Figure 1 (click to enlarge)
Targeted Imaging: The discipline of medical imaging is expanding beyond an assessment of anatomical structure to functional imaging and an assessment of the nature and extent of disease. This advancement is made possible by recent discoveries in molecular science, which provide the opportunity to design targeted contrast agents. Targeted imaging using ultrasound relies on contrast agents to localize a specific molecular signature or physiologic system. Current ultrasound contrast agents are encapsulated microbubbles and have demonstrated effectiveness in cardiology and radiology. These contrast agents become identifiers of a specific molecular signature either by their preferential uptake by a physiologic system or by specific targeting of the agent through incorporation of adhesion molecules into the microbubble shell. Targeted ultrasound contrast agents provide an opportunity to image physiology or pathology that might be otherwise difficult to distinguish from the surrounding tissue without targeted contrast enhancement. Figure 1 demonstrates the adhesion of a microbubble to a activated neutrophil, a cell involved in the inflammatory response. Currently, our lab is collaborating with ImaRx Therapeutics (www.ImaRx.com) in the study of targeted contrast agents.
Figure 1 (click to enlarge)
Drug Delivery: The technology that makes targeted imaging possible also permits targeted drug delivery. Targeted drug desirable because many chemotherapy agents are systemically toxic, and therefore treatment only of the pathologic site may limit many of the side effects usually associated with chemotherapy. A drug delivery vehicle is a microbubble designed to carry a drug payload and to be localized in a specific diseased site. High-intensity pulses of ultrasound fragment these microbubbles. The intended result is the delivery of the therapy agent directly to the affected site. Figure 2 illustrates the disruption of a drug-carrier agent by a pulse of ultrasound.
Figure 2 (click to enlarge)
Sonothrombolysis: In this therapeutic technique, microbubbles are used to help disrupt blood clots non-invasively. Research has demonstrated that the cavitation, or rapid expansion and contraction, of micobubbles when they are exposed to a high-intensity acoustic pulse can erode hard objects such as clots.
Analysis of the behavior of ultrasound contrast agents is accomplished using high-speed optical and acoustical analysis. Through these analysis techniques, imaging parameters required for detection or destruction of microbubbles can be determined. Figure 3 illustrates optical photography of an ultrasound contrast agent insonified with a high-pressure acoustic pulse. Seven frame images portray two-dimensional images of the microbubble, while a "streak" image (bottom) displays the oscillation of the bubble over time. This image sequence illustrates that this microbubble is completely destroyed with an ultrasound pulse.
Figure 3 (click to enlarge)
Additional Information:
molecular signature of cancer: the vascular integrin αvB3: Angiogenesis, the formation of new microvessels, has been shown to be necessary for tumor growth above 1-2 mm3 (Folkman et al. 1995). Tumor-induced angiogenesis is not self-limited and continues indefinitely unless the entire tumor is eradicated or the host dies. Angiogenesis is a complex process that is mediated by factors produced by the tumor cells, the blood, and the stroma of the host tissue. Tumors and surrounding cells produce and release angiogenic growth factors, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor bFGF, that diffuse into the nearby tissues. These angiogenic growth factors bind to specific receptors located on nearby endothelial cells, activating these cells and prompting a cascade of cellular events that lead to endothelial cell growth and motility, and the eventual development of new blood vessels. The integrin αvΒ3 is a crucial part of this process (Brooks et al., 1994). Thus, αvΒ3 is a prime candidate for molecular targeting.
ultrasound contrast agents: Ultrasonic contrast agents (UCAs) are encapsulated microbubbles on the order of 1 to 10 μm in diameter. UCAs are filled with air, or a gas with a lower water solubility than air, such as a perfluorocarbon. The shell, designed to reduce diffusion into the blood, can be stiff (e.g. denatured albumin) or more flexible (phospholipid), and the shell thickness can vary from 10 to 200 nm. Both the shell and the gas core affect the behavior of the microbubble and the resulting clinical application. Encapsulated microbubbles are highly echogenic due to their compressibility. The compressibility of air is 7.65x10-6 m2/N, in comparison with 4.5x10-11 m2/N for water. The compressibility for an encapsulated microbubble falls within this range, as de Jong predicts the compressibility of Albunex® to be 5x10-7 m2 /N (de Jong, 1993) . It is because of their high echogenicity that these microbubbles are useful in increasing the scattered signal from blood. A small amount of contrast agent (ranging from μL to mL) is injected into the bloodstream during an ultrasonic exam. The use of UCAs has been shown to have clinical utility, particularly in cardiology, where contrast echocardiography has already proven to be a powerful diagnostic tool. UCAs have also been shown to be useful as a diagnostic tool in radiology (Goldberg et al. 2001). Microbubbles have been shown to enhance the detection of blood flow in both abdominal and peripheral and vascular structures.
Current ultrasound systems can detect microbubbles using several different
methods. The simplest method consists of detecting the backscatter and resonance
response of an UCA as it is excited by an acoustic pulse. This method relies
on the fact that bubbles are more echogenic than tissue. Other methods take
advantage of nonlinear microbubble oscillations, and detect resulting second
and sub-harmonics (Burns et al. 1992). These harmonics, which are multiples
or fractions of the resonant frequency, can be detected using a clinical imaging
system. Tissue does not exhibit such non-linear oscillations. Ultrasound propagation
at high intensities produces second harmonic frequencies, however, these components
can be minimized by reducing the transmitted intensity. Another imaging strategy
relies on destruction of the UCAs after they have filled a region. By imaging
a region before and after insonation with a pulse destructive to the agents,
and then subtracting the before and after images, one can detect and estimate
reperfusion rates of UCA-filled blood into tissue.
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targeted imaging: Targeted imaging combines the efficacy of a contrast agent with an adhesion molecule to target the contrast directly to the desired region. Site-specific adhesion molecules, such as monoclonal antibodies, peptides, asialoglycoproteins, or polysaccharides are incorporated into the shell of the microbubble or liposome (Lanza et al. 2001). After injection into the bloodstream, the targeted agent accumulates via adhesion receptors at the affected site, enhancing detection with a clinical imaging system. Tumor targeting has recently demonstrated promise with magnetic resonance imaging (MRI) and nuclear medicine, as researchers have demonstrated the success of targeting MRI and nuclear medicine contrast agents to the αvΒ3 integrin, permitting non-invasive monitoring of angiogenesis. Although MRI and nuclear medicine have some advantages over ultrasound, such as usefulness in brain and lung imaging and sensitivity (respectively), the portability, affordability, and dynamic imaging capability of ultrasound emphasize the importance of targeted ultrasound imaging. Recently, the effectiveness of targeted ultrasound contrast agents for inflammation has been demonstrated, in addition to the use of targeted UCAs for imaging thrombus. However, to date, targeted ultrasound contrast agents have not been applied to detection and monitoring of angiogenesis. In ongoing research, we are studying UCAs designed to target the αvΒ3 integrin, which as previously described, is characteristic of malignant tumors, in addition to contrast agents targeted to inflammation and thrombus. These targeted agents are custom designed in cooperation with the UC Davis Chemical Engineering Department and with industry collaborators, such as ImaRx Therapeutics (www.imarx.com).