Charles F. Caskey

Ultrasound contrast microbubbles in imaging and therapy: physical principles and engineering

Microbubble contrast agents and the associated imaging systems have developed over the past 25 years, originating with manually-agitated fluids introduced for intra-coronary injection. Over this period, stabilizing shells and low diffusivity gas materials have been incorporated in microbubbles, extending stability in vitro and in vivo. Simultaneously, the interaction of these small gas bubbles with ultrasonic waves has been extensively studied, resulting in models for oscillation and increasingly sophisticated imaging strategies. Early studies recognized that echoes from microbubbles contained frequencies that are multiples of the microbubble resonance frequency. Although individual microbubble contrast agents cannot be resolved-given that their diameter is on the order of microns-nonlinear echoes from these agents are used to map regions of perfused tissue and to estimate the local microvascular flow rate. Such strategies overcome a fundamental limitation of previous ultrasound blood flow strategies; the previous Doppler-based strategies are insensitive to capillary flow. Further, the insonation of resonant bubbles results in interesting physical phenomena that have been widely studied for use in drug and gene delivery. Ultrasound pressure can enhance gas diffusion, rapidly fragment the agent into a set of smaller bubbles or displace the microbubble to a blood vessel wall. Insonation of a microbubble can also produce liquid jets and local shear stress that alter biological membranes and facilitate transport. In this review, we focus on the physical aspects of these agents, exploring microbubble imaging modes, models for microbubble oscillation and the interaction of the microbubble with the endothelium.

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.

Direct observations of ultrasound microbubble contrast agent interaction with the microvessel wall

Many thousands of contrast ultrasound studies have been conducted in clinics around the world. In addition, the microbubbles employed in these examinations are being widely investigated to deliver drugs and genes. Here, for the first time, the oscillation of these microbubbles in small vessels is directly observed and shown to be substantially different than that predicted by previous models and imaged within large fluid volumes. Using pulsed ultrasound with a center frequency of 1 MHz and peak rarefactional pressure of 0.8 or 2.0 MPa, microbubble expansion was significantly reduced when microbubbles were constrained within small vessels in the rat cecum (p<0.05). A model for microbubble oscillation within compliant vessels is presented that accurately predicts oscillation and vessel displacement within small vessels. As a result of the decreased oscillation in small vessels, a large resting microbubble diameter resulting from agent fusion or a high mechanical index was required to bring the agent shell into contact with the endothelium. Also, contact with the endothelium was observed during asymmetrical collapse, not during expansion. These results will be used to improve the design of drug delivery techniques using microbubbles.

Imaging of angiogenesis using Cadence contrast pulse sequencing and targeted contrast agents.

OBJECTIVES Low-power multipulse contrast ultrasound imaging provides a promising tool to quantify angiogenesis noninvasively when used with contrast agents targeted to vascular markers expressed by the angiogenic endothelium. Targeted ultrasound contrast agents, with a diameter on the order of micrometers, cannot extravasate and therefore are targeted solely to receptors expressed by the vascular endothelium. The aim of this study was to evaluate the potential of a low-power multipulse imaging sequence, Cadence(TM) contrast pulse sequencing (CPS), combined with targeted contrast agents to quantify angiogenesis. MATERIAL AND METHODS Targeted microbubbles were prepared by conjugating echistatin via biotin-avidin linkage to the surface of a phospholipid microbubble shell. The density of echistatin present on the shell was confirmed with flow-cytometry and quantified by total fluorescence. The binding of targeted microbubbles was evaluated in vitro by quantifying the adherence of targeted microbubbles to rat aortic endothelial cells, compared with control (nontargeted) microbubbles. The circulation time and adherence of targeted microbubbles was evaluated in vivo in a Matrigel model in rats and compared with control microbubbles using CPS in addition to a destructive ultrasound pulse. RESULTS Using only the low-power CPS pulse, the echo intensity produced in the neovasculature of the Matrigel pellet was significantly greater with targeted microbubbles than with the control contrast agent (p < 0.001). Combining CPS with the destructive pulse, the processed image was significantly different in intensity (p < 0.001) and spatial extent between targeted and control agents (p < 0.001). When the morphology of the histological sample and ultrasound image correlated, the microvessel density count and the percentage of the circular area enhanced by ultrasound were correlated (p < 0.05). CONCLUSION Low-power multipulse imaging in combination with targeted echistatin-bearing microbubbles facilitated a noninvasive, quantitative evaluation of early angiogenesis during real-time imaging. The addition of high-intensity destructive pulses facilitated estimation of the spatial extent of angiogenesis.

Microbubble oscillation in tubes with diameters of 12, 25, and 195 microns

Ultrasound contrast agents are often used to measure flow rate in the microvasculature; however, the oscillation of these agents in capillary-sized tubes has not been directly observed. Here, oscillations of microbubbles are examined in microvessel phantoms with diameters similar to those of capillaries. High-speed camera images demonstrate the effects of ultrasonic pressure and tube diameter and length on microbubble expansion and fragmentation occurrence. Microbubble displacement due to radiation force is also demonstrated in a phantom microvessel.

Microbubble tunneling in gel phantoms

Insonified microbubbles were observed in vessels within a gel with a Youngs modulus similar to that of tissue, demonstrating shape instabilities, liquid jets, and the formation of small tunnels. In this study, tunnel formulation occurred in the direction of the propagating ultrasound wave, where radiation pressure directed the contact of the bubble and gel, facilitating the activity of the liquid jets. Combinations of ultrasonic parameters and microbubble concentrations that are relevant for diagnostic imaging and drug delivery and that lead to tunnel formation were applied and the resulting tunnel formation was quantified.

Noninvasive thermometry assisted by a dual-function ultrasound transducer for mild hyperthermia

Mild hyperthermia is increasingly important for the activation of temperature-sensitive drug delivery vehicles. Noninvasive ultrasound thermometry based on a 2-D speckle tracking algorithm was examined in this study. Here, a commercial ultrasound scanner, a customized co-linear array transducer, and a controlling PC system were used to generate mild hyperthermia. Because the co-linear array transducer is capable of both therapy and imaging at widely separated frequencies, RF image frames were acquired during therapeutic insonation and then exported for off-line analysis. For in vivo studies in a mouse model, before temperature estimation, motion correction was applied between a reference RF frame and subsequent RF frames. Both in vitro and in vivo experiments were examined; in the in vitro and in vivo studies, the average temperature error had a standard deviation of 0.7°C and 0.8°C, respectively. The application of motion correction improved the accuracy of temperature estimation, where the error range was -1.9 to 4.5°C without correction compared with -1.1 to 1.0°C following correction. This study demonstrates the feasibility of combining therapy and monitoring using a commercial system. In the future, real-time temperature estimation will be incorporated into this system.

Multimodal imaging enables early detection and characterization of changes in tumor permeability of brain metastases

Our goal was to develop strategies to quantify the accumulation of model therapeutics in small brain metastases using multimodal imaging, in order to enhance the potential for successful treatment. Human melanoma cells were injected into the left cardiac ventricle of immunodeficient mice. Bioluminescent, MR and PET imaging were applied to evaluate the limits of detection and potential for contrast agent extravasation in small brain metastases. A pharmacokinetic model was applied to estimate vascular permeability. Bioluminescent imaging after injecting d-luciferin (molecular weight (MW) 320 D) suggested that tumor cell extravasation had already occurred at week 1, which was confirmed by histology. 7T T1w MRI at week 4 was able to detect non-leaky 100 μm sized lesions and leaky tumors with diameters down to 200 μm after contrast injection at week 5. PET imaging showed that (18)F-FLT (MW 244 Da) accumulated in the brain at week 4. Gadolinium-based MRI tracers (MW 559 Da and 2.066 kDa) extravasated after 5 weeks (tumor diameter 600 μm), and the lower MW agent cleared more rapidly from the tumor (mean apparent permeabilities 2.27 × 10(-5)cm/s versus 1.12 × 10(-5)cm/s). PET imaging further demonstrated tumor permeability to (64)Cu-BSA (MW 65.55 kDa) at week 6 (tumor diameter 700 μm). In conclusion, high field T1w MRI without contrast may improve the detection limit of small brain metastases, allowing for earlier diagnosis of patients, although the smallest lesions detected with T1w MRI were permeable only to d-luciferin and the amphipathic small molecule (18)F-FLT. Different-sized MR and PET contrast agents demonstrated the gradual increase in leakiness of the blood tumor barrier during metastatic progression, which could guide clinicians in choosing tailored treatment strategies.

Leveraging the power of ultrasound for therapeutic design and optimization

Contrast agent-enhanced ultrasound can facilitate personalized therapeutic strategies by providing the technology to measure local blood flow rate, to selectively image receptors on the vascular endothelium, and to enhance localized drug delivery. Ultrasound contrast agents are micron-diameter encapsulated bubbles that circulate within the vascular compartment and can be selectively imaged with ultrasound. Microbubble transport-based estimates of local blood flow can quantify changes resulting from anti-angiogenic therapies and facilitate differentiation of angiogenic mechanisms. Microbubbles that are conjugated with targeting ligands attach to endothelial surface receptors that are upregulated in disease, providing high signal-to-noise ratio images of pathological vasculature. In addition to imaging applications, microbubbles can be used to enhance localized gene and drug delivery, either by changing membrane and vascular permeability or by carrying and locally releasing cargo. Our goal in this review is to provide an overview of the use of contrast-enhanced ultrasound methodologies in the design and evaluation of therapeutic strategies with emphases on quantitative blood flow mapping, molecular imaging, and enhanced drug delivery.

Magnetic resonance thermometry at 7T for real-time monitoring and correction of ultrasound induced mild hyperthermia.

While Magnetic Resonance Thermometry (MRT) has been extensively utilized for non-invasive temperature measurement, there is limited data on the use of high field (≥7T) scanners for this purpose. MR-guided Focused Ultrasound (MRgFUS) is a promising non-invasive method for localized hyperthermia and drug delivery. MRT based on the temperature sensitivity of the proton resonance frequency (PRF) has been implemented in both a tissue phantom and in vivo in a mouse Met-1 tumor model, using partial parallel imaging (PPI) to speed acquisition. An MRgFUS system capable of delivering a controlled 3D acoustic dose during real time MRT with proportional, integral, and derivative (PID) feedback control was developed and validated. Real-time MRT was validated in a tofu phantom with fluoroptic temperature measurements, and acoustic heating simulations were in good agreement with MR temperature maps. In an in vivo Met-1 mouse tumor, the real-time PID feedback control is capable of maintaining the desired temperature with high accuracy. We found that real time MR control of hyperthermia is feasible at high field, and k-space based PPI techniques may be implemented for increasing temporal resolution while maintaining temperature accuracy on the order of 1°C.