Ultrasound-induced cavitation and passive acoustic mapping: SonoTran platform performance and short-term safety in a large-animal model.

Ultrasound-induced cavitation is currently under investigation for several potential applications in cancer treatment. Among these, the use of low-intensity ultrasound, coupled with the systemic administration of various cavitation nuclei, has been found to enhance the delivery of co-administered therapeutics into solid tumors. Effective pharmacological treatment of solid tumors is often hampered, among various factors, by the limited diffusion of drugs from the bloodstream into the neoplastic mass and through it, and SonoTran holds the potential to tackle this clinical limitation by increasing the amount of drug and its distribution within the ultrasound-targeted tumor tissue. Here we use a clinically ready system (SonoTran Platform) composed of a dedicated ultrasound device (SonoTran System) capable of instigating, detecting and displaying cavitation events in real time by passive acoustic mapping and associated cavitation nuclei (SonoTran Particles), to instigate cavitation in target tissues and illustrate its performance and safety in a large-animal model. This study found that cavitation can be safely triggered and mapped at different tissue depths and in different organs. No adverse effects were associated with infusion of SonoTran Particles, and ultrasound-induced cavitation caused no tissue damage in clinically targetable organs (e.g., liver) for up to 1 h. These data provide evidence of cavitation initiation and monitoring performance of the SonoTran System and the safety of controlled cavitation in a large-animal model using a clinic-ready platform technology.

Improved therapeutic antibody delivery to xenograft tumors using cavitation nucleated by gas-entrapping nanoparticles.

Aims: Testing ultrasound-mediated cavitation for enhanced delivery of the therapeutic antibody cetuximab to tumors in a mouse model. Methods: Tumors with strong EGF receptor expression were grown bilaterally. Cetuximab was coadministered intravenously with cavitation nuclei, consisting of either the ultrasound contrast agent Sonovue or gas-stabilizing nanoscale SonoTran Particles. One of the two tumors was exposed to focused ultrasound. Passive acoustic mapping localized and monitored cavitation activity. Both tumors were then excised and cetuximab concentration was quantified. Results: Cavitation increased tumoral cetuximab concentration. When nucleated by Sonovue, a 2.1-fold increase (95% CI 1.3- to 3.4-fold) was measured, whereas SonoTran Particles gave a 3.6-fold increase (95% CI 2.3- to 5.8-fold). Conclusions: Ultrasound-mediated cavitation, especially when nucleated by nanoscale gas-entrapping particles, can noninvasively increase site-specific delivery of therapeutic antibodies to solid tumors.

Image quality evaluation of ultrasound imaging systems: advanced B-modes.

The Quality assurance of ultrasound clinical imaging systems is essential for maintaining their performance to the highest level and for complying with the requirements by various regulatory and accrediting agencies. Although there is no standardization yet, most of the quality assessment procedures available in literature are proposed for B-mode and Doppler imaging. However, ultrasound imaging systems offer a variety of advanced imaging modes, besides B-mode and Doppler, which are primarily aimed at improving image quality. This study presents computer-based methods for evaluating image quality for the advanced imaging modes of ultrasound imaging systems: harmonic imaging, spatial compounding imaging, adaptive speckle reduction, and tissue aberration correction. The functions and parameters proposed for evaluating image quality are: grayscale mapping function, image contrast, contrast-to-noise ratio (CNR), and high-contrast spatial resolution. We present our computer-based methods for evaluating image quality of these modes with a number of probe and scanner combinations, which were employed to image targets in ultrasound phantoms. The functions and parameters here proposed in image quality performance evaluation are: grayscale mapping function, image contrast, CNR, and high-contrast spatial resolution. We show that these quantities could be useful in developing standardized methods for evaluating the advanced ultrasound imaging modes, especially when the advanced mode resulted in subtle visual differences.

Simultaneous Passive Acoustic Mapping and Magnetic Resonance Thermometry for Monitoring of Cavitation-Enhanced Tumor Ablation in Rabbits Using Focused Ultrasound and Phase-Shift Nanoemulsions.

Thermal ablation of solid tumors via focused ultrasound (FUS) is a non-invasive image-guided alternative to conventional surgical resection. However, the usefulness of the technique is limited in vascularized organs because of convection of heat, resulting in long sonication times and unpredictable thermal lesion formation. Acoustic cavitation has been found to enhance heating but requires use of exogenous nuclei and sufficient acoustic monitoring. In this study, we employed phase-shift nanoemulsions (PSNEs) to promote cavitation and incorporated passive acoustic mapping (PAM) alongside conventional magnetic resonance imaging (MRI) thermometry within the bore of a clinical MRI scanner. Simultaneous PAM and MRI thermometry were performed in an in vivo rabbit tumor model, with and without PSNE to promote cavitation. Vaporization and cavitation of the nanoemulsion could be detected using PAM, which led to accelerated heating, monitored with MRI thermometry. The maximum heating assessed from MRI was well correlated with the integrated acoustic emissions, illustrating cavitation-enhanced heating. Examination of tissue revealed thermal lesions that were larger in the presence of PSNE, in agreement with the thermometry data. Using fixed exposure conditions over 94 sonications in multiple animals revealed an increase in the mean amplitude of acoustic emissions and resulting temperature rise, but with significant variability between sonications, further illustrating the need for real-time monitoring. The results indicate the utility of combined PAM and MRI for monitoring of tumor ablation and provide further evidence for the ability of PSNEs to promote cavitation-enhanced lesioning.

Scalp sensor for simultaneous acoustic emission detection and electroencephalography during transcranial ultrasound.

Focused ultrasound is now capable of noninvasively penetrating the intact human skull and delivering energy to specific areas of the brain with millimeter accuracy. The ultrasound energy is supplied in high-intensities to create brain lesions or at low-intensities to produce reversible physiological interventions. Conducting acoustic emission detection (AED) and electroencephalography (EEG) during transcranial focused ultrasound may lead to several new brain treatment and research applications. This study investigates the feasibility of using a novel scalp senor for acquiring concurrent AED and EEG during clinical transcranial ultrasound. A piezoelectric disk is embedded in a plastic cup EEG electrode to form the sensor. The sensor is coupled to the head via an adhesive/conductive gel-dot. Components of the sensor prototype are tested for AED and EEG signal quality in a bench top investigation with a functional ex vivo skull phantom.

A dual-mode hemispherical sparse array for 3D passive acoustic mapping and skull localization within a clinical MRI guided focused ultrasound device.

Previous work has demonstrated that passive acoustic imaging may be used alongside MRI for monitoring of focused ultrasound therapy. However, past implementations have generally made use of either linear arrays originally designed for diagnostic imaging or custom narrowband arrays specific to in-house therapeutic transducer designs, neither of which is fully compatible with clinical MR-guided focused ultrasound (MRgFUS) devices. Here we have designed an array which is suitable for use within an FDA-approved MR-guided transcranial focused ultrasound device, within the bore of a 3 Tesla clinical MRI scanner. The array is constructed from 5 × 0.4 mm piezoceramic disc elements arranged in pseudorandom fashion on a low-profile laser-cut acrylic frame designed to fit between the therapeutic elements of a 230 kHz InSightec ExAblate 4000 transducer. By exploiting thickness and radial resonance modes of the piezo discs the array is capable of both B-mode imaging at 5 MHz for skull localization, as well as passive reception at the second harmonic of the therapy array for detection of cavitation and 3D passive acoustic imaging. In active mode, the array was able to perform B-mode imaging of a human skull, showing the outer skull surface with good qualitative agreement with MR imaging. Extension to 3D showed the array was able to locate the skull within ±2 mm/2° of reference points derived from MRI, which could potentially allow registration of a patient to the therapy system without the expense of real-time MRI. In passive mode, the array was able to resolve a point source in 3D within a ±10 mm region about each axis from the focus, detect cavitation (SNR ~ 12 dB) at burst lengths from 10 cycles to continuous wave, and produce 3D acoustic maps in a flow phantom. Finally, the array was used to detect and map cavitation associated with microbubble activity in the brain in nonhuman primates.

A versatile method for the preparation of particle-loaded microbubbles for multimodality imaging and targeted drug delivery.

Microbubbles are currently in clinical use as ultrasound contrast agents and under active investigation as mediators of ultrasound therapy. To improve the theranostic potential of microbubbles, nanoparticles can be attached to the bubble shell for imaging, targeting and/or enhancement of acoustic response. Existing methods for fabricating particle-loaded bubbles, however, require the use of polymers, oil layers or chemical reactions for particle incorporation; embed/attach the particles that can reduce echogenicity; impair biocompatibility; and/or involve multiple processing steps. Here, we describe a simple method to embed nanoparticles in a phospholipid-coated microbubble formulation that overcomes these limitations. Magnetic nanoparticles are used to demonstrate the method with a range of different microbubble formulations. The size distribution and yield of microbubbles are shown to be unaffected by the addition of the particles. We further show that the microbubbles can be retained against flow using a permanent magnet, can be visualised by both ultrasound and magnetic resonance imaging (MRI) and can be used to transfect SH-SY5Y cells with fluorescent small interfering RNA under the application of a magnetic field and ultrasound field.

Combined passive acoustic mapping and magnetic resonance thermometry for monitoring phase-shift nanoemulsion enhanced focused ultrasound therapy.

Focused ultrasound (FUS) has the potential to enable precise, image-guided noninvasive surgery for the treatment of cancer in which tumors are identified and destroyed in a single integrated procedure. However, success of the method in highly vascular organs has been limited due to heat losses to perfusion, requiring development of techniques to locally enhance energy absorption and heating. In addition, FUS procedures are conventionally monitored using MRI, which provides excellent anatomical images and can map temperature, but is not capable of capturing the full gamut of available data such as the acoustic emissions generated during this inherently acoustically-driven procedure. Here, we employed phase-shift nanoemulsions (PSNE) embedded in tissue phantoms to promote cavitation and hence temperature rise induced by FUS. In addition, we incorporated passive acoustic mapping (PAM) alongside simultaneous MR thermometry in order to visualize both acoustic emissions and temperature rise, within the bore of a full scale clinical MRI scanner. Focal cavitation of PSNE could be resolved using PAM and resulted in accelerated heating and increased the maximum elevated temperature measured via MR thermometry compared to experiments without nanoemulsions. Over time, the simultaneously acquired acoustic and temperature maps show translation of the focus of activity towards the FUS transducer, and the magnitude of the increase in cavitation and focal shift both increased with nanoemulsion concentration. PAM results were well correlated with MRI thermometry and demonstrated greater sensitivity, with the ability to detect cavitation before enhanced heating was observed. The results suggest that PSNE could be beneficial for enhancement of thermal focused ultrasound therapies and that PAM could be a critical tool for monitoring this process.

Ultrasound-Enhanced siRNA Delivery Using Magnetic Nanoparticle-Loaded Chitosan-Deoxycholic Acid Nanodroplets.

Small interfering RNA (siRNA) has significant therapeutic potential but its clinical translation has been severely inhibited by a lack of effective delivery strategies. Previous work has demonstrated that perfluorocarbon nanodroplets loaded with magnetic nanoparticles can facilitate the intracellular delivery of a conventional chemotherapeutic drug. The aim of this study is to determine whether a similar agent can provide a means of delivering siRNA, enabling efficient transfection without degradation of the molecule. Chitosan-deoxycholic acid nanoparticles containing perfluoropentane and iron oxide (d 0 = 7.5 ± 0.35 nm) with a mean hydrodynamic diameter of 257.6 ± 10.9 nm are produced. siRNA (AllStars Hs cell death siRNA) is electrostatically bound to the particle surface and delivery to lung cancer cells and breast cancer cells is investigated with and without ultrasound exposure (500 kHz, 1 MPa peak-to-peak focal pressure, 40 cycles per burst, 1 kHz pulse repetition frequency, 10 s duration). The results show that siRNA functionality is not impaired by the treatment protocol and that the nanodroplets are able to successfully promote siRNA uptake, leading to significant apoptosis (52.4%) 72 h after ultrasound treatment.

Passive Acoustic Mapping with the Angular Spectrum Method.

In the present proof of principle study, we evaluated the homogenous angular spectrum method for passive acoustic mapping (AS-PAM) of microbubble oscillations using simulated and experimental data. In the simulated data we assessed the ability of AS-PAM to form 3D maps of a single and multiple point sources. Then, in the two dimensional limit, we compared the 2D maps from AS-PAM with alternative frequency and time domain passive acoustic mapping (FD- and TD-PAM) approaches. Finally, we assessed the ability of AS-PAM to visualize microbubble activity in vivo with data obtained during 8 different experiments of FUS-induced blood-brain barrier disruption in 3 nonhuman primates, using a clinical MR-guided FUS system. Our in silico results demonstrate AS-PAM can be used to perform 3D passive acoustic mapping. 2D AS-PAM as compared to FD- PAM and TD-PAM is 10 and 200 times faster respectively and has similar sensitivity, resolution, and localization accuracy, even when the noise was 10-fold higher than the signal. In-vivo, the AS-PAM reconstructions of emissions at frequency bands pertinent to the different types of microbubble oscillations were also found to be more sensitive than TD-PAM. AS-PAM of harmonic-only components predicted safe blood-brain barrier disruption, whereas AS-PAM of broadband emissions correctly identified MR-evident tissue damage. The disparity (3.2 mm) in the location of the cavitation activity between the three methods was within their resolution limits. These data clearly demonstrate that AS-PAM is a sensitive and fast approach for PAM, thus providing a clinically relevant method to guide therapeutic ultrasound procedures.

Breast Ultrasound Technology and Performance Evaluation of Ultrasound Equipment: B-Mode.

Ultrasound (US) has become increasingly important in imaging and image-guided interventional procedures. In order to ensure that the imaging equipment performs to the highest level achievable and thus provides reliable clinical results, a number of quality control (QC) methods have been developed. Such QC is increasingly required by accrediting agencies and professional organizations; however, these requirements typically do not include detailed procedures for how the tests should be performed. In this paper, a detailed overview of QC methods for general and breast US imaging using computer-based objective methods is described. The application of QC is then discussed within the context of a common clinical application (US-guided needle biopsy) as well as for research applications, where QC may not be mandated, and thus is rarely discussed. The implementation of these methods will help in finding early stage equipment faults and in optimizing image quality, which could lead to better detection and classification of suspicious findings in clinical applications, as well as improving the robustness of research studies.

Enhancement and Passive Acoustic Mapping of Cavitation from Fluorescently Tagged Magnetic Resonance-Visible Magnetic Microbubbles In Vivo.

Previous work has indicated the potential of magnetically functionalized microbubbles to localize and enhance cavitation activity under focused ultrasound exposure in vitro. The aim of this study was to investigate magnetic targeting of microbubbles for promotion of cavitation in vivo. Fluorescently labelled magnetic microbubbles were administered intravenously in a murine xenograft model. Cavitation was induced using a 0.5-MHz focused ultrasound transducer at peak negative focal pressures of 1.2-2.0 MPa and monitored in real-time using B-mode imaging and passive acoustic mapping. Magnetic targeting was found to increase the amplitude of the cavitation signal by approximately 50% compared with untargeted bubbles. Post-exposure magnetic resonance imaging indicated deposition of magnetic nanoparticles in tumours. Magnetic targeting was similarly associated with increased fluorescence intensity in the tumours after the experiments. These results suggest that magnetic targeting could potentially be used to improve delivery of cavitation-mediated therapy and that passive acoustic mapping could be used for real-time monitoring of this process.

Polymeric Cups for Cavitation-mediated Delivery of Oncolytic Vaccinia Virus.

Oncolytic viruses (OV) could become the most powerful and selective cancer therapies. However, the limited transport of OV into and throughout tumors following intravenous injection means their clinical administration is often restricted to direct intratumoral dosing. Application of physical stimuli, such as focused ultrasound, offers a means of achieving enhanced mass transport. In particular, shockwaves and microstreaming resulting from the instigation of an ultrasound-induced event known as inertial cavitation can propel OV hundreds of microns. We have recently developed a polymeric cup formulation which, when delivered intravenously, provides the nuclei for instigation of sustained inertial cavitation events within tumors. Here we report that exposure of tumors to focused ultrasound after intravenous coinjection of cups and oncolytic vaccinia virus , leads to substantial and significant increases in activity. When cavitation was instigated within SKOV-3 or HepG2 xenografts, reporter gene expression from vaccinia virus was enhanced 1,000-fold (P < 0.0001) or 10,000-fold (P < 0.001), respectively. Similar increases in the number of vaccinia virus genomes recovered from tumors were also observed. In survival studies, the application of cup mediated cavitation to a vaccinia virus expressing a prodrug converting enzyme provided significant (P < 0.05) retardation of tumor growth. This technology could improve the clinical utility of all biological therapeutics including OV.

Nanoparticle-Loaded Protein-Polymer Nanodroplets for Improved Stability and Conversion Efficiency in Ultrasound Imaging and Drug Delivery.

A new formulation of volatile nanodroplets stabilized by a protein and polymer coating and loaded with magnetic nanoparticles is developed. The droplets show enhanced stability and phase conversion efficiency upon ultrasound exposure compared with existing formulations. Magnetic targeting, encapsulation, and release of an anticancer drug are demonstrated in vitro with a 40% improvement in cytotoxicity compared with free drug.

Biologically and Acoustically Compatible Chamber for Studying Ultrasound-Mediated Delivery of Therapeutic Compounds.

Ultrasound (US), in combination with microbubbles, has been found to be a potential alternative to viral therapies for transfecting biological cells. The translation of this technique to the clinical environment, however, requires robust and systematic optimization of the acoustic parameters needed to achieve a desired therapeutic effect. Currently, a variety of different devices have been developed to transfect cells in vitro, resulting in a lack of standardized experimental conditions and difficulty in comparing results from different laboratories. To overcome this limitation, we propose an easy-to-fabricate and cost-effective device for application in US-mediated delivery of therapeutic compounds. It comprises a commercially available cell culture dish coupled with a silicon-based "lid" developed in-house that enables the device to be immersed in a water bath for US exposure. Described here are the design of the device, characterization of the sound field and fluid dynamics inside the chamber and an example protocol for a therapeutic delivery experiment.

Passive acoustic mapping of magnetic microbubbles for cavitation enhancement and localization.

Magnetic targeting of microbubbles functionalized with superparamagnetic nanoparticles has been demonstrated previously for diagnostic (B-mode) ultrasound imaging and shown to enhance gene delivery in vitro and in vivo. In the present work, passive acoustic mapping (PAM) was used to investigate the potential of magnetic microbubbles for localizing and enhancing cavitation activity under focused ultrasound. Suspensions of magnetic microbubbles consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), air and 10 nm diameter iron oxide nanoparticles were injected into a tissue mimicking phantom at different flow velocities (from 0 to 50 mm s(-1)) with or without an applied magnetic field. Microbubbles were excited using a 500 kHz single element focused transducer at peak negative focal pressures of 0.1-1.0 MPa, while a 64 channel imaging array passively recorded their acoustic emissions. Magnetic localization of microbubble-induced cavitation activity was successfully achieved and could be resolved using PAM as a shift in the spatial distribution and increases in the intensity and sustainability of cavitation activity under the influence of a magnetic field. Under flow conditions at shear rates of up to 100 s(-1) targeting efficacy was maintained. Application of a magnetic field was shown to consistently increase the energy of cavitation emissions by a factor of 2-5 times over the duration of exposures compared to the case without targeting, which was approximately equivalent to doubling the injected microbubble dose. These results suggest that magnetic targeting could be used to localize and increase the concentration of microbubbles and hence cavitation activity for a given systemic dose of microbubbles or ultrasound intensity.

Properties, characteristics and applications of microbubbles for sonothrombolysis.

INTRODUCTION: Ultrasound enhancement of thrombolysis (sonothrombolysis) is further potentiated by administration of acoustically active microbubbles, which may be developed into powerful adjuvant therapies for thrombolytic treatment of occlusive conditions such as ischaemic stroke. AREAS COVERED: The role of microbubbles in sonothrombolysis is evaluated based on published in vitro and in vivo evidence and a critical review of clinical trials to date. Microbubble, ultrasound and drug parameters compiled from a broad search of the existing literature are tabulated. Mechanisms of microbubble-enhanced sonothrombolysis are discussed, with particular focus on acoustic cavitation and thermal effects. A number of challenges to widespread clinical adoption are identified. Key factors for future optimisation of treatment and microbubble design are proposed. EXPERT OPINION: Microbubble enhancement of thrombolysis is supported by a broad range of in vitro and in vivo evidence that demonstrates improved lysis compared to conventional drug treatment or ultrasound without microbubbles. Clinically, this is shown by accelerated recanalisation of occluded arteries; however, further research is needed to ensure patient safety. Before such techniques can enter widespread clinical practice, an improved understanding of the role of microbubbles in sonothrombolysis is required, in addition to demonstration of significant improvement over existing treatments and the development of reliable real-time monitoring protocols.