Background

[PubMed]

Ultrasound is the most widely used imaging modality (1), and its role in non-invasive molecular imaging is expanding with ligand-carrying microbubbles (2). Microbubbles are spherical cavities filled with a gas encapsulated in a shell. The shells are made of phospholipids, a surfactant, denatured human serum albumin, or a synthetic polymer. Ligands and antibodies can be incorporated into the shell surface of microbubbles, which are usually 2–8 μm in diameter. Microbubbles provide a strongly reflective interface and resonate to ultrasound waves, and they are used as ultrasound contrast agents in imaging of inflammation, angiogenesis, intravascular thrombus, and tumors (3-5). They also have the potential to be used for drug and gene delivery (6).

Endothelial cells are important cells in inflammatory responses (7, 8). Bacterial lipopolysaccharide, virus, inflammation, and tissue injury increase tumor necrosis factor α, interleukin-1, and other cytokine and chemokine secretion. Leukocyte emigration from blood is dependent on the leukocytes rolling along endothelial cell surfaces and subsequently adhering 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, vascular cell adhesion molecule-1, integrins, and selectins. Chemokines are chemotactic to leukocytes at sites of inflammation and tissue injury (9). Angiogenesis is a process of development and growth of new blood vessels from pre-existing vessels. Tumor growth depends on the formation of new blood vessels from angiogenesis. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) enhance angiogenesis. Expression of VEGF receptors is higher on the cell surface of tumor-derived endothelial cells than on the cell surface of normal endothelial cells.

Microbubbles conjugated to peptides or to antibodies against integrins, cell adhesion molecules, and VEGF receptors have previously been studied for the non-invasive assessment and imaging of angiogenesis (10-13). A peptide specific to tumor vasculature and containing Arg-Arg-Leu (RRL) was identified by screening a peptide display library panned against tumor cells derived from SCC-VII murine squamous cell carcinomas (14). Weller et al. (12) studied ultrasonic imaging of tumor vasculature using microbubbles conjugated with biotinylated c(CGGRRLGGC) (MB_RRL) in mice bearing human tumor xenografts.

Synthesis

[PubMed]

For targeted microbubbles, Weller et al. (12) prepared biotinylated microbubbles by sonication of an aqueous dispersion of decafluorobutane gas, phosphatidylcholine, polyethylene glycol-stearate, and phosphatidylethanolamine-biotin in a 2:1:1 ratio by weight. Microbubbles were combined with streptavidin, washed, and conjugated with MB_RRL or control peptide c(CGGGGGGGC) (MB_control). The microbubbles are 3.2 ± 1.0 μm in diameter. The peptide/microbubble ratio was estimated with flow cytometry to be ~60,000 (10).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Weller et al. (12) reported that MB_RRL or MB_control (3.33 × 10⁶/ml) perfused through the flow chamber coated with endothelial cells at a wall shear rate of 100 s^-1 for 3 min. There was a significantly (P < 0.01) greater number of MB_RRL attached to tumor-activated endothelial cells (2.4 ± 0.6 microbubbles/cell) than to normal endothelial cells (0.8 ± 0.1 microbubbles/cell). MB_control attachment to both activated and normal endothelial cells was minimal (0.3–0.4 microbubbles/cell).

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

R01 HL-58865

References

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Villanueva F.S. , Wagner W.R. , Vannan M.A. , Narula J. Targeted ultrasound imaging using microbubbles. Cardiol Clin. 2004; 22 (2):283–98. [PubMed: 15158940]

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Dijkmans P.A. , Juffermans L.J. , Musters R.J. , van Wamel A. , ten Cate F.J. , van Gilst W. , Visser C.A. , de Jong N. , Kamp O. Microbubbles and ultrasound: from diagnosis to therapy. Eur J Echocardiogr. 2004; 5 (4):245–56. [PubMed: 15219539]

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Cybulsky M.I. , Gimbrone M.A. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science. 1991; 251 (4995):788–91. [PubMed: 1990440]

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Lowe J.B. Glycosylation in the control of selectin counter-receptor structure and function. Immunol Rev. 2002; 186 :19–36. [PubMed: 12234359]

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Vanderslice P. , Woodside D.G. Integrin antagonists as therapeutics for inflammatory diseases. Expert Opin Investig Drugs. 2006; 15 (10):1235–55. [PubMed: 16989599]

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Villanueva F.S. , Jankowski R.J. , Klibanov S. , Pina M.L. , Alber S.M. , Watkins S.C. , Brandenburger G.H. , Wagner W.R. Microbubbles targeted to intercellular adhesion molecule-1 bind to activated coronary artery endothelial cells. Circulation. 1998; 98 (1):1–5. [PubMed: 9665051]

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Weller G.E. , Lu E. , Csikari M.M. , Klibanov A.L. , Fischer D. , Wagner W.R. , Villanueva F.S. Ultrasound imaging of acute cardiac transplant rejection with microbubbles targeted to intercellular adhesion molecule-1. Circulation. 2003; 108 (2):218–24. [PubMed: 12835214]

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Weller G.E. , Villanueva F.S. , Klibanov A.L. , Wagner W.R. Modulating targeted adhesion of an ultrasound contrast agent to dysfunctional endothelium. Ann Biomed Eng. 2002; 30 (8):1012–9. [PubMed: 12449762]

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Reinhardt M. , Hauff P. , Linker R.A. , Briel A. , Gold R. , Rieckmann P. , Becker G. , Toyka K.V. , Maurer M. , Schirner M. Ultrasound derived imaging and quantification of cell adhesion molecules in experimental autoimmune encephalomyelitis (EAE) by Sensitive Particle Acoustic Quantification (SPAQ). Neuroimage. 2005; 27 (2):267–78. [PubMed: 15905104]

14.

Brown C.K. , Modzelewski R.A. , Johnson C.S. , Wong M.K. A novel approach for the identification of unique tumor vasculature binding peptides using an E. coli peptide display library. Ann Surg Oncol. 2000; 7 (10):743–9. [PubMed: 11129422]