From bedside to portable and wearable: development of a conformable ultrasound patch for deep breast tissue imaging.

A breakthrough study from Du et al. has developed a wearable, ultrasound imaging patch for standardized and reproducible breast tissue imaging. The technology utilizes a honeycomb patch design to facilitate guided movement of the ultrasound array, enabling comprehensive, multiangle breast imaging. The system was validated in vitro and in vivo with a single human subject and has the potential for early-stage breast cancer detection. This study addressed the current limitations of wearable ultrasound technologies, including imaging over large, curvilinear organs and integration of superior piezoelectric materials for high-performance ultrasound arrays. The transition of ultrasound from the bedside to portable and wearable devices will pave the way for integration with big data collection, such as artificial intelligence (AI)-based diagnosis and personalized ultrasonographic profile generation, for rapid and objective measurements. This advancement is especially important in the context of breast cancer, where early diagnosis and assessment of medical therapy responses are paramount to patient care.

Slow-Flow Ultrasound Localization Microscopy Using Recondensation of Perfluoropentane Nanodroplets.

Ultrasound localization microscopy (ULM) is an emerging, super-resolution imaging technique for detailed mapping of the microvascular structure and flow velocity via subwavelength localization and tracking of microbubbles. Because microbubbles rely on blood flow for movement throughout the vascular space, acquisition times can be long in the smallest, low-flow microvessels. In addition, detection of microbubbles in low-flow regions can be difficult because of minimal separation of microbubble signal from tissue. Nanoscale, phase-change contrast agents (PCCAs) have emerged as a switchable, intermittent or persisting contrast agent for ULM via acoustic droplet vaporization (ADV). Here, the focus is on characterizing the spatiotemporal contrast properties of less volatile perfluoropentane (PFP) PCCAs. The results indicate that at physiological temperature, nanoscale PFP PCCAs with diameters less than 100 nm disappear within microseconds after ADV with high-frequency ultrasound (16 MHz, 5- to 6-MPa peak negative pressure) and that nanoscale PFP PCCAs have an inherent deactivation mechanism via immediate recondensation after ADV. This "blinking" on-and-off contrast signal allowed separation of flow in an in vitro flow phantom, regardless of flow conditions, although with a need for some replenishment at very low flow conditions to maintain count rate. This blinking behavior allows for rapid spatial mapping in areas of low or no flow with ULM, but limits velocity tracking because there is no stable bubble formation with nanoscale PFP PCCAs.

Safety evaluation of a clinical focused ultrasound system for neuronavigation guided blood-brain barrier opening in non-human primates.

An emerging approach with potential in improving the treatment of neurodegenerative diseases and brain tumors is the use of focused ultrasound (FUS) to bypass the blood-brain barrier (BBB) in a non-invasive and localized manner. A large body of pre-clinical work has paved the way for the gradual clinical implementation of FUS-induced BBB opening. Even though the safety profile of FUS treatments in rodents has been extensively studied, the histological and behavioral effects of clinically relevant BBB opening in large animals are relatively understudied. Here, we examine the histological and behavioral safety profile following localized BBB opening in non-human primates (NHPs), using a neuronavigation-guided clinical system prototype. We show that FUS treatment triggers a short-lived immune response within the targeted region without exacerbating the touch accuracy or reaction time in visual-motor cognitive tasks. Our experiments were designed using a multiple-case-study approach, in order to maximize the acquired data and support translation of the FUS system into human studies. Four NHPs underwent a single session of FUS-mediated BBB opening in the prefrontal cortex. Two NHPs were treated bilaterally at different pressures, sacrificed on day 2 and 18 post-FUS, respectively, and their brains were histologically processed. In separate experiments, two NHPs that were earlier trained in a behavioral task were exposed to FUS unilaterally, and their performance was tracked for at least 3 weeks after BBB opening. An increased microglia density around blood vessels was detected on day 2, but was resolved by day 18. We also detected signs of enhanced immature neuron presence within areas that underwent BBB opening, compared to regions with an intact BBB, confirming previous rodent studies. Logistic regression analysis showed that the NHP cognitive performance did not deteriorate following BBB opening. These preliminary results demonstrate that neuronavigation-guided FUS with a single-element transducer is a non-invasive method capable of reversibly opening the BBB, without substantial histological or behavioral impact in an animal model closely resembling humans. Future work should confirm the observations of this multiple-case-study work across animals, species and tasks.

Effect of Overpressure on Acoustic Emissions and Treated Tissue Histology in ex Vivo Bulk Ultrasound Ablation.

Bulk ultrasound ablation is a thermal therapy approach in which tissue is heated by unfocused or weakly focused sonication (average intensities on the order of 100 W/cm2) to achieve coagulative necrosis within a few minutes exposure time. Assessing the role of bubble activity, including acoustic cavitation and tissue vaporization, in bulk ultrasound ablation may help in making bulk ultrasound ablation safer and more effective for clinical applications. Here, two series of ex vivo ablation trials were conducted to investigate the role of bubble activity and tissue vaporization in bulk ultrasound ablation. Fresh bovine liver tissue was ablated with unfocused, continuous-wave ultrasound using ultrasound image-ablate arrays sonicating at 31 W/cm2 (0.9 MPa amplitude) for either 20 min at a frequency of 3.1 MHz or 10 min at 4.8 MHz. Tissue specimens were maintained at a static overpressure of either 0.52 or 1.2 MPa to suppress bubble activity and tissue vaporization or at atmospheric pressure for control groups. A passive cavitation detector was used to record subharmonic (1.55 or 2.4 MHz), broadband (1.2-1.5 MHz) and low-frequency (5-20 kHz) acoustic emissions. Treated tissue was stained with 2% triphenyl tetrazolium chloride to evaluate thermal lesion dimensions. Subharmonic emissions were significantly reduced in overpressure groups compared with control groups. Correlations observed between acoustic emissions and lesion dimensions were significant and positive for the 3.1-MHz series, but significant and negative for the 4.8-MHz series. The results indicate that for bulk ultrasound ablation, where both acoustic cavitation and tissue vaporization are possible, bubble activity can enhance ablation in the absence of tissue vaporization, but can reduce thermal lesion dimensions in the presence of vaporization.

Displacement Imaging for Focused Ultrasound Peripheral Nerve Neuromodulation.

Focused ultrasound (FUS) is an emerging technique for neuromodulation due to its noninvasive application and high depth penetration. Recent studies have reported success in modulation of brain circuits, peripheral nerves, ion channels, and organ structures. In particular, neuromodulation of peripheral nerves and the underlying mechanisms remain comparatively unexplored in vivo. Lack of methodologies for FUS targeting and monitoring impede further research in in vivo studies. Thus, we developed a method that non-invasively measures nerve engagement, via tissue displacement, during FUS neuromodulation of in vivo nerves using simultaneous high frame-rate ultrasound imaging. Using this system, we can validate, in real-time, FUS targeting of the nerve and characterize subsequent compound muscle action potentials (CMAPs) elicited from sciatic nerve activation in mice using 0.5 to 5 ms pulse durations and 22 - 28 MPa peak positive stimulus pressures at 4 MHz. Interestingly, successful motor excitation from FUS neuromodulation required a minimum interframe nerve displacement of 18 µm without any displacement incurred at the skin or muscle levels. Moreover, CMAPs detected in mice monotonically increased with interframe nerve displacements within the range of 18 to 300 µm . Thus, correlation between nerve displacement and motor activation constitutes strong evidence FUS neuromodulation is driven by a mechanical effect given that tissue deflection is a result of highly focused acoustic radiation force.

A Clinical System for Non-invasive Blood-Brain Barrier Opening Using a Neuronavigation-Guided Single-Element Focused Ultrasound Transducer.

Focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening is currently being investigated in clinical trials. Here, we describe a portable clinical system with a therapeutic transducer suitable for humans, which eliminates the need for in-line magnetic resonance imaging (MRI) guidance. A neuronavigation-guided 0.25-MHz single-element FUS transducer was developed for non-invasive clinical BBB opening. Numerical simulations and experiments were performed to determine the characteristics of the FUS beam within a human skull. We also validated the feasibility of BBB opening obtained with this system in two non-human primates using U.S. Food and Drug Administration (FDA)-approved treatment parameters. Ultrasound propagation through a human skull fragment caused 44.4 ± 1% pressure attenuation at a normal incidence angle, while the focal size decreased by 3.3 ± 1.4% and 3.9 ± 1.8% along the lateral and axial dimension, respectively. Measured lateral and axial shifts were 0.5 ± 0.4 mm and 2.1 ± 1.1 mm, while simulated shifts were 0.1 ± 0.2 mm and 6.1 ± 2.4 mm, respectively. A 1.5-MHz passive cavitation detector transcranially detected cavitation signals of Definity microbubbles flowing through a vessel-mimicking phantom. T1-weighted MRI confirmed a 153 ± 5.5 mm3 BBB opening in two non-human primates at a mechanical index of 0.4, using Definity microbubbles at the FDA-approved dose for imaging applications, without edema or hemorrhage. In conclusion, we developed a portable system for non-invasive BBB opening in humans, which can be achieved at clinically relevant ultrasound exposures without the need for in-line MRI guidance. The proposed FUS system may accelerate the adoption of non-invasive FUS-mediated therapies due to its fast application, low cost and portability.

Fast qualitative two-dimensional mapping of ultrasound fields with acoustic cavitation-enhanced ultrasound imaging.

Characterization of ultrasound fields is a routine procedure for both diagnostic and therapeutic ultrasound. Quantitative field mapping with a calibrated hydrophone and multi-axis positioning system can be difficult and time consuming. In this study, the use of acoustic cavitation field mapping as a qualitative surrogate to acoustic pressure field mapping, albeit without acoustic pressure values is demonstrated. This technique allows for fast qualitative mapping of ultrasound fields and thereby functionality of the corresponding transducers, in a matter of seconds. In addition, this technique could be used to rapidly image in vivo acoustic cavitation fields during therapeutic ultrasound applications.

Control of Acoustic Cavitation for Efficient Sonoporation with Phase-Shift Nanoemulsions.

Acoustic cavitation can be used to temporarily disrupt cell membranes for intracellular delivery of large biomolecules. Termed sonoporation, the ability of this technique for efficient intracellular delivery (i.e., >50% of initial cell population showing uptake) while maintaining cell viability (i.e., >50% of initial cell population viable) has proven to be very difficult. Here, we report that phase-shift nanoemulsions (PSNEs) function as inertial cavitation nuclei for improvement of sonoporation efficiency. The interplay between ultrasound frequency, resultant microbubble dynamics and sonoporation efficiency was investigated experimentally. Acoustic emissions from individual microbubbles nucleated from PSNEs were captured using a broadband passive cavitation detector during and after acoustic droplet vaporization with short pulses of ultrasound at 1, 2.5 and 5 MHz. Time domain features of the passive cavitation detector signals were analyzed to estimate the maximum size (Rmax) of the microbubbles using the Rayleigh collapse model. These results were then applied to sonoporation experiments to test if uptake efficiency is dependent on maximum microbubble size before inertial collapse. Results indicated that at the acoustic droplet vaporization threshold, Rmax was approximately 61.7 ± 5.2, 24.9 ± 2.8, and 12.4 ± 2.1 µm at 1, 2.5 and 5 MHz, respectively. Sonoporation efficiency increased at higher frequencies, with efficiencies of 39.5 ± 13.7%, 46.6 ± 3.28% and 66.8 ± 5.5% at 1, 2.5 and 5 MHz, respectively. Excessive cellular damage was seen at lower frequencies because of the erosive effects of highly energetic inertial cavitation. These results highlight the importance of acoustic cavitation control in determining the outcome of sonoporation experiments. In addition, PSNEs may serve as tailorable inertial cavitation nuclei for other therapeutic ultrasound applications.

Pulse inversion enhances the passive mapping of microbubble-based ultrasound therapy.

Therapeutic ultrasound combined with preformed circulating microbubbles has enabled non-invasive and targeted drug delivery into the brain, tumors, and blood clots. Monitoring the microbubble activity is essential for the success of such therapies; however, skull and tissues limit our ability to detect low acoustic signals. Here, we show that by emitting consecutive therapeutic pulses of inverse polarity, the sensitivity in the detection of weak bubble acoustic signals during blood-brain barrier opening is enhanced compared to therapeutic pulses of the same polarity. Synchronous passive mapping of the cavitation activity was conducted using delay-and-sum beamforming with absolute time delays, which offers superior spatial resolution compared to the existing asynchronous passive imaging techniques. Sonication with pulse inversion allowed filter-free suppression of the tissue signals by up to 8 dB in a tissue-mimicking phantom and by 7 dB in vivo, compared to exposure without pulse inversion, enabling enhanced passive mapping of microbubble activity. Both therapeutic schemes resulted in similar free-field microbubble activation in vitro and efficient blood-brain barrier opening in vivo.

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.

Acoustic Cavitation-Mediated Delivery of Small Interfering Ribonucleic Acids with Phase-Shift Nano-Emulsions.

Localized, targeted delivery of small interfering ribonucleic acid (siRNA) has been the foremost hurdle in the use of siRNA for the treatment of various diseases. Major advances have been achieved in the synthesis of siRNA, which have led to greater target messenger RNA (mRNA) silencing and stability under physiologic conditions. Although numerous delivery strategies have shown promise, there are still limited options for targeted delivery and release of siRNA administered systemically. In this in vitro study, phase-shift nano-emulsions (PSNE) were explored as cavitation nuclei to facilitate free siRNA delivery to cancer cells via sonoporation. A cell suspension containing varying amounts of PSNE and siRNA was exposed to 5-MHz pulsed ultrasound at fixed settings (6.2-MPa peak negative pressure, 5-cycle pulses, 250-Hz pulse repetition frequency (PRF) and total exposure duration of 100 s). Inertial cavitation emissions were detected throughout the exposure using a passive cavitation detector. Successful siRNA delivery was achieved (i.e., >50% cell uptake) with high (>80%) viability. The percentage of cells with siRNA uptake was correlated with the amount of inertial cavitation activity generated from vaporized PSNE. The siRNA remained functional after delivery, significantly reducing expression of green fluorescent protein in a stably transfected cell line. These results indicate that vaporized PSNE can facilitate siRNA entry into the cytosol of a majority of sonicated cells and may provide a non-endosomal route for siRNA delivery.

Prediction and suppression of HIFU-induced vessel rupture using passive cavitation detection in an ex vivo model.

BACKGROUND: Occlusion of blood vessels using high-intensity focused ultrasound (HIFU) is a potential treatment for arteriovenous malformations and other neurovascular disorders. However, attempting HIFU-induced vessel occlusion can also cause vessel rupture, resulting in hemorrhage. Possible rupture mechanisms include mechanical effects of acoustic cavitation and heating of the vessel wall. METHODS: HIFU exposures were performed on 18 ex vivo porcine femoral arteries with simultaneous passive cavitation detection. Vessels were insonified by a 3.3-MHz focused source with spatial-peak, temporal-peak focal intensity of 15,690-24,430 W/cm(2) (peak negative-pressure range 10.92-12.52 MPa) and a 50% duty cycle for durations up to 5 min. Time-dependent acoustic emissions were recorded by an unfocused passive cavitation detector and quantified within low-frequency (10-30 kHz), broadband (0.3-1.1 MHz), and subharmonic (1.65 MHz) bands. Vessel rupture was detected by inline metering of saline flow, recorded throughout each treatment. Recorded emissions were grouped into 'pre-rupture' (0-10 s prior to measured point of vessel rupture) and 'intact-vessel' (>10 s prior to measured point of vessel rupture) emissions. Receiver operating characteristic curve analysis was used to assess the ability of emissions within each frequency band to predict vessel rupture. Based on these measurements associating acoustic emissions with vessel rupture, a real-time feedback control module was implemented to monitor acoustic emissions during HIFU treatment and adjust the ultrasound intensity, with the goal of maximizing acoustic power delivered to the vessel while avoiding rupture. This feedback control approach was tested on 10 paired HIFU exposures of porcine femoral and subclavian arteries, in which the focal intensity was stepwise increased from 9,117 W/cm(2) spatial-peak temporal-peak (SPTP) to a maximum of 21,980 W/cm(2), with power modulated based on the measured subharmonic emission amplitude. Time to rupture was compared between these feedback-controlled trials and paired controller-inactive trials using a paired Wilcoxon signed-rank test. RESULTS: Subharmonic emissions were found to be the most predictive of vessel rupture (areas under the receiver operating characteristic curve (AUROC) = 0.757, p < 10(-16)) compared to low-frequency (AUROC = 0.657, p < 10(-11)) and broadband (AUROC = 0.729, p < 10(-16)) emissions. An independent-sample t test comparing pre-rupture to intact-vessel emissions revealed a statistically significant difference between the two groups for broadband and subharmonic emissions (p < 10(-3)), but not for low-frequency emissions (p = 0.058). In a one-sided paired Wilcoxon signed-rank test, activation of the control module was shown to increase the time to vessel rupture (T - = 8, p = 0.0244, N = 10). In one-sided paired t tests, activation of the control module was shown to cause no significant difference in time-averaged focal intensity (t = 0.362, p = 0.363, N = 10), but was shown to cause delivery of significantly greater total acoustic energy (t = 2.037, p = 0.0361, N = 10). CONCLUSIONS: These results suggest that acoustic cavitation plays an important role in HIFU-induced vessel rupture. In HIFU treatments for vessel occlusion, passive monitoring of acoustic emissions may be useful in avoiding hemorrhage due to vessel rupture, as shown in the rupture suppression experiments.

Synthesis of phase-shift nanoemulsions with narrow size distributions for acoustic droplet vaporization and bubble-enhanced ultrasound-mediated ablation.

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.

Passive imaging with pulsed ultrasound insonations.

Previously, passive cavitation imaging has been described in the context of continuous-wave high-intensity focused ultrasound thermal ablation. However, the technique has potential use as a feedback mechanism for pulsed-wave therapies, such as ultrasound-mediated drug delivery. In this paper, results of experiments and simulations are reported to demonstrate the feasibility of passive cavitation imaging using pulsed ultrasound insonations and how the images depend on pulsed ultrasound parameters. The passive cavitation images were formed from channel data that was beamformed in the frequency domain. Experiments were performed in an invitro flow phantom with an experimental echo contrast agent, echogenic liposomes, as cavitation nuclei. It was found that the pulse duration and envelope have minimal impact on the image resolution achieved. The passive cavitation image amplitude scales linearly with the cavitation emission energy. Cavitation images for both stable and inertial cavitation can be obtained from the same received data set.

The potential applications of high-intensity focused ultrasound (HIFU) in vascular neurosurgery.

This review assesses the feasibilty of high-intensity focused ultrasound (HIFU) in neurosurgical applications, specifically occlusion of intact blood vessels. Fourteen articles were examined. In summary, MRI was effective for HIFU guidance whereas MR angiography assessed vessel occlusion. Several studies noted immediate occlusion of blood vessels with HIFU. Long-term data, though scarce, indicated a trend of vessel recanalization and return to pre-treatment diameters. Effective parameters for extracranial vascular occlusion included intensity ranges of 1,690-8,800 W/cm(2), duration <15 seconds, and 0.68-3.3 MHz frequency. A threshold frequency-intensity product of 8,250 MHzW/cm(2) was needed for vascular occlusion with a sensitivity of 70% and a specificity of 86%. Complications include skin burns, hemorrhage, and damage to surrounding structures. With evidence that HIFU can successfully occlude extracranial blood vessels, refinement in applications and demonstrable intracranial occlusion are needed.

Treatment of rabbit liver cancer in vivo using miniaturized image-ablate ultrasound arrays.

In the preclinical studies reported here, VX2 cancer within rabbit liver has been treated by bulk ultrasound ablation employing miniaturized image-ablate arrays. Array probes were constructed with 32 elements in a 2.3 × 20 mm(2) aperture, packaged within a 3.1 mm stainless steel tube with a cooling and coupling balloon for in vivo use. The probes were measured capable of 50% fractional bandwidth for pulse-echo imaging (center frequency 4.4 MHz) with >110 W/cm(2) surface intensity available at sonication frequencies 3.5 and 4.8 MHz. B-scan imaging performance of the arrays was measured to be comparable to larger diagnostic linear arrays, although nearfield image quality was reduced by ringdown artifacts. A series of in vivo ablation procedures was performed using an unfocused 32-element aperture firing at 4.8 MHz with exposure durations 20-70.5 s and in situ spatial average, temporal average intensities 22.4-38.5 W/cm(2). Ablation of a complete tumor cross-section was confirmed by vital staining in seven of 12 exposures, with four exposures ablating an additional margin >1 mm beyond the tumor in all directions. Analysis suggests a threshold ablation effect, with complete ablation of tumor cross-sections for exposures with delivery of >838 J acoustic energy. The results show feasibility for in vivo liver cancer ablation using miniaturized image-ablate arrays suitable for interstitial deployment.