Shukui Zhao

Influence of lipid shell physicochemical properties on ultrasound-induced microbubble destruction

We present the first study of the effects of monolayer shell physicochemical properties on the destruction of lipid-coated microbubbles during insonification with single, one-cycle pulses at 2.25 MHz and low-duty cycles. Shell cohesiveness was changed by varying phospholipid and emulsifier composition, and shell microstructure was controlled by postproduction processing. Individual microbubbles with initial resting diameters between 1 and 10 /spl mu/m were isolated and recorded during pulsing with brightfield and fluorescence video microscopy. Microbubble destruction occurred through two modes: acoustic dissolution at 400 and 600 kPa and fragmentation at 800 kPa peak negative pressure. Lipid composition significantly impacted the acoustic dissolution rate, fragmentation propensity, and mechanism of excess lipid shedding. Less cohesive shells resulted in micron-scale or smaller particles of excess lipid material that shed either spontaneously or on the next pulse. Conversely, more cohesive shells resulted in the buildup of shell-associated lipid strands and globular aggregates of several microns in size; the latter showed a significant increase in total shell surface area and lability. Lipid-coated microbubbles were observed to reach a stable size over many pulses at intermediate acoustic pressures. Observations of shell microstructure between pulses allowed interpretation of the state of the shell during oscillation. We briefly discuss the implications of these results for therapeutic and diagnostic applications involving lipid-coated microbubbles as ultrasound contrast agents and drug/gene delivery vehicles.

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.

Tailoring the Size Distribution of Ultrasound Contrast Agents: Possible Method for Improving Sensitivity in Molecular Imaging

Encapsulated microbubble contrast agents incorporating an adhesion ligand in the microbubble shell are used for molecular imaging with ultrasound. Currently available microbubble agents are produced with techniques that result in a large size variance. Detection of these contrast agents depends on properties related to the microbubble diameter such as resonant frequency, and current ultrasound imaging systems have bandwidth limits that reduce their sensitivity to a polydisperse contrast agent population. For ultrasonic molecular imaging, in which only a limited number of targeted contrast agents may be retained at the site of pathology, it is important to optimize the sensitivity of the imaging system to the entire population of contrast agent. This article presents contrast agents with a narrow size distribution that are targeted for molecular imaging applications. The production of a functionalized, lipid-encapsulated, microbubble contrast agent with a monodisperse population is demonstrated, and we evaluate parameters that influence the size distribution and demonstrate initial acoustic testing.

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.

Asymmetric oscillation of adherent targeted ultrasound contrast agents

With a lipid shell containing biotin, micron-sized bubbles bound to avidin on a porous and flexible cellulose boundary were insonified by ultrasound. The oscillation of these targeted microbubbles was observed by high-speed photography and compared to the oscillation of free-floating microbubbles. Adherent microbubbles were observed to oscillate asymmetrically in the plane normal to the boundary, and nearly symmetrically in the plane parallel to the boundary, with a significantly smaller maximum expansion in each dimension for bound than free bubbles. With sufficient transmitted pressure, a jet was produced traveling toward the boundary.

Acoustic response from adherent targeted contrast agents.

In ultrasonic molecular imaging, encapsulated micron-sized gas bubbles are tethered to a blood vessel wall by targeting ligands. A challenging problem is to detect the echoes from adherent microbubbles and distinguish them from echoes from nonadherent agents and tissue. Echoes from adherent contrast agents are observed to include a high amplitude at the fundamental frequency, and significantly different spectral shape compared with free agents (p <0.0003). Mechanisms for the observed acoustical difference and potential techniques to utilize these differences for molecular imaging are proposed.

Modeling of the acoustic response from contrast agent microbubbles near a rigid wall

In ultrasonic targeted imaging, specially designed encapsulated microbubbles are used, which are capable of selectively adhering to the target site in the body. A challenging problem is to distinguish the echoes from such adherent agents from echoes produced by freely circulating agents. In the present paper, an equation of radial oscillation for an encapsulated bubble near a plane rigid wall is derived. The equation is then used to simulate the echo from a layer of contrast agents localized on a wall. The echo spectrum of adherent microbubbles is compared to that of free, randomly distributed microbubbles inside a vessel, in order to examine differences between the acoustic responses of free and adherent agents. It is shown that the fundamental spectral component of adherent bubbles is perceptibly stronger than that of free bubbles. This increase is accounted for by a more coherent summation of echoes from adherent agents and the acoustic interaction between the agents and the wall. For cases tested, the increase of the fundamental component caused by the above two effects is on the order of 8-9 dB. Bubble aggregates, which are observed experimentally to form near a wall due to secondary Bjerknes forces, increase the intensity of the fundamental component only if they are formed by bubbles whose radii are well below the resonant radius. If the formation of aggregates contributes to the growth of the fundamental component, the increase can exceed 17 dB. Statistical analysis for the comparison between adhering and free bubbles, performed over random space bubble distributions, gives p-values much smaller than 0.05.

Observation of contrast agent response to chirp insonation with a simultaneous optical-acoustical system

Rayleigh-Plesset analysis, ultra-high speed photography, and single bubble acoustical recordings previously were applied independently to characterize the radial oscillation and resulting echoes from a microbubble in response to an ultrasonic pulse. In addition, high-speed photography has shown that microbubbles are destroyed over a single pulse or pulse train by diffusion and fragmentation. In order to develop a single model to characterize microbubble echoes based on oscillatory and destructive characteristics, an optical-acoustical system was developed to simultaneously record the optical image and backscattered echo from each microbubble. Combined observation provides the opportunity to compare predictions for oscillation and echoes with experimental results and identify discrepancies due to diffusion or fragmentation. Optimization of agents and insonating pulse parameters may be facilitated with this system. The mean correlation of the predicted and experimental radius-time curves and echoes exceeds 0.7 for the parameters studied here. An important application of this new system is to record and analyze microbubble response to a long pulse in which diffusion is shown to occur over the pulse duration. The microbubble response to an increasing or decreasing chirp is evaluated using this new tool. For chirp insonation beginning with the lower center frequency, low-frequency modulation of the oscillation envelope was obvious. However, low-frequency modulation was not observed in the radial oscillation produced by decreasing chirp insonation. Comparison of the echoes from similar sized microbubbles following increasing and decreasing chirp insonation demonstrated that the echoes were not time-reversed replicas. Using a transmission pressure of 620 kPa, the -6 dB echo length was 0.9 and 1.1 /spl mu/s for increasing and decreasing chirp insonation, respectively (P = 0.02). The mean power in the low-frequency portion of the echoes was 8 (mV)/sup 2/ and 13 (mV)/sup 2/ for increasing and decreasing chirp insonation, respectively (P = 0.01).

Physico-chemical properties of the microbubble lipid shell [ultrasound contrast agents]

Targeted microbubbles stabilized by a lipid monolayer are currently being developed in conjunction with ultrasound for diagnostic and therapeutic applications. Targeted microbubbles utilize specific receptor-ligand interactions to adhere to the site of interest - a process which can be enhanced by using ultrasonic radiation force. The monolayer shell is composed of a main phospholipid component and an emulsifier comprising a poly(ethylene glycol) (PEG) headgroup. The targeting ligand is tethered to a lipid in the monolayer via a PEG spacer. Using epi-fluorescence microscopic techniques, we show here for the first time that the targeting ligand is not uniformly distributed over the microbubble surface, but instead clusters into the expanded phase interdomain region. We also demonstrate for the first time the relationships between composition, microstructure and lipid shedding properties during ultrasonic insonation of the microbubble. These results have implications for the rational design of lipid-coated microbubbles as targeted ultrasound contrast agents.

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.