Alexander L. Klibanov

Optical and acoustical observations of the effects of ultrasound on contrast agents

Optimal use of encapsulated microbubbles for ultrasound contrast agents and drug delivery requires an understanding of the complex set of phenomena that affect the contrast agent echo and persistence. With the use of a video microscopy system coupled to either an ultrasound flow phantom or a chamber for insonifying stationary bubbles, we show that ultrasound has significant effects on encapsulated microbubbles. In vitro studies show that a train of ultrasound pulses can alter the structure of an albumin-shelled bubble, initiate various mechanisms of bubble destruction or produce aggregation that changes the echo spectrum. In this analysis, changes observed optically are compared with those observed acoustically for both albumin and lipid-shelled agents. We show that, when insonified with a narrowband pulse at an acoustic pressure of several hundred kPa, a phospholipid-shelled bubble can undergo net radius fluctuations of at least 15%; and an albumin-shelled bubble initially demonstrates constrained expansion and contraction. If the albumin shell contains air, the shell may not initially experience surface tension; therefore, the echo changes more significantly with repeated pulsing. A set of observations of contrast agent destruction is presented, which includes the slow diffusion of gas through the shell and formation of a shell defect followed by rapid diffusion of gas into the surrounding liquid. These observations demonstrate that the low-solubility gas used in these agents can persist for several hundred milliseconds in solution. With the transmission of a high-pulse repetition rate and a low pressure, the echoes from, contrast agents can be affected by secondary radiation force. Secondary radiation force is an attractive force for these experimental conditions, creating aggregates with distinct echo characteristics and extended persistence. The scattered echo from an aggregate is several times stronger and more narrowband than echoes from individual bubbles.

Acoustic radiation force in vivo: a mechanism to assist targeting of microbubbles

The goal of targeted imaging is to produce an enhanced view of physiological processes or pathological tissue components. Contrast agents may improve the specificity of imaging modalities through selective targeting, and this may be particularly significant when using ultrasound (US) to image inflammatory processes or thrombi. One means of selective targeting involves the attachment of contrast agents to the desired site with the use of a specific binding mechanism. Because molecular binding mechanisms are effective over distances on the order of nanometers, targeting effectiveness would be greatly increased if the agent is initially concentrated in a particular region, and if the velocity of the agent is decreased as it passes the potential binding site. Ultrasonic transmission produces a primary radiation force that can manipulate microbubbles with each acoustic pulse. Observations demonstrate that primary radiation force can displace US contrast agents from the center of the streamline to the wall of a 200-microm cellulose vessel in vitro. Here, the effects of radiation force on contrast agents in vivo are presented for the first time. Experimental results demonstrate that radiation force can displace a contrast agent to the wall of a 50-microm blood vessel in the mouse cremaster muscle, can significantly reduce the velocity of flowing contrast agents, and can produce a reversible aggregation. Acoustic radiation force presents a means to localize and concentrate contrast agents near a vessel wall, which may assist the delivery of targeted agents.

Targeted delivery of gas-filled microspheres, contrast agents for ultrasound imaging

Gas-filled microbubbles, with the size of several micrometres, are strong scatterers of ultrasound waves used in diagnostic imaging. Application of these microbubbles as ultrasound contrast materials is discussed, in view of the design of materials capable of selectively targeting the diseased tissues/organs. Methods of preparation, mechanisms of action, biodistribution and stability in vitro and in vivo are reviewed. Targeted microbubbles with various ligands (antibodies, peptides) attached to the shells have been prepared and tested in vitro and in vivo. Examples of specific application in diagnostic imaging and possible therapeutic use are discussed.

Microbubble persistence in the microcirculation during ischemia/reperfusion and inflammation is caused by integrin- and complement-mediated adherence to activated leukocytes

BACKGROUND Albumin microbubbles that are used for contrast echocardiography persist within the myocardial microcirculation after ischemia/reperfusion (I-R). The mechanism responsible for this phenomenon is unknown. METHODS AND RESULTS Intravital microscopy of the microcirculation of exteriorized cremaster muscle was performed in 12 wild-type mice during intravenous injections of fluorescein-labeled microbubbles composed of albumin, anionic lipids, or cationic lipids. Injections were performed at baseline and after 30 to 90 minutes of I-R in 8 mice and 2 hours after intrascrotal tumor necrosis factor-alpha (TNF-alpha) in 4 mice. Microbubble adherence at baseline was uncommon (<2/50 high-power fields). After I-R, adherence increased (P<0.05) to 9+/-5 and 5+/-4 per 50 high-power fields for albumin and anionic lipid microbubbles, respectively, due to their attachment to leukocytes adherent to the venular endothelium. TNF-alpha produced even greater microbubble binding, regardless of the microbubble shell composition. The degree of microbubble attachment correlated (r=0.84 to 0.91) with the number of adhered leukocytes. Flow cytometry revealed that microbubbles preferentially attached to activated leukocytes. Albumin microbubble attachment was inhibited by blocking the leukocyte beta(2)-integrin Mac-1, whereas lipid microbubble binding was inhibited when incubations were performed in complement-depleted or heat-inactivated serum rather than control serum. CONCLUSIONS Microvascular attachment of albumin and lipid microbubbles in the setting of I-R and TNF-alpha-induced inflammation is due to their beta(2)-integrin- and complement-mediated binding to activated leukocytes adherent to the venular wall. Thus, microbubble persistence on contrast ultrasonography may be useful for the detection and monitoring of leukocyte adhesion in inflammatory diseases.

Optical observation of contrast agent destruction

Fragmentation of an ultrasound contrast agent on the time scale of microseconds provides opportunities for the advancement of microvascular detection, blood flow velocity estimation, and targeted drug delivery. Images captured by high-speed imaging systems show destruction of a microbubble during compression. Peak wall velocity of −700 m/s and peak acceleration of 1.2×1012 m/s2 is observed for insonation with a peak pressure of −1.1 MPa and a center frequency of 2.4 MHz. Theoretical calculations of wall velocity and acceleration using a modified Rayleigh–Plesset model predict a peak negative wall velocity of −680 m/s and peak acceleration of 2×1012 m/s2.

Targeting and ultrasound imaging of microbubble-based contrast agents.

Preparation and characterization of targeted microbubbles (ultrasound contrast agents) is described. Specific ligands were attached to the microbubble shell, and ligand-coated microbubbles were selectively attached to various targets, using either an avidin-biotin model system or an antigen-antibody system for targeting to live activated endothelial cells. Firm attachment of microbubbles to the target was achieved. Forces necessary to detach microbubbles from the target were estimated to exceed dozens of pN. Microbubbles were bound to the target even in the rapidly moving stream of the aqueous medium. Down to 20 ng of the ultrasound contrast material on the target surface could be detected by the ultrasound imaging with a commercial medical imaging system. At high bubble density on the target surface, strong ultrasound image attenuation was observed.

Direct video-microscopic observation of the dynamic effects of medical ultrasound on ultrasound contrast microspheres.

RATIONALE AND OBJECTIVES Ultrasound can cause destruction of microbubble contrast agents used to enhance medical ultrasound imaging. This study sought to characterize the dynamics of this interaction by direct visual observation of microbubbles during insonification in vitro by a medical ultrasound imaging system. METHODS Video microscopy was used to observe air-filled sonicated albumin microspheres adsorbed to a solid support during insonation. RESULTS Deflation was not observed at lowest transmit power settings. At higher intensities, gas left the microparticle gradually, apparently dissolving into the surrounding medium. Deflation was slower for higher microsphere surface densities. Intermittent ultrasound imaging (0.5 Hz refresh rate) caused slower deflation than continuous imaging (33 Hz). CONCLUSIONS Higher concentrations of microbubbles, lower ultrasound transmit power settings, and intermittent imaging each can reduce the rate of destruction of microspheres resulting from medical ultrasound insonation.

Destruction of contrast agent microbubbles in the ultrasound field: the fate of the microbubble shell and the importance of the bubble gas content.

Disappearance of ultrasound (US) contrast agents (gasfilled microbubbles) after contrast agent administration in the bloodstream occurs by way of several mechanisms. First, microbubbles are filtered and captured by various organs, including uptake by Kupffer cells in the liver (1,2). Second, gas diffuses from the microbubble core and dissolves in the surrounding medium, leaving the nonechogenic shell behind (3). The third process is the most rapid and important for the practice of diagnostic imaging by US. It is the destruction of microbubbles by the acoustic field of the US medical imaging system. It has been shown that the rate of microbubble destruction in the ultrasound field depends on the ultrasound frequency and pressure (4–7). During imaging, the microbubble contrast agent can be destroyed by a single pulse of the ultrasound, if the ultrasound mechanical index (MI) value is high enough (7,8). This phenomenon has been applied successfully for the imaging of tissue perfusion and perfusion defects (8,9). Thus, the mechanisms of microbubble contrast agent disappearance in the ultrasound field are important and need to be investigated in detail. We have previously reported a microbubble destruction/microscopy study performed with an air-filled microbubble agent, sonicated human serum albumin (Albunex; Mallinckrodt, St Louis, Mo) (10). More recent contrast agents filled with insoluble fluorinated gases, such as Optison (Mallinckrodt), demonstrate extended in vivo circulation and stability (11). It is of interest to evaluate the stability of these microbubbles in the field of the US medical imager. The nature of the gas and the gas exchange may play an important role in the bubble destruction. Here, we describe video microscopic studies performed in vitro for immobilized microbubbles during their insonification. Fluorescence microscopy was used to evaluate the behavior of microbubble shells during the destruction of microbubbles.

Microbubble-endothelial cell interactions as a basis for assessing endothelial function.

Clinical signs and symptoms of coronary artery disease are predated in decades by endothelial dysfunction, an aberration in the vascular lining that permits the development and propagation of atherosclerotic lesions and vasomotor dysfunction in the arterial circulation. These ultimately lead to acute and chronic coronary ischemic syndromes. Other pathophysiologic scenarios encountered in clinical cardiology practice, such as cardiac transplant rejection and the period following coronary angioplasty or cardiac surgery, also are associated with endothelial dysfunction. Endothelial dysfunction parallels coronary risk factors and is potentially reversible, rendering early identification of the phenomenon a clinically important endpoint. Current methods for detecting endothelial dysfunction are limited, however. Myocardial contrast echocardiography using microbubbles targeted to bind to cell surface markers uniquely expressed by dysfunctional endothelial cells may offer an approach to the noninvasive detection of endothelial disease using clinical ultrasound imaging techniques. This article will discuss the concept of targeted ultrasound imaging and present preliminary studies in this area as applied to endothelial assessment. Potential applications to other disease states, both diagnostic and therapeutic, also are discussed.