Microbubbles with a disteroylphosphatidylcholine, disteroylphosphatidylethanolamine-polyethyleneglycol (PEG) 2000-pyridyldithio propionate-PEG 40 stearate shell conjugated to cyclic arginine-glycine-aspartic acid-d-tyrosine-lysine (cRGD) pentapeptide


Chopra A.

Publication Details



In vitro Rodents



Ultrasonography (ultrasound) is a technique for the noninvasive imaging of tumors because it is easy to use, is relatively inexpensive compared to other imaging modalities, does not use radionuclides or x-rays, and produces real-time images (1, 2). Imaging with this modality may involve the use of ultrasound contrast agents (UCA) based on microbubbles (MB), which are made up of a thin, biodegradable, lipid or polymeric shell filled with various types of gases, such as perfluorocarbon, sulphur hexafluoride, decafluorobutane, etc (3, 4). Because of their size and structural features, the MB cannot permeate the extracellular spaces, so they stay in the vascular circulation until the core gas diffuses into the blood and the remaining shell is metabolized (2). When exposed to a narrow range of ultrasound frequencies (3–5 MHz), the gas in the MB resonates with the sound; this in turn causes the MB to oscillate, which generates a signature acoustic echo (signal) that can be captured with a transducer and converted into a signal to generate an image. The application of ultrasound in medicine has been discussed in detail elsewhere (5).

Investigators have recently become interested in the use of targeted UCA for the detection of malignant tumors because these agents can be directed to bind to specific molecules that are overexpressed on the surface of cells in cancerous tissues (4). An additional advantage of using targeted UCA with ultrasonography is that malignant lesions can be visualized noninvasively, whereas tumors can be overlooked during a visual ex vivo examination of tissues obtained after a biopsy. Tumors with a malignant phenotype are known to show elevated angiogenic activity (development of new vasculature from old blood vessels), and endothelial cells in the vasculature of these lesions show increased expression of certain cell surface molecules such as αvβ3 integrins (6). The αvβ3 integrins are heterodimeric transmembrane cell adhesion molecules that are recognized biomarkers of angiogenesis, tumor progression, and metastasis, and they are overexpressed in a variety of cancers (6). Integrins are targeted by a variety of antagonist drugs that can prevent tumor progression (6), and they are also used with imaging agents, including UCA, for the noninvasive visualization of tumors (4).

Most targeted MB have traditionally been prepared with either avidin or biotin as the coupling agents, and these MB could not be used in the clinical setting because of their potential immunogenicity (4). In an effort to alleviate this problem, lipid-based (liposomal) MB with pyridyldithio propionate (PDP) on the surface were prepared and conjugated to a cyclic arginine-glycine-aspartic acid motif (cRGD) containing pentapeptide (such peptides are known to have a high affinity for αvβ3 integrins (7)) to generate cRGD-MB. The cRGD-MB were evaluated for use in imaging the cancerous lesion vasculature in mice bearing tumors generated with bEnd.3 cells (mouse endothelial cells that express αvβ3 integrins as confirmed with flow cytometry) (4).

Other Sources of Information

MICAD chapters related to integrins

Homo sapiens integrin alpha V, transcript variant 1, protein and mRNA sequences

Integrin alpha V in Gene database, Gene ID: 3685

Homo sapiens integrin beta 3, protein and mRNA sequences

Integrin beta 3 in Gene database, Gene ID: 374209

Clinical trials involving integrins

Integrins in Online Mendelian Inheritance in Man (OMIM) database

Integrin signaling pathways in Pathway Interaction Database



MB with a disteroylphosphatidylcholine, disteroylphosphatidylethanolamine-polyethylene glycol (PEG) 2000-PDP-PEG 40 stearate shell (PDP-MB) were prepared as described by Anderson et al. (4). In some preparations, ~2% moles of Dil, a commercially available fluorescent membrane probe, was included in the reaction mixture for the detection of MB during ex vivo fluorescence microscopy. The PDP-MB were conjugated with the cRGD pentapeptide as detailed elsewhere (4). For some studies, MB containing a fluorescein isothiocyanate (FITC) derivative of cRGD (FITC-cRGD-MB) were prepared as described above. Control MB containing a non-binding scrambled cRAD pentapeptide (arginine-alanine-aspartic acid) were also prepared as described above.

The mean diameters of the control MB and the targeted MB were 2.75 ± 0.02 μm and 2.71 ± 0.01 μm, respectively, and <2% of the MB had a diameter >8.0 μm. The concentrations of the control MB and the targeted MB in the final preparations were reported to be 133 ± 8.0 × 107/mL and 148 ± 1.1 × 107/mL, respectively. Fluorescence spectroscopy of the FITC-cRGD-MB preparation revealed that 8.2 ± 1.6 × 106 cRGD peptides were conjugated to each targeted MB.

In Vitro Studies: Testing in Cells and Tissues


Parallel plate flow chamber assays showed that the adhesion of cRGD-MB to recombinant αvβ3 integrin substrates was significantly higher (37.5 ± 9.2 MB/field of view (FOV); P < 0.01) than adhesion to a casein surface (0.7 ± 0.5 MB/FOV) or to an αvβ3 integrin surface blocked with an anti-αvβ3 integrin antibody (3.9 ± 1.5 MB/FOV) (4). The cRAD-MB exhibited minimal binding to any one of these surfaces.

In another study in which bEnd.3 cells were exposed to cRGD-MB alone, the MB showed >5-fold higher binding than cRAD-MB, PDP-MB, and cells treated with antibodies blocking either the αv or the β3 integrin subunit (4). This indicated that the cRGD-MB bound specifically to the αvβ3 integrin receptors.

Animal Studies



Ultrasound imaging was performed on Friend leukemia virus B–sensitive inbred mice bearing metastatic breast cancer Met-1 cell tumors as described by Anderson et al. (4). The animals were administered cRGD-MB and either cRAD-MB (n = 6 mice) or PDP-MB (n = 4 mice) via bolus through the tail vein, and images were acquired in a random order from the animals in each group. There was a 5-min delay between each imaging session, and a high-power ultrasound pulse was introduced to destroy MB from the previous study. Ultrasound imaging was performed with a protocol that could distinguish between the contrast echoes obtained from the target-bound and the free circulating MB as detailed elsewhere (4). Application of a high-powered ultrasound destruction sequence at 7 min after injection was shown to minimize the signal obtained from any circulating MB; this time point was selected for comparison of images obtained from the different MB treatment groups. Images obtained from mice treated with cRGD-MB clearly showed the tumor size and border and were easily distinguished from baseline images obtained before treatment with the UCA. The accumulation of targeted MB in the tumor vasculature of these animals was confirmed ex vivo with confocal microscopy using Dil-cRGD-MB as described by Anderson et al. (4).

In another study, the binding specificity of cRGD-MB was investigated in mice bearing Met-1 cell tumors after the animals (n = 6) were treated with a monoclonal antibody (mAb) that blocked the αvβ3 integrins (4). The mice were administered cRGD-MB to establish baseline adhesion of the UCA in the tumors, and the animals were injected with ~700 pmol anti-αvβ3 integrin mAb at 5 min after injection of cRGD-MB. At 30 min after the mAb treatment, the animals were again injected with cRGD-MB, and ultrasound imaging was performed to assess binding of the MB to the target. The tumor image intensity obtained from the mAb-treated animals was reported to be reduced by 3.2-fold compared to the intensity observed before the mAb treatment (baseline image). This indicated that the cRGD-MB bound specifically to the αvβ3 integrins.

Administration of the soluble cRGD pentapeptide (~1 μmol) to the mice (n = 10) 30 min prior to the cRGD-MB injection did not block binding of the UCA to the tumor vasculature (4). The investigators have proposed several reasons for this observation, including rapid clearance of the peptide from the intravascular spaces due to its low molecular weight (~500 kDa), which may have reduced the local concentration of cRGD such that it was insufficient to block the αvβ3 integrin receptors (4).

On the basis of these results, the investigators concluded that the cRGD-MB had good binding specificity for the αvβ3 integrins and was a suitable UCA to visualize tumor angiogenesis (4).

Other Non-Primate Mammals


No publications are currently available.

Non-Human Primates


No publications are currently available.

Human Studies


No publications are currently available.

Supplemental Information


No information is currently available.

NIH Support

Supported by National Institutes of Health grants IR43CA137913, 2R44EB007857, R01CA134659, R01CA112356, and R01CA103828.


Eisenbrey J.R., Forsberg F. Contrast-enhanced ultrasound for molecular imaging of angiogenesis. Eur J Nucl Med Mol Imaging. 2010;37 Suppl 1:S138–46. [PubMed: 20461376]
Wilson S.R., Burns P.N. Microbubble-enhanced US in body imaging: what role? Radiology. 2010;257(1):24–39. [PubMed: 20851938]
Deshpande N., Needles A., Willmann J.K. Molecular ultrasound imaging: current status and future directions. Clin Radiol. 2010;65(7):567–81. [PMC free article: PMC3144865] [PubMed: 20541656]
Anderson C.R., Hu X., Zhang H., Tlaxca J., Decleves A.E., Houghtaling R., Sharma K., Lawrence M., Ferrara K.W., Rychak J.J. Ultrasound molecular imaging of tumor angiogenesis with an integrin targeted microbubble contrast agent. Invest Radiol. 2011;46(4):215–24. [PMC free article: PMC3075480] [PubMed: 21343825]
Moore C.L., Copel J.A. Point-of-care ultrasonography. N Engl J Med. 2011;364(8):749–57. [PubMed: 21345104]
Desgrosellier J.S., Cheresh D.A. Integrins in cancer: biological implications and therapeutic opportunities. Nat Rev Cancer. 2010;10(1):9–22. [PMC free article: PMC4383089] [PubMed: 20029421]
Liu S. Radiolabeled cyclic RGD peptides as integrin alpha(v)beta(3)-targeted radiotracers: maximizing binding affinity via bivalency. Bioconjug Chem. 2009;20(12):2199–213. [PMC free article: PMC2795072] [PubMed: 19719118]