conjugated with Cypate as Cyp-GRD to study in vivo biodistribution of the tracer in tumor-bearing mice (18). Cypate is a NIR fluorescent dye with an absorbance maximum at 778 nm and an emission maximum at 805 nm with a high extinction coefficient of 224,000 (mol/L)−1cm−1. Cyp-GRD was found to have a high and long-lasting accumulation in αvβ3-positve A549 human non-small cell lung carcinomas in nude mice. The binding of Cyp-GRD to the integrin receptor was found to be specific both in vitro and in vivo.



A detailed synthesis of Cypate was reported by Ye et al. (16). Cypate was conjugated to peptides prepared on solid support by standard fluorenylmethoxycarbonyl peptide synthesis (18). The conjugated peptides were subsequently cleaved from the solid resin and purified by high-performance liquid chromatography (HPLC) in good yields and high purity (>95%).

In Vitro Studies: Testing in Cells and Tissues


A549 cells were validated to have αv and β3 integrins by immunohistochemistry and Western blot analysis (18). The β3 subunit was expressed at a higher level than the αv subunit, suggesting that β3 may also be associated with another α subunit.

Fluorescence microscopy was used to study cellular distribution of Cyp-GRD in A549 cells (18). Cyp-GRD (1 μm) was internalized into the cytoplasm and not the nucleus by the cells at 37º C after 30 min of incubation, whereas the tracer remained on the cell surface at 4º C. The binding of the Cyp-GRD was inhibited by 10 μm cyclo(Arg-Gly-Asp-D-Phe-Val), an RGD antagonist with little fluorescence that was visible in A549 cells. The binding and internalization of Cyp-GRD were also inhibited by anti-β3 antibody (10 μm) and to a lesser extent by anti-αv antibody (10 μm), suggesting an important role for the β3 subunit in the binding and internalization of Cyp-GRD in A549 cells. Cyp-GRD was not toxic to A549 cells up to 100 μm as measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Animal Studies



Biodistribution studies of Cyp-GRD were evaluated in nude mice bearing an A549 subcutaneous xenograft model (18). Whole-body small animal images by reflectance planar fluorescence were obtained at various time points after injection of Cyp-GRD (0.3 μmol/kg). The tumor uptake of Cyp-GRD was visible at 8 h after the tracer had been cleared from the blood and non-targeted tissues. A maximal uptake in the tumor was reached at 24 h. The tracer uptake in the tumor could be blocked (>80%) by co-injection of cyclo(Arg-Gly-Asp-D-Phe-Val) (0.3 μmol/kg), whereas the blocking resulted in higher fluorescence intensity in the liver and kidneys. Use of an optical technique provided noninvasive imaging of tumor cells in mice.

The biodistribution of Cyp-GRD, Cypate-Gly-Arg-Gly-Asp-Ser-Pro-Lys (Cyp-RGD), and Cypate-cyclo(Arg-Gly-Asp-D-Phe-Val-Lys) (Cyp-cyclo-RGD) was studied in A549 tumor-bearing mice by ex vivo tissue fluorescence intensity measurements at 24 h after injection (18). Cyp-GRD uptake in tumor was the highest, followed to a lesser extent by Cyp-cyclo-RGD. The uptake of Cyp-RGD in the tumor was minimal. Cyp-cyclo-RGD and Cyp-RGD were markedly retained in the liver and kidneys as compared with Cyp-GRD. Cyp-GRD had a minimal uptake in non-targeted tissues. Furthermore, 111In-DOTA-GRD was not retained by the tumor. Therefore, there seems to be a synergic effect of Cypate- and GRD-containing peptide for binding to the αvβ3 integrin.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

R01 CA109754-01, R01 CA109754-02, R01 CA109754-03, R01CA109754, R21 CA100972-01, R33 CA100972-02, R33 CA100972-04, R33 CA100972


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