Susannah H. Bloch

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.

Optical observation of lipid- and polymer-shelled ultrasound microbubble contrast agents

High-speed optical experiments demonstrate that the behavior of a polymer-shelled microbubble contrast agent in response to an acoustic pulse is qualitatively and quantitatively different from that of a lipid-shelled agent. The lipid-shelled agent expands in response to a two-cycle pulse, and at pressures approaching 1 MPa, both the shell and its contents fragment. The polymer-shelled agent remains largely intact at pressures up to 1.5 MPa and exhibits a different destruction mechanism: the polymer shell does not oscillate significantly in response to ultrasound; instead, a gas bubble is extruded and ejected through a shell defect while the shell appears to remain largely intact.

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.

Targeted imaging using ultrasound contrast agents

The applications of ultrasound contrast agents have recently expanded from blood pool enhancement to include passive targeting of physiological systems (in particular, the lymphatic and reticuloendothelial systems) and molecular imaging of factors expressed in angiogenesis, atherosclerosis, and inflammation. This article summarizes the progress made in targeted imaging using ultrasound with an emphasis on the opportunities this research provides for both clinical and research applications. We begin with a summary of current ultrasound contrast technology and then review the latest research in the use of targeted ultrasound contrast agents.

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.

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.

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 (r2 > 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 (r2 = 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.

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 tip to 50 /spl mu/m 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.

Increasing binding efficiency of ultrasound targeted agents with radiation force

We demonstrate both theoretically and experimentally that ultrasound radiation force can significantly increase the binding efficiency of targeted contrast agents without increasing non-specific adhesion of agents to the target surface. The radial oscillation of a microbubble was determined using a previously developed model, and then displacement and translational velocity were predicted by solving the trajectory equation of the microbubble. Theoretical evaluation showed that a microbubble can be easily displaced across a vessel by radiation force. Experiments with an avidin-coated tube and biotin-targeted microbubbles clearly demonstrated the effect of radiation force in increasing the efficiency of specific binding. Under control conditions, only sporadic binding to the vessel wall was observed. With radiation force, targeted agents adhered to the vessel wall at 20 times the rate of control experiments. An experiment with microbubbles targeted to /spl alpha//sub v//spl beta//sub 3/ expressing cells showed similar results.

Sonothrombolysis with phospholipid-coated perfluoropropane microbubbles

Compared to other modalities that might be employed for molecular imaging, ultrasound has some unique features. It is portable; highly adapted to the surgical environment; can be used for cavitation, heating, and tissue ablation; and is exquisitely sensitive to microbubbles. Using microbubbles as a contrast agent, ultrasound imaging can detect a single microbubble. Ultrasound in concert with bubbles can be used in NanoInvasive™ therapy and drug delivery. An example of NanoInvasive therapy is sonothrombolysis with microbubbles, or SonoLysis™.