Improved Therapeutic Delivery Targeting Clinically Relevant Orthotopic Human Pancreatic Tumors Engrafted in Immunocompromised Pigs Using Ultrasound-Induced Cavitation: A Pilot Study.

Pancreatic tumors can be resistant to drug penetration due to high interstitial fluid pressure, dense stroma, and disarrayed vasculature. Ultrasound-induced cavitation is an emerging technology that may overcome many of these limitations. Low-intensity ultrasound, coupled with co-administered cavitation nuclei consisting of gas-stabilizing sub-micron scale SonoTran Particles, is effective at increasing therapeutic antibody delivery to xenograft flank tumors in mouse models. Here, we sought to evaluate the effectiveness of this approach in situ using a large animal model that mimics human pancreatic cancer patients. Immunocompromised pigs were surgically engrafted with human Panc-1 pancreatic ductal adenocarcinoma (PDAC) tumors in targeted regions of the pancreas. These tumors were found to recapitulate many features of human PDAC tumors. Animals were intravenously injected with the common cancer therapeutics Cetuximab, gemcitabine, and paclitaxel, followed by infusion with SonoTran Particles. Select tumors in each animal were targeted with focused ultrasound to induce cavitation. Cavitation increased the intra-tumor concentrations of Cetuximab, gemcitabine, and paclitaxel by 477%, 148%, and 193%, respectively, compared to tumors that were not targeted with ultrasound in the same animals. Together, these data show that ultrasound-mediated cavitation, when delivered in combination with gas-entrapping particles, improves therapeutic delivery in pancreatic tumors under clinically relevant conditions.

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.

Dual-Array Passive Acoustic Mapping for Cavitation Imaging With Enhanced 2-D Resolution.

Passive acoustic mapping (PAM) techniques have been developed for the purposes of detecting, localizing, and quantifying cavitation activity during therapeutic ultrasound procedures. Implementation with conventional diagnostic ultrasound arrays has allowed planar mapping of bubble acoustic emissions to be overlaid with B-mode anatomical images, with a variety of beamforming approaches providing enhanced resolution at the cost of extended computation times. However, no passive signal processing techniques implemented to date have overcome the fundamental physical limitation of the conventional diagnostic array aperture that results in point spread functions with axial/lateral beamwidth ratios of nearly an order of magnitude. To mitigate this problem, the use of a pair of orthogonally oriented diagnostic arrays was recently proposed, with potential benefits arising from the substantially expanded range of observation angles. This article presents experiments and simulations intended to demonstrate the performance and limitations of the dual-array system concept. The key finding of this study is that source pair resolution of better than 1 mm is now possible in both dimensions of the imaging plane using a pair of 7.5-MHz center frequency conventional arrays at a distance of 7.6cm. With an eye toward accelerating computations for real-time applications, channel count reductions of up to a factor of eight induce negligible performance losses. Modest sensitivities to sound speed and relative array position uncertainties were identified, but if these can be kept on the order of 1% and 1 mm, respectively, then the proposed methods offer the potential for a step improvement in cavitation monitoring capability.

Passive Acoustic Mapping Using Data-Adaptive Beamforming Based on Higher Order Statistics.

Sources of nonlinear acoustic emissions, particularly those associated with cavitation activity, play a key role in the safety and efficacy of current and emerging therapeutic ultrasound applications, such as oncological drug delivery, blood-brain barrier opening, and histotripsy. Passive acoustic mapping (PAM) is the first technique to enable real-time and non-invasive imaging of cavitation activity during therapeutic ultrasound exposure, through the recording and passive beamforming of broadband acoustic emissions using an array of ultrasound detectors. Initial limitations in PAM spatial resolution led to the adoption of optimal data-adaptive beamforming algorithms, such as the robust capon beamformer (RCB), that provide improved interference suppression and calibration error mitigation compared to non-adaptive beamformers. However, such approaches are restricted by the assumption that the recorded signals have a Gaussian distribution. To overcome this limitation and further improve the source resolvability of PAM, we propose a new beamforming approach termed robust beamforming by linear programming (RLPB). Along with the variance, this optimization-based method uses higher-order-statistics of the recorded signals, making no prior assumption on the statistical distribution of the acoustic signals. The RLPB is found via numerical simulations to improve resolvability over time exposure acoustics and RCB. In vitro experimentation yielded improved resolvability with respect to the source-to-array distance on the order of 22% axially and 13% transversely relative to RCB, whilst successfully accounting for array calibration errors. The improved resolution and decreased dependence on accurate calibration of RLPB is expected to facilitate the clinical translation of PAM for diagnostic, including super-resolution, and therapeutic ultrasound applications.

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.

Ultrasound-mediated cavitation does not decrease the activity of small molecule, antibody or viral-based medicines.

The treatment of cancer using nanomedicines is limited by the poor penetration of these potentially powerful agents into and throughout solid tumors. Externally controlled mechanical stimuli, such as the generation of cavitation-induced microstreaming using ultrasound (US), can provide a means of improving nanomedicine delivery. Notably, it has been demonstrated that by focusing, monitoring and controlling the US exposure, delivery can be achieved without damage to surrounding tissue or vasculature. However, there is a risk that such stimuli may disrupt the structure and thereby diminish the activity of the delivered drugs, especially complex antibody and viral-based nanomedicines. In this study, we characterize the impact of cavitation on four different agents, doxorubicin (Dox), cetuximab, adenovirus (Ad) and vaccinia virus (VV), representing a scale of sophistication from a simple small-molecule drug to complex biological agents. To achieve tight regulation of the level and duration of cavitation exposure, a "cavitation test rig" was designed and built. The activity of each agent was assessed with and without exposure to a defined cavitation regime which has previously been shown to provide effective and safe delivery of agents to tumors in preclinical studies. The fluorescence profile of Dox remained unchanged after exposure to cavitation, and the efficacy of this drug in killing a cancer cell line remained the same. Similarly, the ability of cetuximab to bind its epidermal growth factor receptor target was not diminished following exposure to cavitation. The encoding of the reporter gene luciferase within the Ad and VV constructs tested here allowed the infectivity of these viruses to be easily quantified. Exposure to cavitation did not impact on the activity of either virus. These data provide compelling evidence that the US parameters used to safely and successfully delivery nanomedicines to tumors in preclinical models do not detrimentally impact on the structure or activity of these nanomedicines.

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.

Sum-of-harmonics method for improved narrowband and broadband signal quantification during passive monitoring of ultrasound therapies.

Passive Acoustic Mapping (PAM) enables real-time monitoring of ultrasound therapies by beamforming acoustic emissions emanating from the ultrasound focus. Reconstruction of the narrowband or broadband acoustic emissions component enables mapping of different physical phenomena, with narrowband emissions arising from non-linear propagation and scattering, non-inertial cavitation or tissue boiling, and broadband (generally, of significantly lower amplitude) indicating inertial cavitation. Currently, accurate classification of the received signals based on pre-defined frequency-domain comb filters cannot be guaranteed because varying levels of leakage occur as a function of signal amplitude and the choice of windowing function. This work presents a time-domain parametric model aimed at enabling accurate estimation of the amplitude of time-varying narrowband components in the presence of broadband signals. Conversely, the method makes it possible to recover a weak broadband signal in the presence of a dominant harmonic or other narrowband component. Compared to conventional comb filtering, the proposed sum-of-harmonics method enables PAM of cavitation sources that better reflect their physical location and extent.

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.

Ultrasound-Propelled Nanocups for Drug Delivery.

Ultrasound-induced bubble activity (cavitation) has been recently shown to actively transport and improve the distribution of therapeutic agents in tumors. However, existing cavitation-promoting agents are micron-sized and cannot sustain cavitation activity over prolonged time periods because they are rapidly destroyed upon ultrasound exposure. A novel ultrasound-responsive single-cavity polymeric nanoparticle (nanocup) capable of trapping and stabilizing gas against dissolution in the bloodstream is reported. Upon ultrasound exposure at frequencies and intensities achievable with existing diagnostic and therapeutic systems, nanocups initiate and sustain readily detectable cavitation activity for at least four times longer than existing microbubble constructs in an in vivo tumor model. As a proof-of-concept of their ability to enhance the delivery of unmodified therapeutics, intravenously injected nanocups are also found to improve the distribution of a freely circulating IgG mouse antibody when the tumor is exposed to ultrasound. Quantification of the delivery distance and concentration of both the nanocups and coadministered model therapeutic in an in vitro flow phantom shows that the ultrasound-propelled nanocups travel further than the model therapeutic, which is itself delivered to hundreds of microns from the vessel wall. Thus nanocups offer considerable potential for enhanced drug delivery and treatment monitoring in oncological and other biomedical applications.

Passive acoustic mapping utilizing optimal beamforming in ultrasound therapy monitoring.

Passive acoustic mapping (PAM) is a promising imaging method that enables real-time three-dimensional monitoring of ultrasound therapy through the reconstruction of acoustic emissions passively received on an array of ultrasonic sensors. A passive beamforming method is presented that provides greatly improved spatial accuracy over the conventionally used time exposure acoustics (TEA) PAM reconstruction algorithm. Both the Capon beamformer and the robust Capon beamformer (RCB) for PAM are suggested as methods to reduce interference artifacts and improve resolution, which has been one of the experimental issues previously observed with TEA. Simulation results that replicate the experimental artifacts are shown to suggest that bubble interactions are the chief cause. Analysis is provided to show that these multiple bubble artifacts are generally not reduced by TEA, while Capon-based methods are able to reduce the artifacts. This is followed by experimental results from in vitro experiments and in vivo oncolytic viral therapy trials that show improved results in PAM, where RCB is able to more accurately localize the acoustic activity than TEA.

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.

Non-invasive and real-time passive acoustic mapping of ultrasound-mediated drug delivery.

New classes of biologically active materials, such as viruses, siRNA, antibodies and a wide range of engineered nanoparticles have emerged as potent agents for diagnosing and treating diseases, yet many of these agents fail because there is no effective route of delivery to their intended targets. Focused ultrasound and its ability to drive microbubble-seeded cavitation have been shown to facilitate drug delivery. However, cavitation is difficult to control temporally and spatially, making prediction of therapeutic outcomes deep in the body difficult. Here, we utilized passive acoustic mapping in vivo to understand how ultrasound parameters influence cavitation dynamics and to correlate spatial maps of cavitation to drug delivery. Focused ultrasound (center frequency: 0.5 MHz, peak-rarefactional pressure: 1.2 MPa, pulse length: 25 cycles or 50,000 cycles, pulse repetition interval: 0.02, 0.2, 1 or 3 s, number of pulses: 80 pulses) was applied to murine xenograft-model tumors in vivo during systemic injection of microbubbles with and without cavitation-sensitive liposomes or type 5 adenoviruses. Analysis of in vivo cavitation dynamics through several pulses revealed that cavitation was more efficiently produced at a lower pulse repetition frequency of 1 Hz than at 50 Hz. Within a pulse, inertial cavitation activity was shown to persist but reduced to 50% and 25% of its initial magnitude in 4.3 and 29.3 ms, respectively. Both through several pulses and within a pulse, the spatial distribution of cavitation was shown to change in time due to variations in microbubble distribution present in tumors. Finally, we demonstrated that the centroid of the mapped cavitation activity was within 1.33  ±  0.6 mm and 0.36 mm from the centroid location of drug release from liposomes and expression of the reporter gene encoded by the adenovirus, respectively. Thus passive acoustic mapping not only unraveled key mechanisms whereby a successful outcome is achieved, but also a predicted drug delivery outcome.

Thin-film sparse boundary array design for passive acoustic mapping during ultrasound therapy.

A new 2-D hydrophone array for ultrasound therapy monitoring is presented, along with a novel algorithm for passive acoustic mapping using a sparse weighted aperture. The array is constructed using existing polyvinylidene fluoride (PVDF) ultrasound sensor technology, and is utilized for its broadband characteristics and its high receive sensitivity. For most 2-D arrays, high-resolution imagery is desired, which requires a large aperture at the cost of a large number of elements. The proposed array's geometry is sparse, with elements only on the boundary of the rectangular aperture. The missing information from the interior is filled in using linear imaging techniques. After receiving acoustic emissions during ultrasound therapy, this algorithm applies an apodization to the sparse aperture to limit side lobes and then reconstructs acoustic activity with high spatiotemporal resolution. Experiments show verification of the theoretical point spread function, and cavitation maps in agar phantoms correspond closely to predicted areas, showing the validity of the array and methodology.

Passive cavitation mapping with temporal sparsity constraint.

The spatial resolution of cavitation maps generated from passive recordings of cavitation emissions is compromised by the bandlimited nature of the recordings. Deconvolution based on the assumption that cavitation consists of a sparse series of discrete events allows the recovery of frequency components that are not only outside the frequency band of the receivers, but may also have been attenuated by the medium before being detectable. In the current work, two sparse deconvolution techniques, matching pursuit and basis pursuit, were applied to simulated and experimental cavitation recordings before they were beamformed to provide passive maps of cavitation activity. Matching pursuit was shown to reduce the maximal diameter of the point spread function by almost a third, at the cost of greater susceptibility to inter-source interference. In contrast, although basis pursuit causes an almost 20% increase in the maximal diameter of the point spread function, its application to experimental data appears to enhance the ability of passive mapping to resolve multiple sources.