Research

Typical lipid-based microbubbles contain two species: a main phospholipid component (e.g., long chain phosphatidylcholine) and an emulsifying agent (e.g., PEG-lipid). Our recent fluorescence microscopy and FTIR results have indicated that these components phase separate within the monolayer shell into phospholipid-rich domains surrounded by a PEG-lipid enriched matrix. A third phase, the collapsed state, also has been observed. Thus, the shell is not homogeneous as previously asserted. However, the complexity of the shell should lend better functionality through rational design (e.g., ligand clusters and compartmentalization). Furthermore, amphiphilic drugs or pro-drugs can be directly incorporated into the shell and targeted to specific sites in the vasculature, such as tumor angiogenesis, for efficient release using ultrasound. We are currently working on several strategies to incorporate amphiphilic drugs into the lipid shell and deliver them to tumor cells both in vitro and in vivo.

Primary and secondary radiation forces result from pressure gradients in the incident and scattered ultrasonic fields. These forces and their dependence on experimental parameters are described, and the theory for primary radiation force is extended to consider a pulsed traveling wave. Both primary and secondary radiation forces are shown to have a significant effect on the flow of microbubbles through a small vessel during insonation. The primary radiation force produces displacement of microspheres across a 100 micron vessel radius for a small transmitted acoustic pressure. The displacement produced by primary radiation force is shown to display the expected linear dependence on the pulse repetition frequency and a nonlinear dependence on transmitted pressure. The secondary radiation force produces a reversible attraction and aggregation of microspheres with a significant attraction over a distance of approximately 100 microns. The magnitude of the secondary radiation force is proportional to the inverse of the squared separation distance, and thus two aggregates accelerate as they approach one another. We show that this force is sufficient to produce aggregates that remain intact for a physiologically appropriate shear rate. Brief interruption of acoustic transmission allows an immediate disruption of the aggregate.

Diagram of experimental system. (click to enlarge)

Displacement of a stream of bubbles due to radiation force, as a function of the acoustic pressure, for a transmitted center frequency 4 MHz, PRF 1250 Hz, peak velocity 7 mm/s. (a) Acoustic pressure 133 kPa. (b) Increase in acoustic pressure to 168 kPa. (c) Increase in acoustic pressure to 211 kPa. (click to enlarge)

Ultrasound at frequencies greater than 20 MHz allows us to explore blood flow in peripheral tissues such as the eye and skin with spatial resolution currently unattainable with clinical ultrasound systems and with much greater penetration depth than optical methods. We have developed the first high frequency ultrasound system capable of mapping blood flow in the microcirculation in real-time with spatial resolution down to 40 m and with sub-mm/sec velocity detection. The lack of arrays operating at frequencies above 20 MHz necessitates mechanical scanning of a fixed-focus transducer for image formation. The conventional method involves acquiring groups of echoes at independent lines-of-sight (LOS), which requires that the transducer be stopped at each LOS. Scanning with this method is prohibitively slow, with frame acquisition times on the order of 1 minute. We proposed a significantly faster method (Kruse et al., 1998), which takes advantage of the difference in the spatial variations of echoes from tissue and blood as compared to the finite size of the resolution cell. In this approach, which we call swept-scanning, echoes are acquired from very closely spaced LOS at the pulse-repetition-frequency (PRF), which results in frame acquisition times on the order of 1 second. Using this technique, we have primarily studied blood flow in the eye with application to the treatment of glaucoma. We are currently studying the effects of various glaucoma drug treatment protocols on anterior segment blood flow with collaborators Jack Coleman, MD and Ron Silverman, PhD (Weill Medical College of Cornell University, Dept. of Ophthalmology, New York, NY).

38 MHz color flow images from the rabbit eye (adapted from Silverman, et al., 1998). Image (a) shows a 3D reconstruction of the major arterial circle (MAC) of the iris, which is approximately 200 m in diameter. (click to enlarge)

Image (b) shows M-mode images from a single line-of-sight directed through the MAC. From left to right, an image of RF echo data, a color flow image recorded before applying topical atropine, and after applying atropine. Time points of peak systole are denoted by arrows. There is a clear increase in blood flow after the application of atropine. This preliminary study demonstrates our system's sensitivity to changes in blood flow caused by a vasoactive drug. (click to enlarge)

The most significant problem in high-resolution color flow mapping is the differentiation of echoes from low velocity blood flow and tissue echoes with similar velocity magnitudes. Our signal processing methods take advantage of the limited depth-of-focus of high frequency ultrasound transducers over which tissue motion is typically constant. Using this characteristic, we developed an eigendecomposition-based adaptive clutter filter for rejection of time-varying tissue echoes (Kruse and Ferrara, 2002). Additionally, we introduced a method for estimating the velocity of the tissue motion component in order to correct post-clutter filter velocity estimates.

25-MHz color flow images of the human finger produced using an eigendecomposition-based clutter filter and tissue velocity correction (adapted from Kruse and Ferrara, 2002). The leftmost images show a 1.2 mm finger vein without tissue motion (a) and with motion (b). The rightmost images show small vessels down to 100 m in the nail bed of the index finger without tissue motion (c) and with motion (d). Each image is 4 mm laterally and 3 mm in depth. The full-scale velocity range is ±15.4 mm/s. Note the absence of color flash or bands of constant velocity over depth. These images show that vivo blood flow velocities below 1 mm/sec in vessels down to 100 microns can be reliably detected despite maximum tissue velocities of 2 mm/sec and accelerations of 8 cm/sec2. (click to enlarge)

We are exploring new techniques for assessing microvascular blood flow and perfusion using ultrasound contrast agents, which are encapsulated microbubbles. When acoustically driven, microbubbles exhibit strong scattering relative to blood, non-linear oscillations, destruction, and interactions between neighboring bubbles. By taking advantage of these characteristics, together with increased resolution offered by our high frequency system, we hope to make sensitive high-resolution maps of blood perfusion in vivo. Currently, we are involved in a collaborative research project with Dr. Michele Lim (Dept. of Ophthalmology, UC Davis, Sacramento, CA) to look at blood flow in the ciliary processes of the eye as a function of laser cytophotocoagulation treatment, which is used to treat advanced cases of glaucoma.

A movie clip of 25 MHz power Doppler images acquired sequentially from the anterior segment of a rabbit eye, showing an intravenous bolus injection of ultrasound contrast agent wash in and washout of the iris and ciliary processes. The image size is 4 mm laterally by 3 mm in depth and displays 40 dB of dynamic range. The movie is played back at approximately 38x the actual frame rate of 0.4 fps. The large bright vessel near the middle is the major arterial circle of the iris. Note that the bottom edges of the ciliary processes are clearly enhanced by the contrast agent. (click to enlarge)

Targeted Deposition of a Model Drug Using Ultrasound Radiation Force and Molecular Interactions

A drug delivery vehicle which targets vasculature using both ultrasound radiation force and specific molecular interactions has been developed. Ultrasound radiation force results from the nonlinear oscillation of small gas bubbles in an ultrasonic field, and can deflect a particle over distances on the order of millimeters with clinically-relevant parameters. The vehicles consist of a 1-micron lipid-shelled bubble coated with targeted nanoparticles that can carry a drug. In our model system, neutravidin-coated beads bind biotinylated microbubbles non-uniformly, associating with microbubble lipid domains. At a fluid shear stress of 3 dyn/cm2, ultrasound radiation force (1.3 second push pulse at 3MHz and 150 kPa peak negative pressure (PNP) and a 5 cycle break pulse at 1.5 MHz and 1.1 MPa PNP) directed these vehicles to the vessel surface and allowed the local deposition of 40 nm beads on the tube wall. Accumulation of beads on biotinylated tubes was molecularly-specific and dependent upon vehicle concentration and length of insonation. Using a concentration of vehicles commensurate with standard contrast agent treatments (106/ml), we observed that bound beads were rarely detected in the absence of ultrasound. There was a 3-fold increase in bead adhesion between 2 and 8 minutes of insonation, while blocking neutravidin reduced bead adhesion 7-fold. Additionally, portions of the microbubble lipid shell remained attached to the adherent beads. This method of delivery enables the targeted deposition of a particle and may be easily altered for use in many diverse diseases.

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.

Schematic of an acoustically active liposphere depicting the location of the oil layer in which therapeutics can be dissolved, the lipid shell and the gas interior (thickness of lipid/oil layer not to scale with diameter of AAL.). The mean outer diameter of the AALs used in our study is 1.6µm. The thickness of the oil and lipid layer was previously measured by May et al. [6], using scanning electron microscopy, and was found to be between 500 nm and 1000 nm, depending on the diameter of the entire agent. (click to enlarge)

Sample optical microscopy images in excised rat cecum demonstrating adherent drug-vehicle complex to a capillary wall after multiple pulse sequences (top). A transillumination image acquired after insonation of the vessel showing that the vessel is clear of intact vehicles (bottom). The corresponding epifluorescence image in which the fluorescent model drug has adhered selectively to the far endothelial wall. (click to enlarge)

D. N. Stephens, K. K. Shung, J. Cannata, J. Z. Zhao, R. Chia, H. Nguyen, K. Thomenius, A. Dentinger, D. G. Wildes, X. Chen, M. O'Donnell, R. I. Lowe, J. Pemberton, G. H. Burch, D. J. Sahn, “Clinical Application and Technical Challenges for Intracardiac Ultrasound Imaging,” in Proc. IEEE Ultrason. Symp., 2004, pp. 772 - 777. (invited talk in Montreal, August 26, 2004)

K. Thomenius, A. Dentinger, K. K. Shung, R. Chia, D. N. Stephens, M. O'Donnell, R. I. Lowe, C. H. Davies, G. H. Burch, D. J. Sahn, "A new high frequency intracardiac imaging technology with improved resolution and steerability facilitates motion mapping and EP electrodes for rhythm analysis," presented at the American Heart Association Annual Scientific Sessions, Nov. 7 – 10, 2004, New Orleans, La.

D. N. Stephens, K. Thomenius, K. Kirk Shung, R. Chia, A. Dentinger, X. Chen, M. O'Donnell, J. Pemberton, C. H. Davies, D. J. Sahn, “The Effects of VOO Ventricular Pacing on Ventricular Function Using a New Intracardiac Imaging System Developed to Guide Multi-Site Pacing," presented at the American College of Cardiology Annual Scientific Sessions, March 6-9, 2005, Orlando, Fl.

A. Dentinger, K. Thomenius,K. K. Shung, J. Cannata, R. Chia, D. N. Stephens, X. Chen, M. O'Donnell, J. Pemberton, C. H. Davies, G. Burch, S. Balaji, D. J. Sahn, “A New Intracardiac Ultrasound Imaging System With High Resolution, High Frame Rate Motion Mapping and EP Recording Capability," presented at the American College of Cardiology Annual Scientific Sessions, March 6-9, 2005, Orlando, Fl.

D. N. Stephens, K. K. Shung, J. Cannata, J. Z. Zhao, R. Chia, H. Nguyen, K. Thomenius, A. Dentinger, D. G. Wildes, X. Chen, M. O'Donnell, R. I. Lowe, J. Pemberton, G. H. Burch, D. J. Sahn, “Clinical Application and Technical Challenges for Intracardiac Ultrasound Imaging,” an invited talk presented at the American Institute of Ultrasound in Medicine (AIUM), Annual Convention and 50th Anniversary, Orlando, Florida, June 22, 2005.

Xiaokui Li, James Pemberton, Kai Thomenius, Aaron Dentinger, Robert I. Lowe, Kirk Shung, Raymond Chia, Douglas N. Stephens,Matthew O'Donnell, Seshadri Balaji, David J. Sahn, “Intracardiac Imaging Technology with Improved Resolution and Steering Ability Facilitates Motion Mapping and Integrated Electrophysiology Electrodes for Rhythm Analysis and Treatment,” in review by the Journal of the American Society of Echocardiography.

The goal of our project is to develop a technique to locally increase the permeability of the vasculature in order to facilitate the delivery of a therapeutic agent to a target site. In our studies, we illustrate a technique to increase the permeability of the vessel wall in the capillary bed using low frequency ultrasound in conjunction with a contrast agent, using a model that allows optical observation.

Although the exact mechanism has yet to be isolated, the introduction of ultrasound contrast agent can lower the threshold for acoustic intensity over which an increase in vessel permeability is observed.

Before ultrasound application. (click to enlarge)

After ultrasound application. (click to enlarge)

Ultrasound contrast agents enhance echoes from the microvasculature and enable the visualization of flow in smaller vessels. Here, we optically and acoustically investigate microbubble oscillation and echoes following insonation with a 10 MHz center frequency pulse. A high-speed camera system with a temporal resolution of 10 ns, which provides 2-D frame images and streak images, is used in optical experiments. Two confocally-aligned transducers, transmitting at 10 MHz and receiving at 5 MHz (T10R5), are utilized in acoustical experiments in order to detect subharmonic components. Results of a numerical evaluation of the modified Rayleigh-Plesset equation are used to predict the dynamics of a microbubble, and compared to results of in-vitro experiments. From the optical observations of a single microbubble, nonlinear oscillation, destruction and radiation force are observed. The maximum bubble expansion, resulting from insonation with a 20-cycle 10-MHz linear chirp with a peak negative pressure of 3.5 MPa, has been evaluated. For an initial diameter ranging from 1.5 to 5 µm, a maximum diameter less than 8 µm is produced during insonation. Optical and acoustical experiments provide insight into the mechanisms of destruction, including fragmentation and active diffusion. High frequency pulse transmission may provide the opportunity to detect contrast echoes resulting from a single pulse, may be robust in the presence of tissue motion, and may provide the opportunity to incorporate high frequency ultrasound into destruction-replenishment techniques.

(a) plot of driving pulse received by the hydrophone filtered by a bandpass filter from 1 to 15 MHz. The y-axis is pressure in MPa corrected for the frequency response of the hydrophone. (b) time-frequency power spectrum of the filtered driving pulse received by the hydrophone showing 30 dB of dynamic range. (c) spectrum of the signal in (a). (click to enlarge)

Optical evidence of fragmentation of a lipid-shelled microbubble with an initial diameter of 3.6 µm, insonified by a 10 MHz chirp).(a) 2-D image of the microbubble before insonation; (b) streak image of same microbubble under insonation, with overlay of hydrophone recording of transmitted pulse; (c) image of fragmentation after insonation. The scale bar is 5 µm. (click to enlarge)

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 αvβ3 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.

Illustration of the effect of radiation force on targeted imaging with ultrasound. (A) Without radiation force, the majority of the contrast agents fail to contact the target site, and therefore do not bind. (B) Radiation force pushes flowing targeted contrast agents into contact with cells along a vessel wall, where they bind to target receptors. (click to enlarge)

Fluorescence photomicrographs showing a portion of a microvessel flow phantom. Each image is approximately 100 x 60 μm. (A) Fluorescent RGD-targeted microbubbles adherent to αvβ3-expressing HUVEC after 10 radiation force pulses over 100 sec. (B) Minimal adhesion of fluorescent RDG-targeted microbubbles to HUVEC is seen without application of radiation force over the same period. (click to enlarge)

Spectra from echoes scattered from a microvessel in response to imaging pulses of (A) 440 kPa and (B) 690 kPa. Both plots show spectra from water only (dotted line), from freely flowing bubbles (thin solid line), after rinsing with water after applying radiation force pulses (thick solid line), and after rinsing with water with no radiation force application (dashed line). The narrowband spectrum of adherent bubbles after application of radiation force pulses can be observed in both pressures. (click to enlarge)