Optimizing Acoustic Activation of Phase Change Contrast Agents With the Activation Pressure Matching Method: A Review

Sub-micron phase-change contrast agents consist of a liquid perfluo ocarbon core that can be vaporized by ultrasound (acoustic droplet vaporization) to generate contrast with excellent spatial and temporal control. When these agents, commonly referred to as nanodroplets, are formulated with cores of low boiling-point perfluo ocarbons such as decaflu orobutane and octafluo opropane, they can be activated with low-mechanical index imaging pulses for diagnostic applications. Since the utilization of minimum mechanical index is often desirable to avoid unnecessary biological effects, enabling consistent activation of these agents in an acoustic fiel is a challenge because the energy that must be delivered to achieve the vaporization threshold increases with depth due to attenuation. A novel vaporization approach called Activation Pressure Matching has been developed to deliver the same pressure throughout a fiel of view in order to produce uniform nanodroplet vaporization and to limit the amount of energy that is delivered. In this manuscript, we discuss the application of this method with a Versasonics V1 Research Ultrasound System to modulate the output pressure from an ATL L11–5 transducer. Vaporization-pulse spacing optimization can be used in addition to matching the activation pressure through depth, and we demonstrate the feasibility of this approach both in vivo and in vitro. The use of optimized vaporization parameters increases the amount of time a single bolus of nanodroplets can generate useful contrast and provides consistent image enhancement in vivo. Therefore, APM is a useful technique for those wishing to maximize the efficac of phase change contrast agent while minimizing delivered acoustic energy

Adaptive windowing in contrast-enhanced intravascular ultrasound imaging

Intravascular ultrasound (IVUS) is one of the most commonly-used interventional imaging techniques and has seen recent innovations which attempt to characterize the risk posed by atherosclerotic plaques. One such development is the use of microbubble contrast agents to image vasa vasorum, fine vessels which supply oxygen and nutrients to the walls of coronary arteries and typically have diameters less than 200 µm. The degree of vasa vasorum neovascularization within plaques is positively correlated with plaque vulnerability. Having recently presented a prototype dual-frequency transducer for contrast agent-specific intravascular imaging, here we describe signal processing approaches based on minimum variance (MV) beamforming and the phase coherence factor (PCF) for improving the spatial resolution and contrast-to-tissue ratio (CTR) in IVUS imaging. These approaches are examined through simulations, phantom studies, ex vivo studies in porcine arteries, and in vivo studies in chicken embryos. In phantom studies, PCF processing improved CTR by a mean of 4.2 dB, while combined MV and PCF processing improved spatial resolution by 41.7%. Improvements of 2.2 dB in CTR and 37.2% in resolution were observed in vivo. Applying these processing strategies can enhance image quality in conventional B-mode IVUS or in contrast-enhanced IVUS, where signal-to-noise ratio is relatively low and resolution is at a premium

Enhancing Nanoparticle Accumulation and Retention in Desmoplastic Tumors via Vascular Disruption for Internal Radiation Therapy

Aggressive, desmoplastic tumors are notoriously difficult to treat because of their extensive stroma, high interstitial pressure, and resistant tumor microenvironment. We have developed a combination therapy that can significantly slow the growth of large, stroma-rich tumors by causing massive apoptosis in the tumor center while simultaneously increasing nanoparticle uptake through a treatment-induced increase in the accumulation and retention of nanoparticles in the tumor. The vascular disrupting agent Combretastatin A-4 Phosphate (CA4P) is able to increase the accumulation of radiation-containing nanoparticles for internal radiation therapy, and the retention of these delivered radioisotopes is maintained over several days. We use ultrasound to measure the effect of CA4P in live tumor-bearing mice, and we encapsulate the radio-theranostic isotope 177Lutetium as a therapeutic agent as well as a means to measure nanoparticle accumulation and retention in the tumor. This combination therapy induces prolonged apoptosis in the tumor, decreasing both the fibroblast and total cell density and allowing further tumor growth inhibition using a cisplatin-containing nanoparticle

On the Relationship Between Microbubble Fragmentation, Deflation and Broadband Superharmonic Signal Production

Acoustic angiography imaging of microbubble contrast agents utilizes the superharmonic energy produced from excited microbubbles, and enables high-contrast, high-resolution imaging. However, the exact mechanism by which broadband harmonic energy is produced is not fully understood. In order to elucidate the role of microbubble shell fragmentation in superharmonic signal production, simultaneous optical and acoustic measurements were performed on individual microbubbles at transmit frequencies from 1.75 to 3.75 MHz and pressures near the shell fragmentation threshold for microbubbles of varying diameter. High-amplitude, broadband superharmonic signals were produced with shell fragmentation, while weaker signals (approximately 25% of peak amplitude) were observed in the presence of shrinking bubbles. Furthermore, when imaging populations of stationary microbubbles with a dual-frequency ultrasound imaging system, a sharper decline in image intensity with respect to frame number was observed for 1 μm bubbles than for 4 μm bubbles. Finally, in a study of two rodents, increasing frame rate from 4 to 7 Hz resulted in a decrease in mean steady-state image intensity of 27% at 1000 kPa and 29% at 1300 kPa. While the existence of superharmonic signals when bubbles shrink has the potential to prolong the imaging efficacy of microbubbles, parameters such as frame rate and peak pressure must be balanced with expected re-perfusion rate in order to maintain adequate contrast during in vivo imaging

3-D Ultrasound Localization Microscopy for Identifying Microvascular Morphology Features of Tumor Angiogenesis at a Resolution Beyond the Diffraction Limit of Conventional Ultrasound

Angiogenesis has been known as a hallmark of solid tumor cancers for decades, yet ultrasound has been limited in its ability to detect the microvascular changes associated with malignancy. Here, we demonstrate the potential of 'ultrasound localization microscopy' applied volumetrically in combination with quantitative analysis of microvascular morphology, as an approach to overcome this limitation. This pilot study demonstrates our ability to image complex microvascular patterns associated with tumor angiogenesis in-vivo at a resolution of tens of microns - substantially better than the diffraction limit of traditional clinical ultrasound, yet using an 8 MHz clinical ultrasound probe. Furthermore, it is observed that data from healthy and tumor-bearing tissue exhibit significant differences in microvascular pattern and density. Results suggests that with continued development of these novel technologies, ultrasound has the potential to detect biomarkers of cancer based on the microvascular 'fingerprint' of malignant angiogenesis rather than through imaging of blood flow dynamics or the tumor mass itself

Molecular Acoustic Angiography: A New Technique for High-resolution Superharmonic Ultrasound Molecular Imaging

Ultrasound molecular imaging utilizes targeted microbubbles to bind to vascular targets such as integrins, selectins, and other extracellular binding domains. After binding, these microbubbles are typically imaged using low pressures and multi-pulse imaging sequences. In this article, we present an alternative approach for molecular imaging using ultrasound which relies on superharmonic signals produced by microbubble contrast agents. Bound bubbles were insonified near resonance using a low frequency (4 MHz) and superharmonic echoes were received at high frequencies (25–30 MHz). While this approach was observed to produce declining image intensity during repeated imaging in both in vitro and in vivo experiments due to bubble destruction, the feasibility of superharmonic molecular imaging was demonstrated for transmit pressures which are sufficiently high to induce shell disruption in bound microbubbles. This approach was validated using microbubbles targeted to the αvβ3 integrin in a rat fibrosarcoma model (n=5), and combined with superharmonic images of free microbubbles to produce high contrast, high resolution 3D volumes of both microvascular anatomy and molecular targeting. Image intensity over repeated scans and the effect of microbubble diameter were also assessed in vivo, indicating that larger microbubbles yield increased persistence in image intensity. Using ultrasound-based acoustic angiography images rather than conventional B-mode ultrasound to provide the underlying anatomical information facilitates anatomical localization of molecular markers. Quantitative analysis of relationships between microvasculature and targeting information indicated that most targeting occurred within 50 µm of a resolvable vessel (>100 µm diameter). The combined information provided by these scans may present new opportunities for analyzing relationships between microvascular anatomy and vascular targets, subject only to limitations of the current mechanically-scanned system and microbubble persistence to repeated imaging at moderate mechanical indices

Ex Vivo Porcine Arterial and Chorioallantoic Membrane Acoustic Angiography Using Dual-Frequency Intravascular Ultrasound Probes

The presence of blood vessels within a developing atherosclerotic plaque has been shown to be correlated to increased plaque vulnerability and ensuing cardiac events, however, detecting coronary intraplaque neovascularizations poses a significant challenge in the clinic. In this paper, we demonstrate in vivo a new intravascular ultrasound imaging method using a dual-frequency transducer to visualize contrast flow in microvessels with high specificity. This method uses a specialized transducer capable of exciting contrast agents at a low frequency (5.5 MHz) while detecting their nonlinear superhamonics at a much higher frequency (37 MHz). In vitro evaluation of the approach was performed in a microvascular phantom to produce 3D renderings of simulated vessel patterns and to determine image quality metrics as a function of depth. Furthermore, the ability of the system to detect microvessels is demonstrated both ex vivo using porcine arteries and in vivo using the chorioallantoic membrane of a developing chicken embryo with optical confirmation. Dual-frequency contrast specific imaging was able to resolve vessels of a similar size to those found in vulnerable atherosclerotic plaques at clinically relevant depths. The results of this study adds growing support for further evaluation and translation of contrast specific imaging in intravascular ultrasound for the detection of vulnerable plaques in atherosclerosis

Intravascular forward-looking ultrasound transducers for microbubble-mediated sonothrombolysis

Effective removal or dissolution of large blood clots remains a challenge in clinical treatment of acute thrombo-occlusive diseases. Here we report the development of an intravascular microbubble-mediated sonothrombolysis device for improving thrombolytic rate and thus minimizing the required dose of thrombolytic drugs. We hypothesize that a sub-megahertz, forward-looking ultrasound transducer with an integrated microbubble injection tube is more advantageous for efficient thrombolysis by enhancing cavitation-induced microstreaming than the conventional high-frequency, side-looking, catheter-mounted transducers. We developed custom miniaturized transducers and demonstrated that these transducers are able to generate sufficient pressure to induce cavitation of lipid-shelled microbubble contrast agents. Our technology demonstrates a thrombolysis rate of 0.7 ± 0.15 percent mass loss/min in vitro without any use of thrombolytic drugs

Microfluidic Generation of Acoustically Active Nanodroplets

A microfluidic approach for the generation of perfluorocarbon nanodroplets as the primary emulsion with diameters as small as 300-400 nm is described. The system uses a pressure-controlled delivery of all reagents and increased viscosity in the continuous phase to drive the device into an advanced tip-streaming regime, which results in generation of droplets in the sub-micrometer range. Such nanodroplets may be appropriate for emerging biomedical applications

Precision Manufacture of Phase-Change Perfluorocarbon Droplets Using Microfluidics

Liquid perfluorocarbon droplets have been of interest in the medical acoustics community for use as acoustically activated particles for tissue occlusion, imaging, and therapeutics. To date, methods to produce liquid perfluorocarbon droplets typically result in a polydisperse size distribution. Since the threshold of acoustic activation is a function of diameter, there would be benefit from a monodisperse population to preserve uniformity in acoustic activation parameters. Through the use of a microfluidic device with flow focusing technology, the production of droplets of perfluoropentane with a uniform size distribution is demonstrated. Stability studies indicate that these droplets are stable in storage for at least two weeks. Acoustic studies illustrate the thresholds of vaporization as a function of droplet diameter, and a logarithmic relationship is observed between acoustic pressure and vaporization threshold within the size ranges studied. Droplets of uniform size have very little variability in acoustic vaporization threshold. Results indicate that microfluidic technology can enable greater manufacturing control of phase change perfluorocarbons for acoustic droplet vaporization applications