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AlexaFluor 700–Labeled regioselectively addressable functionalized template-[cyclo-(Arg-Gly-Asp-d-Phe-Lys)]4

, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: August 29, 2012.

Chemical name:AlexaFluor 700–Labeled regioselectively addressable functionalized template-[cyclo-(Arg-Gly-Asp-d-Phe-Lys)]4
Abbreviated name:A700-RAFT-RGD
Agent Category:Peptides
Target:Integrin αvβ3
Target Category:Receptors
Method of detection:Optical imaging
Source of signal / contrast:AlexaFluor 700
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No structure available.



AlexaFluor 700 (A700)–Labeled regioselectively addressable functionalized template (RAFT)-[cyclo-(Arg-Gly-Asp-d-Phe-Lys)]4, abbreviated as A700-RAFT-RGD, is a tetrameric Arg-Gly-Asp (RGD)-based peptide conjugate synthesized by Briat et al. for tumor imaging by targeting integrin αvβ3 (1).

Angiogenesis is a process of neovascular development and growth from preexisting vessels. Different from the normal blood vessel system, the neovasculature is chaotic and irregular in tumors with abnormal expression of diverse vascular surface biomarkers. These biomarkers have fewer kinetic compartments that must be crossed for intravenously administered agents to reach them (2, 3). Integrin αvβ3 is one of these biomarkers, and it has been intensively investigated as a target for imaging and antiangiogenic therapy. Integrin αvβ3 is minimally expressed in normal blood vessels but is significantly overexpressed in newly sprouting vasculature in tumors (4, 5). Small peptides are one group of ligands frequently used for targeting purposes, such as RGD, Asp-Gly-Arg, His-Gly-Phe, and Arg-Arg-Leu (3). The RGD tripeptide sequence is an adhesive protein recognition site, presenting in the extracellular matrix and blood. Integrin αvβ3 binds extracellular matrix proteins through the exposed RGD tripeptide sequence. In general, RGD peptides are less selective (binding with 8 of the 24 integrins), degrade rapidly in vivo, and have a relatively low binding affinity (3, 6).

Development of multivalent RGD peptides is one approach to improve their properties. Boturyn et al. synthesized a RAFT-RGD platform based on a cyclic decapeptide scaffold that incorporates and presents in a spatially controlled manner two independent functional domains: a clustered ligand domain for integrin recognition and cell targeting, and a labeling domain for detection and characterization of this binding (7). This platform has been labeled with Cy5, 99mTc, and 64Cu and has been shown to efficiently accumulate in tumors in animal models (8-11). Although the results are promising, approximately 30%–50% of the total dose of the labeled RAFT-RGD has been found to remain trapped in the kidneys several days after injection, which is at least partially due to the tetramerization of RGD peptides (8, 12, 13). Although peptides can be reabsorbed, endocytosed, and degraded by the lysosomes of proximal tubular cells, radiometal-chelated amino acids may be retained in the tubular cells, leading to radiation-induced toxicity and a weak imaging contrast for tumors surrounding the kidneys.

To reduce the renal accumulation of fluorescent and radiolabeled RAFT-RGD, Briat et al. evaluated the effect of Gelofusine on the renal reabsorption of these agents (1). Gelofusine is a gelatin-based plasma expander that consists of succinylated bovine gelatin molecules, a mixture of collagen-derived peptides. Infusion of Gelofusine has been reported to increase the renal excretion of megalin ligands and to decrease the retention of radiolabeled compounds in the cortical proximal tubules (14-16). The results by Briat et al. showed that Gelofusine significantly reduced the renal retention of labeled RAFT-RGD, while it increased the ratio of tumor to healthy tissue (1). The data obtained with 111In-DOTA-RAFT-RGD were summarized in the chapter on 111In-DOTA-RAFT-RGD in MICAD. This chapter summarizes the data obtained with A700-RAFT-RGD and Gelofusine.



Boturyn et al. described the synthesis of the series of RAFT-RGD compounds (7). Briat et al. labeled RAFT-RGD with fluorescent A700 mono-N-hydroxysuccinimide ester by grafting the dye at a lysine side chain (1). The labeling efficiency was not reported nor was the A700 per RGD.

In Vitro Studies: Testing in Cells and Tissues


Boturyn et al. characterized the series of RAFT-RGD compounds and their cellular internalization (7). No data are available for the binding and cellular internalization of A700-RAFT-RGD.

Animal Studies



Ahmadi et al. and Briat et al. investigated the biodistribution of 111In-DOTA-RAFT-RGD in αvβ3-positive mice bearing TS/A-pc (murine mammary carcinoma cells) tumors (1, 13). Briat et al. also analyzed the distribution of A700-RAFT-RGD, which showed results similar to the distribution of 111In-DOTA-RAFT-RGD (no detailed data provided) (1).

Briat et al. further studied the effects of Gelofusine on the biodistribution of A700-RAFT-RGD in mice bearing TS/A-pc tumors (n = 6 mice/group) (Table 1) (1). Each mouse received a tail vein injection of 200 µl (10 nmol dye/mouse) A700-RAFT-RGD. Gelofusine (4%) or phosphate-buffed saline (PBS) was preinjected 2–5 min before A700-RAFT-RGD injection. Fluorescent imaging was performed at 1 h and 24 h after injection, and the mice were then euthanized. Compared with PBS, preinjection of Gelofusine reduced the kidney uptake of A700-RAFT-RGD by 63% as measured from the dissected kidneys and by 31% as measured with noninvasive optical imaging (P < 0.01). The tumor uptake was not significantly affected by Gelofusine (P > 0.05). No blocking studies were reported.

Table 1: Effect of Gelofusine on the biodistribution of A700-RAFT-RGD.

Ratio1 h24 hDissected11 h24 hDissected1
T/M2.02 ± 0.092.18 ± 0.336.87 ± 0.341.95 ± 0.432.03 ± 0.643.80 ± 0.04
T/K0.66 ± 0.131.33 ± 0.010.35 ± 0.121.24 ± 0.261.68 ± 0.370.73 ± 0.41
Kidney210,457 ± 1,470985 ± 2134,601 ± 1,0865,040 ± 580682 ± 1671,698 ± 61

1Data were obtained from dissected organs at 24 h after injection. Other data in the table were obtained directly from the images.

2Data are expressed as the mean of the relative light unit per pixel.

Abbreviations: T/M = tumor/muscle ratio; T/K = tumor/kidney ratio.

Confocal microscopy of the tissue sections of the dissected kidneys showed that A700-RAFT-RGD was mainly trapped in the renal cortex at 24 h after injection (1). There was no effect of Gelofusine on the A700-RAFT-RGD localization into the kidneys or into a particular cell type.

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


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