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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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Biotinylated vascular endothelial growth factor121-Avi-streptavidin-IRDye800

VEGF121-Avib-SA800
, PhD
National for Biotechnology Information, NLM, NIH, Bethesda, MD

Created: ; Last Update: July 7, 2009.

Chemical name:Biotinylated vascular endothelial growth factor121-Avi-streptavidin-IRDye800
Abbreviated name:VEGF121-Avib-SA800
Synonym:
Agent category:Polypeptide
Target:VEGF receptor
Target category:Receptor
Method of detection:Optical, near-infrared (NIR) fluorescence imaging
Source of signal:IRDye800
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about VEGF.

Background

[PubMed]

Optical fluorescence imaging is increasingly being used to monitor biological functions of specific targets in small animals (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 compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is a non-invasive complement to radionuclide imaging in small animals.

Vascular endothelial growth factor (VEGF) consists of at least six isoforms of various numbers of amino acids (121, 145, 165, 183, 189, and 206 amino acids) produced through alternative splicing (4). VEGF121, VEGF165, and VEGF189 are the forms secreted by most cell types and are active as homodimers linked by disulfide bonds. VEGF121 does not bind to heparin like the other VEGF species (5). VEGF is a potent angiogenic factor that induces proliferation, sprouting, migration, and tube formation of endothelial cells. There are three high-affinity tyrosine kinase VEGF receptors (VEGFR-1, Flt-1; VEGFR-2, KDR/Flt-1; and VEGFR-3, Flt-4) on endothelial cells. Several types of non-endothelial cells such as hematopoietic stem cells, melanoma cells, monocytes, osteoblasts, and pancreatic β cells also express VEGF receptors (4).

VEGF receptors were found to be overexpressed in various tumor cells and tumor-associated endothelial cells (6). Inhibition of VEGF receptor function has been shown to inhibit pathological angiogenesis as well as tumor growth and metastasis (7, 8). Radiolabeled VEGF tracers have been developed for imaging solid tumors and angiogenesis in humans (9-11). Wang et al. (12) fused the Avi peptide (14 amino acids) to the C-terminus of VEGF121 to allow site-specific biotinylation of the epsilon amine group of a central lysine residue of Avi. The binotinylated VEGF121-Avi (VEGF121-Avib) was able to form a tight complex with streptavidin-IRDye800 (SA800) as VEGF121-Avib-SA800 for NIR imaging of VEGFR expression in vivo to study tumor angiogenesis.

Synthesis

[PubMed]

Wang et al. (12) constructed the fusion gene encoding VEGF121-Avi with DNA recombinant technology. The purified fusion protein was incubated with BirA enzyme for biotinylation for 30 min at 30ºC to form VEGF121-Avib. There was one biotin molecule per VEGF121-Avib protein. SA-IRDye800 contains 2.4 dye molecules per SA molecule.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

In vitro competition binding studies were performed with porcine aortic endothelial cells expressing cloned human VEGFR-2 using 125I-VEGF165 as the radioactive ligand (12). The binding affinity values of VEGF121, VEGF121-Avi, VEGF121-Avib-SA800, and VEGF121-Avi-IRDye800 (IRDye800 chemically conjugated) were 0.72 ± 0.41, 5.18 ± 1.29, 3.09 ± 1.15, and 3,030 ± 790 nM, respectively. The binding affinity of VEGF121-Avib-SA800 was only slightly lower than that of VEGF121, whereas the binding affinity of VEGF121-Avi-IRDye800 was >1,000-fold lower than that of VEGF121.

Animal Studies

Rodents

[PubMed]

Wang et al. (12) performed in vivo NIR fluorescence imaging studies of VEGF121-Avib-SA800, VEGFm-Avib-SA800 (non-active VEGF mutant), VEGF121-Avi-IRDye800 (less active than VEGF121 as binding affinity was decreased with the direct conjugation of IRDye800), and SA800 (0.5 nmol IRDye800 equivalent per mouse) in mice (n = 4/group) bearing VEGFR-2–expressing 67NR murine breast tumors. Sagittal images were obtained at 2, 18, 40, and 66 h after intravenous injection. VEGF121-Avib-SA800 exhibited peak signal intensity at 18 h with a gradual washout. The tumor/muscle ratios for VEGF121-Avib-SA800 were 6–7 at these time points, whereas VEGFm-Avib-SA800, VEGF121-Avi-IRDye800, and SA800 exhibited tumor/muscle ratios of <2 at these time points. No blocking experiment was performed. Ex vivo NIR fluorescence images of various organs were obtained at 66 h after injection. The normalized light intensity signal was 3.5 in the tumor with VEGF121-Avib-SA800. The organs with the highest NIR signal were the kidneys (7.5) and liver (4.9), with little signal in the lung (0.6), spleen (1.0), and heart (1.1). The tumor/muscle ratios were 4, 1, 1, and 1 for VEGF121-Avib-SA800, VEGFm-Avib-SA800, VEGF121-Avi-IRDye800, and SA800, respectively. Immunofluorescence staining confirmed the colocalization of VEGF-Avib-SA-Cy5.5 with the luminal endothelial cells in the tumor.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

NIH Support

P50 CA114747, U54 CA119367

References

1.
Achilefu S. Lighting up tumors with receptor-specific optical molecular probes. Technol Cancer Res Treat. 2004;3(4):393–409. [PubMed: 15270591]
2.
Ntziachristos V., Bremer C., Weissleder R. Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur Radiol. 2003;13(1):195–208. [PubMed: 12541130]
3.
Becker A., Hessenius C., Licha K., Ebert B., Sukowski U., Semmler W., Wiedenmann B., Grotzinger C. Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands. Nat Biotechnol. 2001;19(4):327–31. [PubMed: 11283589]
4.
Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev. 2004;25(4):581–611. [PubMed: 15294883]
5.
Cohen T., Gitay-Goren H., Sharon R., Shibuya M., Halaban R., Levi B.Z., Neufeld G. VEGF121, a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells. J Biol Chem. 1995;270(19):11322–6. [PubMed: 7744769]
6.
Soria J.C., Fayette J., Armand J.P. Molecular targeting: targeting angiogenesis in solid tumors. Ann Oncol. 2004;15 Suppl 4:iv223–7. [PubMed: 15477311]
7.
Ferrara N. Vascular endothelial growth factor as a target for anticancer therapy. Oncologist. 2004;9 Suppl 1:2–10. [PubMed: 15178810]
8.
Hicklin D.J., Ellis L.M. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol. 2005;23(5):1011–27. [PubMed: 15585754]
9.
Li S., Peck-Radosavljevic M., Koller E., Koller F., Kaserer K., Kreil A., Kapiotis S., Hamwi A., Weich H.A., Valent P., Angelberger P., Dudczak R., Virgolini I. Characterization of (123)I-vascular endothelial growth factor-binding sites expressed on human tumour cells: possible implication for tumour scintigraphy. Int J Cancer. 2001;91(6):789–96. [PubMed: 11275981]
10.
Li S., Peck-Radosavljevic M., Kienast O., Preitfellner J., Hamilton G., Kurtaran A., Pirich C., Angelberger P., Dudczak R. Imaging gastrointestinal tumours using vascular endothelial growth factor-165 (VEGF165) receptor scintigraphy. Ann Oncol. 2003;14(8):1274–7. [PubMed: 12881392]
11.
Li S., Peck-Radosavljevic M., Kienast O., Preitfellner J., Havlik E., Schima W., Traub-Weidinger T., Graf S., Beheshti M., Schmid M., Angelberger P., Dudczak R. Iodine-123-vascular endothelial growth factor-165 (123I-VEGF165). Biodistribution, safety and radiation dosimetry in patients with pancreatic carcinoma. Q J Nucl Med Mol Imaging. 2004;48(3):198–206. [PubMed: 15499293]
12.
Wang H., Chen K., Niu G., Chen X. Site-specifically biotinylated VEGF(121) for near-infrared fluorescence imaging of tumor angiogenesis. Mol Pharm. 2009;6(1):285–94. [PubMed: 19099493]
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