Application of ultrasound to selectively localize nanodroplets for targeted imaging and therapy.

Lipid-coated perfluorocarbon nanodroplets are submicrometer-diameter liquid-filled droplets with proposed applications in molecularly targeted therapeutics and ultrasound (US) imaging. Ultrasonic molecular imaging is unique in that the optimal application of these agents depends not only on the surface chemistry, but also on the applied US field, which can increase receptor-ligand binding and membrane fusion. Theory and experiments are combined to demonstrate the displacement of perfluorocarbon nanoparticles in the direction of US propagation, where a traveling US wave with a peak pressure on the order of megapascals and frequency in the megahertz range produces a particle translational velocity that is proportional to acoustic intensity and increases with increasing center frequency. Within a vessel with a diameter on the order of hundreds of micrometers or larger, particle velocity on the order of hundreds of micrometers per second is produced and the dominant mechanism for droplet displacement is shown to be bulk fluid streaming. A model for radiation force displacement of particles is developed and demonstrates that effective particle displacement should be feasible in the microvasculature. In a flowing system, acoustic manipulation of targeted droplets increases droplet retention. Additionally, we demonstrate the feasibility of US-enhanced particle internalization and therapeutic delivery.

Ultrasound assessment of angiogenesis in a matrigel model in rats.

Matrigel, a basement membrane extract, has been extensively used in in vivo angiogenesis. Contrast ultrasound imaging (CUI) of implanted Matrigel plugs with (+bFGF) and without basic fibroblast growth factor (-bFGF) was performed 7 and 14 d after implantation, followed by histologic analysis. Statistically significant differences between +bFGF and -bFGF plugs were apparent at d 7 in both plug size and contrast enhancement (both p < 0.05). Histopathology revealed differences in microvessel density (MVD) between +bFGF and -bFGF at d 7 and d 14. A significant correlation between MVD and both power Doppler contrast-enhanced area (r = 0.65, p < 0.05) and fraction of plug enhanced (r = 0.59, p < 0.05) was present. CUI of Matrigel plugs was shown to be a robust method for distinguishing between two different angiogenic states. Ultrasound measurements of blood flow in the plugs correlated with MVD, a histologic technique used to quantify tumor angiogenesis.

The effect of size on the acoustic response of polymer-shelled contrast agents.

In this technical note, we study three polymer-shelled microbubble contrast agents manufactured by POINT Biomedical Corporation that have identical shell composition and mean volumetric diameters of 0.74 microm, 0.91 microm and 1.33 microm. We investigate the effect of agent size on the amplitude, frequency and probability of acoustic echoes received in response to five-cycle, 2.25-MHz pulses of varying pressure. We find that the amplitude and frequency response from the three agents is not significantly different. However, significant differences among the agents do exist in the probability of response to acoustic interrogation: at a pressure of 1.06 MPa, an echo from the 1.33 microm agent is 5 times as likely as an echo from the 0.91 microm agent and 18 times as likely as an echo from the 0.74 microm agent. We hypothesize that there exists an effective pressure-dependent threshold diameter above which single polymer-shelled agents respond to acoustic interrogation.

Contrast-enhanced computed tomography and ultrasound for the evaluation of tumor blood flow.

OBJECTIVE: We evaluated implanted rat mammary adenocarcinoma tumors during a 5-week period using ultrasound, computed tomography (CT), and histology. MATERIALS AND METHODS: Contrast-enhanced ultrasound with a destruction-replenishment imaging scheme was used to derive estimates of blood volume and flow. These ultrasound-derived measures of microvascular physiology were compared with contrast-enhanced CT-derived measures of perfusion and vascular volume made by the Mullani-Gould formula and Patlak analysis, respectively. RESULTS: The tumor cross-sectional area and necrotic core cross-sectional area determined by the 3 methods were correlated (r>0.8, P<0.001, n=15). The spatial integral of perfusion estimated by CT correlated with the spatial integral of flow from ultrasound (P<0.05). The contrast-enhanced tumor area calculated from the ultrasound analysis was highly correlated with the contrast-enhanced area estimated by CT images (r=0.89, P<0.001, n=15). However, the fraction of the tumor area enhanced by the CT contrast agent was significantly larger than either the fraction enhanced by ultrasound contrast agent or than the viable area as estimated from histology slides. CONCLUSION: Destruction-replenishment ultrasound provides valuable information about the spatial distribution of blood flow and vascular volume in tumors and ultrasound analysis compares favorably with a validated contrast-enhanced CT method.

Radiation-force assisted targeting facilitates ultrasonic molecular imaging.

Ultrasonic molecular imaging employs contrast agents, such as microbubbles, nanoparticles, or liposomes, coated with ligands specific for receptors expressed on cells at sites of angiogenesis, inflammation, or thrombus. Concentration of these highly echogenic contrast agents at a target site enhances the ultrasound signal received from that site, promoting ultrasonic detection and analysis of disease states. In this article, we show that acoustic radiation force can be used to displace targeted contrast agents to a vessel wall, greatly increasing the number of agents binding to available surface receptors. We provide a theoretical evaluation of the magnitude of acoustic radiation force and show that it is possible to displace micron-sized agents physiologically relevant distances. Following this, we show in a series of experiments that acoustic radiation force can enhance the binding of targeted agents: The number of biotinylated microbubbles adherent to a synthetic vessel coated with avidin increases as much as 20-fold when acoustic radiation force is applied; the adhesion of contrast agents targeted to alpha(v)beta3 expressed on human umbilical vein endothelial cells increases 27-fold within a mimetic vessel when radiation force is applied; and finally, the image signal-to-noise ratio in a phantom vessel increases up to 25 dB using a combination of radiation force and a targeted contrast agent, over use of a targeted contrast agent alone.

A method for radiation-force localized drug delivery using gas-filled lipospheres.

We have developed a method using ultrasound and acoustically active lipospheres (AALs) that might be used to deliver bioactive substances to the vascular endothelium. The AALs consist of a small gas bubble surrounded by a thick oil shell and enclosed by an outermost lipid layer. The AALs are similar to ultrasound contrast agents: they can be nondestructively deflected using ultrasound radiation force, and fragmented with high-intensity ultrasound pulses. The lipid-oil complex might be used to carry bioactive substances at high concentrations. An optimized sequence of ultrasound pulses can deflect the AALs toward a vessel wall then disrupt them, painting their contents across the vascular endothelium. This paper presents results from a series of in vitro and ex vivo experiments demonstrating localization of a fluorescent model drug. In experiments using a human melanoma cell (A2085) monolayer, a specific radiation force-fragmentation ultrasound pulse sequence increased cell fluorescence more than 10-fold over no ultrasound or fragmentation pulses alone, and by 50% over radiation force pulses alone. We observe that dye transfer is limited to cells that are in the region of ultrasonic focus, indicating that the application of radiation force pulses to bring the delivery vehicle into proximity with the cell is required for successful adhesion of the vehicle fragments to the cell membrane. We also demonstrate dye transfer from flowing AALs, both in a mimetic vessel and in excised rat cecum. We believe that this method could be successfully used for drug delivery in vivo.

Acoustic signatures of submicron contrast agents.

Previous studies have revealed that hard-shelled submicron contrast agents exhibit large relative expansions and strong acoustical echoes that can be observed experimentally, and predicted by theoretical simulations. In this paper, we study harmonic imaging and pulse-pair imaging techniques designed to assist in the differentiation of these contrast agents from tissue. For harmonic imaging, we apply a high-sensitivity, narrowband strategy that differentiates the microbubble from tissue based on the generation of strong harmonic echoes. For pulse-pair imaging, we apply high spatial resolution, wideband strategies using phase inversion, which relies on the frequency differences observed in response to phase-inverted pulses, and signal subtraction, which takes advantage of the amplitude differences in response to identical pulses. The bubble-to-phantom signal amplitude ratio in the absence of motion approaches 20 dB using phase inversion and 30 dB using signal subtraction; both techniques are robust for up to 50 microm of simulated motion. With the experience gained in these studies, we hope to advance the development of multi-pulse or shaped-pulse techniques that are optimized for specific clinical applications.

Contrast-assisted destruction-replenishment ultrasound for the assessment of tumor microvasculature in a rat model.

Angiogenesis, the development of new blood vessels, is necessary for tumor growth. Anti-angiogenic therapies have recently received attention as a possible cancer treatment. The purpose of this study was to monitor the vascularity of induced tumors in rats using contrast-enhanced ultrasound during anti-angiogenic therapy. Six rats with subcutaneously implanted R3230 murine mammary adenocarcinomas were treated with an orally administered anti-angiogenic agent (SU11657) beginning 28 days after tumor implantation (20 mg/kg BW once daily). Three additional tumor-bearing control rats were treated with an equivalent volume of vehicle alone. Sonographic evaluation of tumor blood flow was performed using a modified Siemens Sonoline Elegra equipped with a 5.0 MHz linear transducer prior to drug administration, during the first 51 hours following initial drug administration, and on days 8 and 15 after initiation of therapy. Tumor volumes were estimated at each time point using a prolate ellipsoid method from linear dimensions measured on the B-mode ultrasound image in the three major axes. A destruction-replenishment technique was used for tumor blood flow evaluation using a constant rate infusion of intravenously delivered ultrasound contrast media (Definity). A destructive pulse was fired first, followed by a chain of non-destructive pulses that allowed for visualization of vascular contrast agent replenishment. Parametric maps of the time required for contrast agent replenishment and the time-integrated intensity were generated for both the tumor and kidney. Following ultrasound examination, contrast-enhanced computed tomography of each tumor was performed in the same imaging plane as that used to acquire the ultrasound images. Fifteen days after the start of treatment, tumors were excised, preserved in 10% formalin, and sectioned in a plane approximating the ultrasound and CT imaging planes. Sections were prepared for light microscopy with H & E, CD31 and factor VIII immunostain to evaluate overall morphology and vessel distribution. Ultrasound measurements of tumor volume, the spatial extent of contrast enhancement, and the time required for contrast replenishment within control tumors were significantly different from those of treated tumors. The time-integrated ultrasound contrast enhancement decreases and the time required for replenishment of the contrast agent within the tumor volume increases over the course of anti-angiogenic therapy. Parametric maps of integrated intensity are shown to correlate with the regions of viable tumor demonstrated on H & E and regions of elevated contrast intensity on CT. Contrast-enhanced ultrasound imaging of implanted tumors provides a tool to assess differences in the microcirculation of treated and control tumors in studies of anti-angiogenic agents.