Background

[PubMed]

Ultrasound is widely used imaging modality (1) and is expanding its role in noninvasive molecular imaging with ligand-carrying microbubbles (2). Microbubbles are comprised of spherical cavities filled by a gas encapsulated in a shell. The shells are made of phospholipids, surfactant, denatured human serum albumin or synthetic polymer. Ligands and antibodies can be incorporated into the shell surface of microbubbles. Microbubbles are usually 2 to 8 μm in size. They 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 (3-5). They are also potentially 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 (TNFα), interleukin-1 (IL-1) and other cytokine and chemokine secretion. Leukocyte emigration from blood is dependent on their rolling along endothelial cell surfaces and subsequently adherence 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 to leukocytes to 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 (9).

ICAM-1 is found on cell surface of endothelial cells and other vascular cells, such as smooth muscle cells and fibroblasts (10-14). It binds to counter-receptors on the cell-surface of leukocytes. IL-1 and TNFα increase ICAM-1 and other cell adhesion molecule expression on the vascular endothelial cells, leading to leukocyte adhesion to the activated endothelium. Microbubbles targeted with antibody against ICAM-1 are being developed as a noninvasive agent for ICAM-1 expression in vascular endothelial cells of dysfunctional endothelium (15-18).

Synthesis

[PubMed]

For targeted microbubbles, Weller et al. (17) prepared biotinylated microbubbles by sonication of an aqueous dispersion of decafluorobutane gas, phosphatidylcholine, polyethyleneglycol-(PEG-) stearate, and phosphatidylethanolamine-biotin in a 2:1:1 ratio by weight. Microbubbles were combined with streptavidin, washed, and conjugated with biotinylated mouse monoclonal antibody against ICAM-1(MB_ICAM-1) or isotype control monoclonal antibody (MB_iso). Control lipid microbubbles (MB_c) were also prepared. The microbubbles are about 3.4 ± 1.2 microns in diameter. An antibody to microbubble ratio was estimated to be 60,000 ± 5,000 by flow cytometry. Alternatively, the primary amino groups of antibody were covalently conjugated to the carboxylic groups on the microbubble shell, which has been activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (15).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Villanueva et al. (15) reported that MB_ICAM-1 (1^_10 x 10⁸/ml) perfused through the flow chamber coated with endothelial cells at a wall shear rate of 25 s^-1. There was a significantly greater number of MB_ICAM-1 attached to IL-1 activated endothelial cells (8.0 ± 3.5 microbubbles/cell) than to normal endothelial cells (0.21 ± 0.09 microbubbles/cell). The number of adherent MB_ICAM-1 to activated endothelial cell decreased to 2.6 ± 0.3 and 0.8 ± 0.4 at 100 and 1000 s^-1, respectively. The number of MB_ICAM-1 adherent to normal endothelial cell was only 0.1 at 1000 s^-1. MB_c and MB_iso attachment to both activated and normal endothelial cells were minimal (0.03-0.05) at 25 s^-1 and there were no significant changes at higher wall shear rates.

Weller et al. (16, 17, 19) confirmed that microbubble shell antibody density and wall shear rate are critical parameters controlling microbubble targeted adhesion. Microbubble adhesion was significantly greater with greater anti-ICAM-1 antibody density of the microbubbles and greater ICAM-1 expression on the cell surface of the endothelial cells. On the other hand, microbubble adhesion to endothelial cells was inversely proportional to the wall shear rate. Therefore, accumulation and retention of MB_ICAM-1 is possible under physiologic flow conditions and is strongly influenced by shear stress and surface density of the target receptor.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

HL58865

References

1.

Wells P.N. Physics and engineering: milestones in medicine. Med Eng Phys. 2001;23(3):147–53. [PubMed: 11410379]

2.

Liang H.D., Blomley M.J. The role of ultrasound in molecular imaging. Br J Radiol. 2003;76(Spec No 2):S140–50. [PubMed: 15572336]

3.

Klibanov A.L. Ligand-carrying gas-filled microbubbles: ultrasound contrast agents for targeted molecular imaging. Bioconjug Chem. 2005;16(1):9–17. [PubMed: 15656569]

4.

Lindner J.R. Microbubbles in medical imaging: current applications and future directions. Nat Rev Drug Discov. 2004;3(6):527–32. [PubMed: 15173842]

5.

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]

6.

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]

7.

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

8.

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

9.

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

10.

Bochner B.S., Luscinskas F.W., Gimbrone M.A. Jr, Newman W., Sterbinsky S.A., Derse-Anthony C.P., Klunk D., Schleimer R.P. Adhesion of human basophils, eosinophils, and neutrophils to interleukin 1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules. J Exp Med. 1991;173(6):1553–7. [PMC free article: PMC2190849] [PubMed: 1709678]

11.

Kume N., Cybulsky M.I., Gimbrone M.A. Jr. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J Clin Invest. 1992;90(3):1138–44. [PMC free article: PMC329976] [PubMed: 1381720]

12.

Leung K.H. Release of soluble ICAM-1 from human lung fibroblasts, aortic smooth muscle cells, dermal microvascular endothelial cells, bronchial epithelial cells, and keratinocytes. Biochem Biophys Res Commun. 1999;260(3):734–9. [PubMed: 10403835]

13.

Luscinskas F.W., Cybulsky M.I., Kiely J.M., Peckins C.S., Davis V.M., Gimbrone M.A. Jr. Cytokine-activated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving both endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1. J Immunol. 1991;146(5):1617–25. [PubMed: 1704400]

14.

Nagel T., Resnick N., Atkinson W.J., Dewey C.F. Jr, Gimbrone M.A. Jr. Shear stress selectively upregulates intercellular adhesion molecule-1 expression in cultured human vascular endothelial cells. J Clin Invest. 1994;94(2):885–91. [PMC free article: PMC296171] [PubMed: 7518844]

15.

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]

16.

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]

17.

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]

18.

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]

19.

Weller G.E., Villanueva F.S., Tom E.M., Wagner W.R. Targeted ultrasound contrast agents: in vitro assessment of endothelial dysfunction and multi-targeting to ICAM-1 and sialyl Lewisx. Biotechnol Bioeng. 2005;92(6):780–8. [PubMed: 16121392]

20.

Reinhardt M., Hauff P., Briel A., Uhlendorf V., Linker R.A., Maurer M., Schirner M. Sensitive particle acoustic quantification (SPAQ): a new ultrasound-based approach for the quantification of ultrasound contrast media in high concentrations. Invest Radiol. 2005;40(1):2–7. [PubMed: 15597013]