Transcranial Blood-Brain Barrier Opening in Alzheimer's Disease Patients Using a Portable Focused Ultrasound System with Real-Time 2-D Cavitation Mapping.

Background: Focused ultrasound (FUS) in combination with microbubbles has recently shown great promise in facilitating blood-brain barrier (BBB) opening for drug delivery and immunotherapy in Alzheimer's disease (AD). However, it is currently limited to systems integrated within the MRI suites or requiring post-surgical implants, thus restricting its widespread clinical adoption. In this pilot study, we investigate the clinical safety and feasibility of a portable, non-invasive neuronavigation-guided FUS (NgFUS) system with integrated real-time 2-D microbubble cavitation mapping. Methods: A phase 1 clinical study with mild to moderate AD patients (N=6) underwent a single session of microbubble-mediated NgFUS to induce transient BBB opening (BBBO). Microbubble activity under FUS was monitored with real-time 2-D cavitation maps and dosing to ensure the efficacy and safety of the NgFUS treatment. Post-operative MRI was used for BBB opening and closure confirmation as well as safety assessment. Changes in AD biomarker levels in both blood serum and extracellular vesicles (EVs) were evaluated, while changes in amyloid-beta (Aß) load in the brain were assessed through 18F-Florbetapir PET. Results: BBBO was achieved in 5 out of 6 subjects with an average volume of 983±626 mm3 following FUS at the right frontal lobe both in white and gray matter regions. The outpatient treatment was completed within 34.8±10.7 min. Cavitation dose significantly correlated with the BBBO volume (R2>0.9, N=4), demonstrating the portable NgFUS system's capability of predicting opening volumes. The cavitation maps co-localized closely with the BBBO location, representing the first report of real-time transcranial 2-D cavitation mapping in the human brain. Larger opening volumes correlated with increased levels of AD biomarkers, including Aß42 (R2=0.74), Tau (R2=0.95), and P-Tau181 (R2=0.86), assayed in serum-derived EVs sampled 3 days after FUS (N=5). From PET scans, subjects showed a lower Aß load increase in the treated frontal lobe region compared to the contralateral region. Reduction in asymmetry standardized uptake value ratios (SUVR) correlated with the cavitation dose (R2>0.9, N=3). Clinical changes in the mini-mental state examination over 6 months were within the expected range of cognitive decline with no additional changes observed as a result of FUS. Conclusion: We showed the safety and feasibility of this cost-effective and time-efficient portable NgFUS treatment for BBBO in AD patients with the first demonstration of real-time 2-D cavitation mapping. The cavitation dose correlated with BBBO volume, a slowed increase in pathology, and serum detection of AD proteins. Our study highlights the potential for accessible FUS treatment in AD, with or without drug delivery.

Characterization of the responses of brain macrophages to focused ultrasound-mediated blood-brain barrier opening.

The opening of the blood-brain barrier (BBB) by focused ultrasound (FUS) coupled with intravenously injected microbubbles can be leveraged as a form of immunotherapy for the treatment of neurodegenerative disorders. However, how FUS BBB opening affects brain macrophages is not well understood. Here by using single-cell sequencing to characterize the distinct responses of microglia and central nervous system-associated macrophages (CAMs) to FUS-mediated BBB opening in mice, we show that the treatment remodels the immune landscape via the recruitment of CAMs and the proliferation of microglia and via population size increases in disease-associated microglia. Both microglia and CAMs showed early and late increases in population sizes, yet only the proliferation of microglia increased at both timepoints. The population of disease-associated microglia also increased, accompanied by the upregulation of genes associated with gliogenesis and phagocytosis, with the depletion of brain macrophages significantly decreasing the duration of BBB opening.

Using a novel rapid alternating steering angles pulse sequence to evaluate the impact of theranostic ultrasound-mediated ultra-short pulse length on blood-brain barrier opening volume and closure, cavitation mapping, drug delivery feasibility, and safety.

Background: Focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening is a noninvasive, safe and reversible technique for targeted drug delivery to the brain. Most preclinical systems developed to perform and monitor BBB opening are comprised of a separate geometrically focused transducer and passive cavitation detector (PCD) or imaging array. This study builds upon previous work from our group developing a single imaging phased array configuration for simultaneous BBB opening and monitoring called theranostic ultrasound (ThUS), leveraging ultra-short pulse lengths (USPLs) and a novel rapid alternating steering angles (RASTA) pulse sequence design for simultaneous bilateral sonications with target-specific USPL. The RASTA sequence was further employed to evaluate the impact of USPL on BBB opening volume, power cavitation imaging (PCI) pixel intensity, BBB closing timeline, drug delivery efficiency, and safety. Methods: A P4-1 phased array transducer driven by a Verasonics Vantage ultrasound system was operated using a custom script to run the RASTA sequence which consisted of interleaved steered, focused transmits and passive imaging. Contrast-enhanced magnetic resonance imaging (MRI) confirmed initial opening volume and closure of the BBB by longitudinal imaging through 72 hours post-BBB opening. For drug delivery experiments, mice were systemically administered a 70 kDa fluorescent dextran or adeno-associated virus serotype 9 (AAV9) for fluorescence microscopy or enzyme-linked immunosorbent assay (ELISA) to evaluate ThUS-mediated molecular therapeutic delivery. Additional brain sections were also H&E-stained to evaluate histological damage, and IBA1- and GFAP-stained to elucidate the effects of ThUS-mediated BBB opening on stimulation of key cell types involved in the neuro-immune response, microglia and astrocytes. Results: The ThUS RASTA sequence induced distinct BBB openings simultaneously in the same mouse where volume, PCI pixel intensity, level of dextran delivery, and AAV reporter transgene expression were correlated with brain hemisphere-specific USPL, consistent with statistically significant differences between 1.5, 5, and 10-cycle USPL groups. BBB closure after ThUS required 2-48 hours depending on USPL. The potential for acute damage and neuro-immune activation increased with USPL, but such observable damage was nearly reversed 96 hours post-ThUS. Conclusion: ThUS is a versatile single-array technique which exhibits the potential for investigating a variety of non-invasive therapeutic delivery applications in the brain.

Transcranial Theranostic Ultrasound for Pre-Planning and Blood-Brain Barrier Opening: A Feasibility Study Using an Imaging Phased Array In Vitro and In Vivo.

Focused ultrasound (FUS) for blood-brain barrier (BBB) opening is a safe, reversible and non-invasive strategy for targeted drug delivery to the brain, however extensive pre-planning strategies are necessary for successful FUS-mediated BBB opening through the structurally complex primate skull. OBJECTIVE: This study aims to demonstrate a pre-planning pipeline consisting of transcranial simulations and in vitro experimentation used to inform synchronous BBB opening and power cavitation imaging (PCI) with a single theranostic ultrasound (TUS) phased array. METHODS: Acoustic wave propagation simulation findings of pressure attenuation and focal shift through clinical-CT and micro-CT-based primate skull models were compared, while the latter were used to determine the impact of beam steering angle on focal shift and pressure attenuation. In vitro experimentation with a channel phantom enabled characterization of skull-induced receive focal shift (RFS), while in vivo BBB opening and PCI using in silico and in vitro pre-planning information was conducted using a custom Verasonics/MATLAB script. RESULTS: Simulations confirmed steering angle dependent transcranial focal shift and pressure attenuation, while in vitro experiments revealed minimal (0.30-1.50 mm) skull-induced RFS. In vivo rodent experiments with overlaid primate skull fragments demonstrated successful TUS-mediated BBB opening and spatially correlated power cavitation images (PCI) with regions of BBB opening on T1-weighted magnetic resonance images (MRI). CONCLUSION: We demonstrated the feasibility for TUS-mediated BBB opening in vivo using in silico and in vitro pre-planning information. SIGNIFICANCE: TUS as an ultrasound-guided modality for BBB opening could serve as a promising alternative to current FUS-mediated BBB opening configurations in the clinic.

Temporal stability of lipid-shelled microbubbles during acoustically-mediated blood-brain barrier opening.

Non-invasive blood-brain barrier (BBB) opening using focused ultrasound (FUS) is being tested as a means to locally deliver drugs into the brain. Such FUS therapies require injection of preformed microbubbles, currently used as contrast agents in ultrasound imaging. Although their behavior during exposure to imaging sequences has been well described, our understanding of microbubble stability within a therapeutic field is still not complete. Here, we study the temporal stability of lipid-shelled microbubbles during therapeutic FUS exposure in two timescales: the short time scale (i.e., µs of low-frequency ultrasound exposure) and the long time scale (i.e., days post-activation). We first simulated the microbubble response to low-frequency sonication, and found a strong correlation between viscosity and fragmentation pressure. Activated microbubbles had a concentration decay constant of 0.02 d-1 but maintained a quasi-stable size distribution for up to 3 weeks (< 10% variation). Microbubbles flowing through a 4-mm vessel within a tissue-mimicking phantom (5% gelatin) were exposed to therapeutic pulses (fc: 0.5 MHz, peak-negative pressure: 300 kPa, pulse length: 1 ms, pulse repetition frequency: 1 Hz, n=10). We recorded and analyzed their acoustic emissions, focusing on emitted energy and its temporal evolution, alongside the frequency content. Measurements were repeated with concentration-matched samples (107 microbubbles/ml) on day 0, 7, 14, and 21 after activation. Temporal stability decreased while inertial cavitation response increased with storage time both in vitro and in vivo, possibly due to changes in the shell lipid content. Using the same parameters and timepoints, we performed BBB opening in a mouse model (n=3). BBB opening volume measured through T1-weighted contrast-enhanced MRI was equal to 19.1 ± 7.1 mm3, 21.8 ± 14 mm3, 29.3 ± 2.5 mm3, and 38 ± 20.1 mm3 on day 0, 7, 14, and 21, respectively, showing no significant difference over time (p-value: 0.49). Contrast enhancement was 24.9 ± 1.7 %, 23.7 ± 11.7 %, 28.9 ± 5.3 %, and 35 ± 13.4 %, respectively (p-value: 0.63). In conclusion, the in-house made microbubbles studied here maintain their capacity to produce similar therapeutic effects over a period of 3 weeks after activation, as long as the natural concentration decay is accounted for. Future work should focus on stability of commercially available microbubbles and tailoring microbubble shell properties towards therapeutic applications.

Optimization of Ultrasound-Mediated Drug Delivery for Treatment of Onychomycosis.

BACKGROUND: Onychomycosis is a fungal nail disorder that does not have a successful cure due to the poor permeability of topical anti-fungal drugs through the nail. This study utilizes ultrasound to increase the permeability of the nail to the topical drugs currently used in clinic. The first aim of this study was to optimize ultrasonic parameters within the temperature increase limits set by the American Institute of Ultrasound in Medicine (AIUM) and the British Medical Ultrasound Society (BMUS). The second aim of the study was to evaluate the optimized parameters for a cause of action of either cavitation (the creation of micrometer pores in the nail barrier) or acoustic streaming (a steady fluid motion which may help push the drug through the nail). METHODS: Porcine and human nails are used in the five studies. PZFlex Modeling Software is used to model the temperature increase in the toe as a result of ultrasonic application and these results were used to develop the three parameters tested throughout the rest of the studies. The three parameters tested were 1 min of continuous ultrasonic application, 3 min of 50% ultrasonic application and 5 min of 50% ultrasonic application. In order to address the second aim of our research work, these three parameters were tested for the presence of streaming and cavitation. RESULTS: At the three tested parameters, the most permeation of the nail occurs with 1 min of continuous application of ultrasound to the nail. It was also found that there was limited cavitation and significant streaming at all three parameters. This suggests that streaming may be the main mechanism-of-action in ultrasound-mediated drug delivery through the nail. CONCLUSION: The parameter of 1 min of continuous ultrasonic testing will continue to be employed as the testing is moved to a rabbit model of onychomycosis.

Ultrasound-Enhanced Drug Delivery for Treatment of Onychomycosis.

OBJECTIVES: The aim of our study was to determine the effectiveness of using ultrasound (US) to increase the permeability of the nail, with the goal of improving outcomes in the treatment of onychomycosis. METHODS: Porcine nails were used because of their similarity to human nails. A hydrophilic blue dye was used as a drug-mimicking compound. Two sets of experiments were performed: luminosity experiments to assess the dye levels inside the nail after US and sham treatments and diffusion cell experiments for determination of changes in nail permeability due to US application. In both sets of experiments, planar US transducers were used to sonicate the nails at frequencies of 400, 600, and 800 kHz and 1 MHz, an intensity of 1 W/cm2 , and a duration of 5 min in a continuous mode. Modeling studies were also performed to assess the safety of US application to the human toe for later clinical studies. RESULTS: In the luminosity experiments, application of US at frequencies of 600 and 800 kHz led to statistically significant results (P < .05), with an increase in dye delivery into the nail of up to 95% compared to control values. The diffusion cell results found statistical significance (P < .05) at all applied frequencies, with up to a 70% increase in the nail permeability compared to the control. Safety modeling studies found a maximal temperature increase of 4.4 °C in the bone. CONCLUSIONS: Our proposed US method may offer an alternative for improved treatment of onychomycosis. The current maximal temperature increase was found to be at the safety limit, and so pulsing and other alternatives will be investigated to minimize this temperature increase.