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

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 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 αβ integrins (6). The αβ 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 αβ 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 αβ integrins as confirmed with flow cytometry) (4).