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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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Microbubbles conjugated with anti-vascular cell adhesion molecule-1 nanobody cAbVCAM1-5

MB-cAbVCAM1-5
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD
Corresponding author.

Created: ; Last Update: June 14, 2012.

Chemical name:Microbubbles conjugated with anti-vascular cell adhesion molecule-1 nanobody cAbVCAM1-5
Abbreviated name:MB-cAbVCAM1-5, µB-cAbVCAM1-5
Synonym:
Agent category:Antibody
Target:Vascular cell adhesion molecule-1 (VCAM-1)
Target category:Adhesion molecule
Method of detection:Ultrasound (US)
Source of signal:Microbubbles
Activation:No
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Click on protein, nucleotide (RefSeq), and gene for more information about VCAM-1.

Background

[PubMed]

Ultrasound is a widely used imaging modality (1), and its role in noninvasive molecular imaging with ligand-carrying microbubbles (MBs) is expanding (2). MBs are comprised of spherical cavities encapsulated in a shell and filled with a gas. The shells are made of phospholipids, surfactants, denatured human serum albumin, or synthetic polymers. Ligands and antibodies can be incorporated into the MB shell surface. MBs are usually 2–8 μm in size. MBs of this size provide a strongly reflective interface and resonate to ultrasound waves. They are used as ultrasound contrast agents in imaging of inflammation, angiogenesis, intravascular thrombus, and tumors (2-4). They also can potentially be used for drug and gene delivery (5).

Endothelial cells are important cells in inflammatory responses (6, 7). Bacterial lipopolysaccharide (LPS), virus, inflammation, and tissue injury increase tumor necrosis factor α (TNFα), interleukin-1 (IL-1), and other cytokine and chemokine secretion. Leukocyte emigration from blood is dependent on their ability to adhere to endothelial cell surfaces. Inflammatory mediators and cytokines induce chemokine secretion from endothelial cells and other vascular cells and increase their expression of cell surface adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), integrins, and selectins. Chemokines are chemotactic toward leukocytes and toward sites of inflammation and tissue injury. The movements of leukocytes through endothelial junctions into the extravascular space are highly orchestrated through various interactions with different adhesion molecules on endothelial cells (8).

VCAM-1 is found in very low amounts or non-detectable on the cell surface of resting endothelial cells and other vascular cells, such as smooth muscle cells and fibroblasts (9-13). VCAM-1 binds to very late antigen-4 (VLA-4) integrin on the cell surface of leukocytes. IL-1 and TNFα increase expression of VCAM-1 and other cell adhesion molecules on the vascular endothelial cells, which leads to leukocyte adhesion to the activated endothelium. Furthermore, VCAM-1 expression was also induced by oxidized low-density lipoproteins under atherogenic conditions (14). Overexpression of VCAM-1 by atherosclerotic lesions plays an important role in their progression toward vulnerable plaques, which may erode and rupture. MBs targeted with monoclonal antibody against VCAM-1 are being developed as a noninvasive agent for VCAM-1 expression in vascular endothelial cells during different stages of inflammation in atherosclerosis (15). Nanobodies are the smallest intact antigen-binding fragments (15 kDa) isolated from heavy-chain camelid antibodies with efficient and specific tumor targeting (16-18). Hernot et al. (19) prepared an anti-VCAM-1 nanobody (cAbVCAM1-5) that was site-specifically biotinylated in bacteria. cAbVCAM1-5 was coupled to biotinylated lipid MBs via streptavidin-biotin bridging to form MB-cAbVCAM1-5 for use with in vivo ultrasound imaging of VCAM-1 expression.

Synthesis

[PubMed]

For targeted MBs, Hernot et al. (19) prepared biotinylated MBs by sonication of an aqueous dispersion of decafluorobutane gas, phosphatidylcholine, polyoxyethylene-40-stearate, and phosphatidylethanolamine-polyethylene glycol(PEG3400)-biotin. MBs were combined with streptavidin, washed, and conjugated with 62.5 pmol/107 MBs biotinylated nanobody cAbVCAM1-5 against VCAM-1 (MBcAbVCAM1-5) or control nanobody against eGFP (MBcAbGFP4). The targeted MBs were about 2.2 ± 0.2 μm in diameter with a polydispersity index of 1.32. An antibody to MB ratio was estimated with flow cytometry to be 288,000 ± 38,000. There were ~10,000 nanobodies per μm2 of the MB surface.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Binding experiments with biotinylated cAbVCAM1-5 were performed with the use of a Biacore sensor chip immobilized with mouse VCAM-1 protein (20). The Kd value of biotinylated cAbVCAM1-5 was calculated to be 7.2 nM as compared with 10.7 nM for the non-biotinylated nanobody.

Hernot et al. (19) performed perfusion of MBcAbVCAM1-5 (2.5 × 106/ml) through the flow chamber coated with endothelial cells at a wall shear rate of 1.0–5.0 dynes/cm2. There was a significantly greater number of MBcAbVCAM1-5 attached to TNF_activated mouse endothelial cells than to normal endothelial cells at 1.0 dynes/cm2 (P < 0.01). The attachment of MBcAbVCAM1-5 to activated endothelial cells decreased with increasing shear rates, with still significant MB adhesion at 5.0 dynes/cm2 (P < 0.05). The adhesion of MBcAbVCAM1-5 to the activated endothelial cells was inhibited by 70% with excess non-biotinylated cABVCAM1-5 (P < 0.01). No attachment of control MBcAbGFP4 to normal and activated endothelial cells was observed. Flow cytometry showed binding of cAbVCAM1-5 to activated mouse endothelial cells but not to non-activated cells.

Animal Studies

Rodents

[PubMed]

Hernot et al. (19) determined MBcAbVCAM1-5 attachment to the tumor vasculatures in mice (n = 4) bearing MC38 subcutaneous tumors. Contrast-enhanced ultrasound molecular imaging of the tumors was performed for ~12 minutes after intravenous injection of 3.75 × 107 MBcAbVCAM1-5 or MBcAbGFP4. Contrast-enhanced signals peaked at 20 s after injection of MBs. The tumor signal of MBcAbVCAM1-5 declined more slowly than that of MBcAbGFP4. The tumor signal (expressed as % of maximal intensity at 20 s) of MBcAbVCAM1-5 (44 ± 9%) was 100% higher than that of MBcAbGFP4 (22 ± 10%) (P < 0.05) at 10 min after injection. After destruction of the MBs with two high-intensity ultrasound pulses at 10 min, the signals completely disappeared, indicating that the majority of unbound circulating MBs had cleared from the blood. Immunohistochemical analysis of the tumor sections showed expression of VCAM-1 in the tumor vasculature as well as on the tumor cells. No blocking studies were performed with cAbVCAM1-5.

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

R21/33 CA102880

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