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Purpose:The blood–brain barrier (BBB) limits the efficacy of drug therapies for glioblastoma (GBM). Preclinical data indicate that low-intensity pulsed ultrasound (LIPU) can transiently disrupt the BBB and increase intracerebral drug concentrations.Patients and Methods:A first-in-man, single-arm, single-center trial (NCT02253212) was initiated to investigate the transient disruption of the BBB in patients with recurrent GBM. Patients were implanted with a 1-MHz, 11.5-mm diameter cranial ultrasound device (SonoCloud-1, CarThera). The device was activated monthly to transiently disrupt the BBB before intravenous carboplatin chemotherapy.Results:Between 2014 and 2016, 21 patients were registered for the study and implanted with the SonoCloud-1; 19 patients received at least one sonication. In 65 ultrasound sessions, BBB disruption was visible on T1w MRI for 52 sonications. Treatment-related adverse events observed were transient and manageable: a transient edema at H1 and at D15. No carboplatin-related neurotoxicity was observed. Patients with no or poor BBB disruption (n = 8) visible on MRI had a median progression-free survival (PFS) of 2.73 months, and a median overall survival (OS) of 8.64 months. Patients with clear BBB disruption (n = 11) had a median PFS of 4.11 months, and a median OS of 12.94 months.Conclusions:SonoCloud-1 treatments were well tolerated and may increase the effectiveness of systemic drug therapies, such as carboplatin, in the brain without inducing neurotoxicity.See related commentary by Sonabend and Stupp, p. 3750

Supplementary Table 1. The acoustic pressure levels are determined by a calibration of the implants in water prior to implantation in patients. The 95% confidence interval given in the table denotes the measurement uncertainty inherent to this process. Acoustic pressure levels reported previously (19) were re-evaluated using enhanced acoustic calibration methods with reduced uncertainty. The former values are given in the pold column to ease comparison with our previous publication. Supplementary Table 2. BBB disruption grade and acoustic pressure. Data are n (%). Supplementary Table 3. The secondary outcomes of the trial were calculated based on the grading assigned to post-contrast MR images evaluating the level of BBB disruption. Greater amounts of post-sonication BBB disruption resulted in a trend towards longer PFS and OS.

Therapeutic antibodies targeting immune checkpoints have shown limited efficacy in clinical trials in glioblastoma (GBM) patients. Ultrasound-mediated blood-brain barrier opening (UMBO) using low-intensity pulsed ultrasound improved drug delivery to the brain. We explored the safety and the efficacy of UMBO plus immune checkpoint inhibitors in preclinical models of GBM. A Blood-brain barrier (BBB) opening was performed using a 1 MHz preclinical ultrasound system in combination with 10µl/g microbubbles. Brain penetration of immune checkpoint inhibitors was determined, and immune cell populations were evaluated using flow cytometry. The impact of repeated treatments on survival was determined. In syngeneic GL261-bearing immunocompetent mice, we showed that UMBO safely and repeatedly open the BBB. BBB opening was confirmed visually and microscopically using Evans’s blue dye and magnetic resonance imaging. UMBO plus anti-PDL-1 was associated with a significant improvement of the overall survival compared to anti-PD-L1 alone. Using mass spectroscopy, we showed that the penetration of therapeutic antibodies can be increased when delivered intravenously compared to non-sonicated brains. Furthermore, we observed an enhancement of activated microglia percentage when combined with anti-PD-L1. Here, we report that the combination of UMBO and anti-PD-L1 dramatically increases GL261-bearing mice's survival compared to their counterparts treated with anti-PD-L1 alone. Our study highlights the BBB as a limitation to overcome to increase the efficacy of anti-PD-L1 in GBM and supports clinical trials combining UMBO and in GBM patients.

Purpose: The blood–brain barrier (BBB) inhibits adequate dosing/penetration of therapeutic agents to malignancies in the brain. Low-intensity pulsed ultrasound (LIPU) is a safe therapeutic method of temporary BBB disruption (BBBD) to enhance chemotherapeutic delivery to the tumor and surrounding brain parenchyma for treatment of glioblastoma. Experimental Design: We investigated if LIPU could enhance therapeutic efficacy of anti–PD-1 in C57BL/6 mice bearing intracranial GL261 gliomas, epidermal growth factor receptor variant III (EGFRvIII) chimeric antigen receptor (CAR) T cells in NSG mice with EGFRvIII-U87 gliomas, and a genetically engineered antigen-presenting cell (APC)-based therapy producing the T-cell attracting chemokine CXCL10 in the GL261-bearing mice. Results: Mice treated with anti–PD-1 and LIPU-induced BBBD had a median survival duration of 58 days compared with 39 days for mice treated with anti–PD-1, and long-term survivors all remained alive after contralateral hemisphere rechallenge. CAR T-cell administration with LIPU-induced BBBD resulted in significant increases in CAR T-cell delivery to the CNS after 24 (P < 0.005) and 72 (P < 0.001) hours and increased median survival by greater than 129%, in comparison with CAR T cells alone. Local deposition of CXCL10-secreting APCs in the glioma microenvironment with LIPU enhanced T-cell glioma infiltration during the therapeutic window (P = 0.004) and markedly enhanced survival (P < 0.05). Conclusions: LIPU increases immune therapeutic delivery to the tumor microenvironment with an associated increase in survival and is an emerging technique for enhancing novel therapies in the brain.

Background Temporary disruption of the blood-brain barrier (BBB) using pulsed ultrasound leads to the clearance of both amyloid and tau from the brain, increased neurogenesis, and mitigation of cognitive decline in pre-clinical models of Alzheimer’s disease (AD) while also increasing BBB penetration of therapeutic antibodies. The goal of this pilot clinical trial was to investigate the safety and efficacy of this approach in patients with mild AD using an implantable ultrasound device. Methods An implantable, 1-MHz ultrasound device (SonoCloud-1) was implanted under local anesthesia in the skull (extradural) of 10 mild AD patients to target the left supra-marginal gyrus. Over 3.5 months, seven ultrasound sessions in combination with intravenous infusion of microbubbles were performed twice per month to temporarily disrupt the BBB. 18F-florbetapir and 18F-fluorodeoxyglucose positron emission tomography (PET) imaging were performed on a combined PET/MRI scanner at inclusion and at 4 and 8 months after the initiation of sonications to monitor the brain metabolism and amyloid levels along with cognitive evaluations. The evolution of cognitive and neuroimaging features was compared to that of a matched sample of control participants taken from the Alzheimer’s Disease Neuroimaging Initiative (ADNI). Results A total of 63 BBB opening procedures were performed in nine subjects. The procedure was well-tolerated. A non-significant decrease in amyloid accumulation at 4 months of − 6.6% (SD = 7.2%) on 18F-florbetapir PET imaging in the sonicated gray matter targeted by the ultrasound transducer was observed compared to baseline in six subjects that completed treatments and who had evaluable imaging scans. No differences in the longitudinal change in the glucose metabolism were observed compared to the neighboring or contralateral regions or to the change observed in the same region in ADNI participants. No significant effect on cognition evolution was observed in comparison with the ADNI participants as expected due to the small sample size and duration of the trial. Conclusions These results demonstrate the safety of ultrasound-based BBB disruption and the potential of this technology to be used as a therapy for AD patients. Research of this technique in a larger clinical trial with a device designed to sonicate larger volumes of tissue and in combination with disease-modifying drugs may further enhance the effects observed. Trial registration ClinicalTrials.gov, NCT03119961

Background Treatment of prostate cancer using endocavitary High Intensity Focused Ultrasound (HIFU) has become more commonplace since the first treatments in the 1990s. The gold standard HIFU strategy to treat prostate cancer is the complete thermal ablation of the entire prostate gland under real-time ultrasound (US) image guidance. A more desirable treatment and the current trend, however, is towards a focal treatment but more accurate and finely tunable thermal lesions are needed along with improved US imaging guidance. In this study, Capacitive Micromachined Ultrasound Transducer (CMUT) technology is being investigated, as they have shown recent promise for US imaging and potential to be used for HIFU therapy. They offer potential advantages over current piezoelectric designs in the context of ultrasound-guided HIFU (USgHIFU) focal therapies. Objective The presented study evaluates the ability of a planar annular array CMUT design to achieve HIFU dynamic focusing and feasibility of generating thermal lesions in biological tissues. Method The proposed CMUT design consists of a 64-element annular array for HIFU delivery with a space in the center that accommodates a high-resolution 256-element linear imaging array. The pressure field simulations of the HIFU portion of the array were performed using the Rayleigh integral method. The bioheat transfer equation was then used to predict lesion formation. The HIFU performances of the proposed CMUT phased-array design were compared to those of the device currently used in the clinic. Partial CMUT prototypes, including the therapeutic part only, were fabricated and experimentally characterized (electromechanical CMUT behavior, ultrasound pressure field distribution and acoustic intensity). Results The planar 64-element annular CMUT design is capable of dynamically focusing a 3 MHz ultrasound beam at distances ranging from 32 to 72 mm, comparable in size and shape to the ones obtained with the clinical device. The simulated ultrasound fields correlated well to experimental measurements. Visual observation and impedance measurements of the CMUT cells allowed direct estimation of the collapse and snapback voltages of the ring-elements. The surface acoustic intensity of the CMUT ring-elements with both AC driving and DC bias voltages can achieve over 6 W/cm2, shown in simulation to be compatible with the generation of thermal lesions. The electro-acoustic efficiency of the CMUT elements increased with increasing DC bias voltages to reach 31%, and remained stable with increasing AC driving voltages. The ultrasound energy could be dynamically focused from this planar CMUT array during several dozen of minutes. Conclusion This work demonstrates the feasibility of utilizing a planar CMUT probe for generating dynamic HIFU focusing and lesioning compatible with the ablation of prostate tissues under endocavitary treatment approach. Future investigations will consist of validating the lesioning capability experimentally both in vitro and in vivo.

The treatment of primary brain tumors, especially malignant gliomas, remains challenging. The failure of most treatments for this disease is partially explained by the blood-brain barrier (BBB), which prevents circulating molecules from entering the brain parenchyma. Ultrasound-induced BBB disruption (US-BBBD) has recently emerged as a promising strategy to improve the delivery of therapeutic agents to brain tumors. A large body of preclinical studies has demonstrated that the association of low-intensity pulsed ultrasound with intravenous microbubbles can transiently open the BBB in a localized manner. The safety of this technique has been assessed in numerous preclinical studies in both small and large animal models. A large panel of therapeutic agents have been delivered to the brain in preclinical models, demonstrating both tumor control and increased survival. This technique has recently entered clinical trials with encouraging preliminary data. In this review, we describe the mechanisms and histological effects of US-BBBD and summarize the preclinical studies published to date. We furthermore provide an overview of the current clinical development and future potential of this promising technology.

Introduction: In contrast to brain metastases, gliomas have few antigen-presenting cells (APCs) in the tumor microenvironment, which may limit T cell effector responses. We hypothesized that: (1) an APC could be modified to express a T cell attracting chemokine that could enhance localized immune activation, and (2) that ultrasound-based blood-brain barrier (BBB) opening would enhance delivery of these APCs at the site of tumor antigens. Methods: Murine F4/80 and CD11c expressing APCs were isolated from the bone marrow of C57BL/6 mice and modified via lentiviral transduction to encode the cDNA of immune cell chemokines CXCL9 or CXCL10 and methionine-deficient green fluorescent protein (co-expression) for cell tagging. C57BL/6 mice were implanted with GL261 cells and treated with 1 x 106 CXCL9 or CXCL10 over-expressing APCs intracranially (i.c.), or intravenously (i.v.) with or without ultrasound-based BBB opening. To determine how the CXCL10 expressing APCs were altering the tumor microenvironment, immune cells were isolated from the glioma implanted hemisphere of all treatment groups and analyzed by multicolor flow cytometry. Results: Transduced APCs produced 3,392 pg/ml and 2,987 pg/ml of CXCL9 or CXCL10, respectively. Mice tolerated the treatments well without any adverse events, behavioral changes, or neurological toxicity. Survival analysis of mice treated with CXCL9 expressing APCs using i.c., or i.v. with and without BBB opening ultrasound showed no significant survival benefit compared with PBS-treated control mice. Mice treated i.c. with CXCL10-expressing APCs had significantly improved survival relative to the PBS control group (p Conclusions: The delivery of CXCL10-secreting APCs to the glioma microenvironment with ultrasound-based BBB opening was superior to delivery with direct i.c. injection. BBB disruption may have triggered more diffuse dispersal of the APCs throughout the tumor microenvironment or positioned these cells in closer proximity to the T cells emigrating from the vascular space into the localized glioma microenvironment. Citation Format: Aria Sabbagh, Kevin Beccaria, Xiaoyang Ling, Anantha Marisetty, Martina Ott, Ling-Yuan Kong, Dexing Fang, Jun Wei, Carole Desseaux, Guillaume Bouchoux, Michael Canney, Alexandre Carpentier, Amy B. Heimberger. Deposition of genetically engineered T cell attracting antigen presenting cells in the glioma microenvironment with low intensity pulsed ultrasound-based blood-brain barrier opening triggers therapeutic responses in preclinical glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1489.

International audience; The blood-spinal cord barrier (BSCB) considerably limits the delivery and efficacy of treatments for spinal cord diseases. The blood-brain barrier can be safely opened with low-intensity pulsed ultrasound when microbubbles are simultaneously administered intravenously. This technique was tested on the BSCB in a rabbit model in this work. Twenty-three segments of spinal cord were sonicated with a 1-MHz unfocused pulsed ultrasound device and compared with non-sonicated segments. BSCB disruption was assessed using Evan's blue dye (EBD) extravasation. Tolerance was assessed by histologic analysis. An increased EBD concentration indicating BSCB disruption was clearly observed in sonicated segments compared with controls (p = 0.004). On one animal, which received 10 sonications, repetitive BSCB disruptions revealed no evidence of cumulative toxicity. BSCB can be disrupted using an unfocused pulsed ultrasound device in combination with microbubbles without neurotoxicity even in case of repeated sonications.

BACKGROUND The blood-brain barrier (BBB) limits penetration of drug therapies to the brain and may account for the limited effectiveness of chemotherapies in patients with primary brain cancer. An implantable ultrasound (US) device, SonoCloud, was developed to temporarily disrupt the BBB in patients with recurrent glioblastoma (rGBM) prior to carboplatin chemotherapy administration to enhance brain drug concentrations. BBB disruption was investigated with a single emitter device, SonoCloud-1 (SC1) and a three-emitter device, SonoCloud-3 (SC3) in a safety and feasibility study in rGBM patients at the Hôpitaux Universitaires La Pitié Salpêtrière in Paris, France (NCT02253212). MATERIAL AND METHODS The SC1 implant consisted of a 1 MHz, 10-mm diameter ultrasound transducer encapsulated in a biocompatible housing while the SC3 consisted of (3) separate SC1 emitters implanted in a triangular pattern to disrupt the BBB over a 3x larger volume. Patients were implanted with the SonoCloud device during tumor debulking or in a dedicated surgical procedure. The devices were activated monthly by connecting the device to an external generator via a transdermal needle. Intravenous injection of SonoVue® microbubbles was performed with device activation to temporarily disrupt the BBB. Magnetic resonance imaging (MRI) was performed after sonications with gadolinium contrast to verify BBB disruption followed by carboplatin infusion at AUC4-6. Patients received treatments until disease progression. RESULTS Between 2014 and 2016, 21 patients were registered for the study and implanted with the SC1; 19 patients received at least one sonication. Six additional patients were implanted and received sonications with the SC3. A total of 89 US sessions were performed to disrupt the BBB - 65 with the SC1 and 24 with the SC3. Treatment-related adverse events observed were transient and manageable. No carboplatin-related neurotoxicity was observed. SC1 patients with no or poor BBB disruption (n=8) visible on MRI had a median progression-free survival (PFS) of 2.73 months, and a median overall survival (OS) of 8.64 months. SC1 patients with clear BBB disruption (n=11) had a median PFS of 4.11 months, and a median OS of 12.94 months. The SC3 device was as well-tolerated as the SC1 device, with SC3 patients receiving between 1–12 monthly sonications. CONCLUSION These results provide the first safety data on the effects of disrupting the BBB in rGBM patients prior to carboplatin chemotherapy using an implantable low intensity pulsed ultrasound device on enlarged areas of brain. This study has now been completed. A new safety study with a larger device, SonoCloud-9, that covers the tumor and surrounding infiltrative regions, has started in France in early 2019 (NCT03744026). Work supported by CarThera and APHP.

Pediatric brain tumors are the most common solid tumor and the first cause of cancer death in childhood, adolescence, and young adulthood. Current treatments are far from optimal in most of these tumors and the prognosis remains dismal for many of them. One of the main causes of the failure of current medical treatments is in part due to the existence of the blood-brain barrier (BBB), which limits drug delivery to tumors. Opening of the BBB with low-intensity pulsed ultrasound (LIPU) has emerged during the last 2 decades as a promising technique for enhancing drug delivery to the brain. In preclinical models, enhanced delivery of a wide range of therapeutic agents, from low-molecular-weight drugs, to antibodies and immune cells, has been observed as well as tumor control and increased survival. This technique has recently entered clinical trials with extracranial and intracranial devices. The safety and feasibility of this technique has furthermore been shown in patients treated monthly for recurrent glioblastoma receiving carboplatin chemotherapy. In this review, the characteristics of the BBB in the most common pediatric brain tumors are reviewed. Then, principles and mechanisms of BBB disruption with ultrasound (US) are summarized and described at the histological and biological levels. Lastly, preclinical studies that have used US-induced BBB opening in tumor models, recent clinical trials, and the potential use of this technology in pediatrics are provided.

Purpose: The blood–brain barrier (BBB) limits the efficacy of drug therapies for glioblastoma (GBM). Preclinical data indicate that low-intensity pulsed ultrasound (LIPU) can transiently disrupt the BBB and increase intracerebral drug concentrations. Patients and Methods: A first-in-man, single-arm, single-center trial (NCT02253212) was initiated to investigate the transient disruption of the BBB in patients with recurrent GBM. Patients were implanted with a 1-MHz, 11.5-mm diameter cranial ultrasound device (SonoCloud-1, CarThera). The device was activated monthly to transiently disrupt the BBB before intravenous carboplatin chemotherapy. Results: Between 2014 and 2016, 21 patients were registered for the study and implanted with the SonoCloud-1; 19 patients received at least one sonication. In 65 ultrasound sessions, BBB disruption was visible on T1w MRI for 52 sonications. Treatment-related adverse events observed were transient and manageable: a transient edema at H1 and at D15. No carboplatin-related neurotoxicity was observed. Patients with no or poor BBB disruption (n = 8) visible on MRI had a median progression-free survival (PFS) of 2.73 months, and a median overall survival (OS) of 8.64 months. Patients with clear BBB disruption (n = 11) had a median PFS of 4.11 months, and a median OS of 12.94 months. Conclusions: SonoCloud-1 treatments were well tolerated and may increase the effectiveness of systemic drug therapies, such as carboplatin, in the brain without inducing neurotoxicity. See related commentary by Sonabend and Stupp, p. 3750

OBJECTIVEOne of the goals in this study was to set up a semiautomatic method to estimate blood-brain barrier disruption obtained in patients with glioblastoma by using an implantable, unfocused, ultrasound device. Another goal was to correlate the probability of significant ultrasound-induced signal enhancement (SUISE) with local acoustic pressure in the brain.METHODSGd-enhanced MR images acquired before and after ultrasound treatments were analyzed prospectively. The image sets were segmented, normalized, and coregistered to evaluate contrast enhancement. The volume of SUISE was calculated with voxels labeled as gray or white matter, in a cylindrical region of interest, and with enhancement above a given threshold. To validate the method, the resulting volumes of SUISE were compared to qualitative grades previously assigned by 3 clinicians for 40 ultrasound treatments in 15 patients. A parametric study was performed to optimize the algorithm prediction of the qualitative grades. The 3D acoustic field in the brain was estimated from measurements in water combined with simulations accounting for ultrasound attenuation in brain and overlaid on each MR image to correlate local acoustic pressure with the probability of SUISE (defined as enhancement > 10%).RESULTSThe algorithm predicted grade 2 or 3 and grade 3 openings with areas under the receiver operating characteristic curve of 0.831 and 0.995, respectively. The probability of SUISE was correlated with local acoustic pressure (R2 = 0.98) and was 3.33 times higher for gray matter than for white matter.CONCLUSIONSAn algorithm for evaluating blood-brain barrier disruption was validated and can be used for future clinical trials to further understand and quantify this technique in humans.Clinical trial registration no.: NCT02253212 (clinicaltrials.gov)

This preliminary study focuses on Capacitive Micro-machined Ultrasound Transducers (CMUTs) for dualmode ultrasound therapy and imaging. A planar probe design is being investigated in the context of Ultrasound-guided High Intensity Focused Ultrasound (USgHIFU) designed for endocavitary HIFU tissue ablation of the prostate gland. The hypothesis is that this CMUT design could offer potential advantages over currently used piezoelectric probes such as ease of miniaturization, increased elements for improved imaging and more control over electronic focusing allowing focal therapy. A planar dual mode CMUT probe consisting of a 3 MHz, 64-element annular array for therapy and 7 MHz, 256-element linear array for imaging were investigated in simulation and experimentally. Numerical models of the pressure field and tissue heating simulations were performed and compared with an existing piezo-based geometrically focused annular array HIFU probe (16 rings) with a hole in the center that possesses a curvilinear imaging array (128 elements). To validate the modelling and the focusing capabilities, pressure field hydrophone measurements were performed. Simulations showed that the planar annular CMUT design could dynamically focus from 3–7cm which matched well with the experimentally obtained pressure field data. Radiation force balance measurements confirmed the desired 5 W/cm2 surface acoustic intensity. This CMUT prototype will require further investigation for determination of the robustness of this technology and the ability to create lesions, yet according to the modelling and experimental validation thus far, this CMUT design shows promise for improved USgHIFU treatment strategies.

Ultrasound in the sub-megahertz range enhances thrombolysis and may be applied transcranially to ischemic stroke patients. The consistency of transcranial insonification needs to be evaluated. Acoustic and thermal simulations based on computed-tomography (CT) scans of 20 patients were performed. An unfocused 120-kHz transducer allowed homogeneous insonification of the thrombus, and positioning based on external landmarks performed similarly to an optimized placement based on CT data. With a weakly focused 500-kHz transducer, the landmark-based positioning underperformed. The predicted inter-patient variation of in situ acoustic pressure was similar with both the 120 and 500-kHz transducers for the optimized placement (18.0-26.4% relative standard deviation). The simulated maximum acoustic pressure in intervening tissues was 2.6 ± 0.6 and 2.0 ± 0.7 times the pressure in the thrombus for the 120-kHz and 500-kHz transducers, respectively. A 1 W/cm(2) insonification of the thrombus caused a 3.8 ± 2.2 °C increase in the bone for the 120-kHz transducer, and a 13.4 ± 3.3 °C increase for the 500-kHz transducer. Contralateral local maxima up to 1.1 times the pressure amplitude in the targeted zone were predicted for the 120-kHz transducer. We established two transducer placement approaches, one based on analysis of a head CT and the other using simple external, visible landmarks. Both approaches allowed consistent insonification of the thrombus.

Background Targeted and triggered release of liposomal drug using heat or ultrasound represents a promising treatment modality able to increase the therapeutic-totoxicity ratio of encapsulated drugs. Purpose To study the ability for high-intensity focused ultrasound to induce liposomal drug release mainly by focused inertial cavitation in vitro and in an animal model. Methods A 1 MHz ultrasound setup has been developed for in vitro and in vivo drug release from a specific liposomal doxorubicin formulation at a target cavitation dose. Results Controlled cavitation at 1 MHz was applied within the tumors 48 hours after liposome injection according to preliminary pharmacokinetic study. A small non-significant therapeutic effect of US-liposomal treatment was observed compared to liposomes alone suggesting no beneficial effect of ultrasound in the current setup. Conclusion The in vitro study provided a suitable ultrasound setup for delivering a cavitation dose appropriate for safe liposomal drug release. However, when converting to an in vivo model, no therapeutic benefit was observed. This may be due to a number of reasons, one of which may be the difficulty in converting in vitro findings to an in vivo model. In light of these findings, we discuss important design features for future studies.

OBJECTIVE: To determine the safety of repeated disruption of the blood-brain barrier (BBB) by low intensity pulsed ultrasound (LIPU) with the Sonocloud(®) device in children with recurrent malignant supra-tentorial brain tumors prior to intra-venous carboplatin chemotherapy. The safety of this procedure is currently being validated in adult patients with recurrent glioblastoma (Carpentier et al. 2016). MATERIAL AND METHODS: This single-center phase I/IIa clinical trial will include 12 to 24 patients aged 5 to 18 years, with recurrent malignant supra-tentorial brain tumors. The Sonocloud(®) device will be implanted into the skull under general anesthesia, during a standard procedure (biopsy or resection). Thanks to a transdermic connexion, sonications will be performed monthly in combination with injection of an ultrasound contrast agent (Sonovue(®)). US parameters will be based on the adult patients study. BBB disruption will be evaluated using multimodal magnetic resonance imaging (MRI). Carboplatin will be injected during the hour following the sonication. Patients will receive a maximum of 6 monthly sonications and will be followed at least for 6 months after the last sonication. EXPECTED RESULTS: The principal criteria will be the absence of grade ≥ 3 neurological toxicity (NCI-CTCAE 4.0 Version, clinical and radiological assessment) linked to the US-induced opening of the BBB after first treatment. Secondary evaluation criterias will include tolerance of repeated sonications, overall survival and progression-free survival, and feasibility of the technique. CONCLUSION: SONOKID is the first clinical trial assessing the feasibility and tolerance of US-induced opening of the BBB in children. SPONSOR Canceropole Ile de France.

Unresectable liver tumors are often treated with interstitial probes that modify tissue temperature, and efficacious treatment relies on image guidance for tissue targeting and assessment. Here, we report the in vivo evaluation of an interstitial applicator with a mechanically oscillating five-element dual-mode transducer. After thoroughly characterizing the transducer, tissue response to high-intensity ultrasound was numerically calculated to select parameters for experimentation in vivo. Using perfused porcine liver, B-mode sector images were formed before and after a 120-s therapy period, and M-mode imaging monitored the therapy axis during therapy. The time-averaged transducer surface intensity was 21 or 27 W/cm (2). Electroacoustic conversion efficiency was maximally 72 +/- 3% and impulse response length was 295 +/- 1.0 ns at -6 dB. The depth of thermal damage measured by gross histology ranged from 10 to 25 mm for 13 insertion sites. For six sites, M-mode data exhibited a reduction in gray-scale intensity that was interpreted as the temporal variation of coagulation necrosis. Contrast ratio analysis indicated that the gray-scale intensity dropped by 7.8 +/- 3.3 dB, and estimated the final lesion depth to an accuracy of 2.3 +/- 2.4 mm. This paper verified that the applicator could induce coagulation necrosis in perfused liver and demonstrated the feasibility of real-time monitoring.

International audience; In some patients at risk of disease recurrence of renal cancers, maximum conservation of the kidney is possible through partial nephrectomy. However, bloodless surgery is difficult to achieve. The article describes an ultrasonic clamp, which optimises energy deposition and monitors lesion development with an echo-based technique. Using this novel apparatus, coagulation necroses have been obtained in vitro on substantial thicknesses (23 to 38 mm) over exposure durations ranging from 10 s to 130 s, and with acoustic intensities of less than 15 W/cm(2) per transducer. When used for coagulation purposes, two transducers situated on opposite arms of the clamp are driven, while for monitoring, only one is used. Lesions are monitored in real time by analysing the echo signal returned by the opposite arm of the clamp. The presence of a lesion is evaluated on the basis of energy changes and echo phase as a function of time. Both kidneys of two pigs (30 to 36 mm thick) were treated in vivo with the clamp, and the partial nephrectomies performed proved to be bloodless.

Thrombo-occlusive disease is a leading cause of morbidity and mortality. In this chapter, the use of ultrasound to accelerate clot breakdown alone or in combination with thrombolytic drugs will be reported. Primary thrombus formation during cardiovascular disease and standard treatment methods will be discussed. Mechanisms for ultrasound enhancement of thrombolysis, including thermal heating, radiation force, and cavitation, will be reviewed. Finally, in-vitro, in-vivo and clinical evidence of enhanced thrombolytic efficacy with ultrasound will be presented and discussed.

We investigated the feasibility of using echo-strain images to visualize the extent of high-intensity ultrasound (US)-induced thermal lesions during their formation. Echo-strain, defined as the relative deformation of the backscattered ultrasonic signal, is due to tissue expansion and to changes in the speed of sound during heating. First, a theoretical framework was developed to predict the influence of these effects on the echo signal. Then, a simulation tool was developed to create simulated echo-strain images in thermal lesions. Finally, experimental echo-strain images were acquired in 10 porcine liver samples in vitro for various exposure durations and ultrasonic intensities (resulting in lesions that extended 3 to 8 mm deep from the surface). For this purpose, radiofrequency (RF) frames were acquired at 8 frames per s while heating. For each consecutive pair of RF frames, an echo-strain image was calculated using standard elastographic processing. The echo-strain images were cumulated and displayed. The experimental echo-strain images were compared with gross pathology. The (isoechoic) lesions were visible both in simulated and in experimental cumulated echo-strain images as apparent expansion areas (tensile echo-strain), whereas surrounding tissues exhibited apparent compression. The tensile echo-strain area underestimated the lesion in simulations, but was representative of the lesion in experiments. High correspondence was found between the lesion depth measured from experimental cumulative echo-strain images ( y ) and from gross pathology ( x ) (Pearson's correlation=0.90, linear regression y = x −0.1 mm, residual error=0 ± 0.9 mm). We hypothesized that significant tissue expansion made the thermal lesions highly visible in the experimental echo-strain images. In two cases, the ultrasonic intensity was too low to induce a lesion, and the corresponding experimental echo-strain images showed no visible lesion. We conclude that cumulative echo-strain images have the potential to monitor the formation of high-intensity US-induced thermal lesions. (E-mail: Souchon@lyon.inserm.fr)

Resume L’application d’ultrasons a des liposomes sonosensibles charges de chimiotherapie offre de nouvelles perspectives dans le traitement du cancer. Dans ce travail, des essais in vitro ont permis de definir un indice de cavitation pour permettre le suivi des traitements. Cet indice permet egalement d’evaluer la sonosensibilite de liposomes prototypes. Des experimentations ont ete conduites chez des rats porteurs de tumeurs sous-cutanees. Le temps de 48 heures apres injection a ete choisi pour l’application des ultrasons. Concernant le traitement, les liposomes ralentissent significativement la dynamique tumorale. Les ultrasons combines aux liposomes semblent contribuer au ralentissement du developpement de la tumeur. Cependant, la grande efficacite des liposomes seuls et la possible faible interaction avec les ultrasons ne permettent pas de garantir que l’effet des ultrasons est significatif.

International audience; The development of endocavitary dual-mode probes is essential for the accurate treatment of many deep seated cancers which require a high imaging resolution and the capacity to selectively treat focal areas in the region of interest. The MULTIP project is aimed at using state-of-art piezoelectric technologies to design dual-mode ultrasonic probes for cancer-foci treatment and monitoring. In order to allow an efficient surgery planning, the technical study has been accompanied by a volume processing study permitting the design of the ultrasonic imaging/therapy process based on high-resolutionhigh-quality MRI images. Several prototypes were designed based on a simulation study and implemented: 1) two successive wide-band dual-mode transducer allowing imaging at high resolution (6 MHz) on a wide field of view, and therapy at 3 MHz with a good transduction efficiency (48% and 70%); 2) a therapy-only transducer matrix adapted to the desired curvature with a high transduction efficiency (70%). Finally, a registration study of MRI volumes on ultrasound volumes has shown that, because of the texture of the ultrasound images, it is more efficient to search at registering the surfaces of the volumes once they have been segmented in each modality, rather than trying to register the two data volumes directly.

The prevalence of stroke worldwide and the paucity of effective therapies have triggered interest in the use of transcranial ultrasound as an adjuvant to thrombolytic therapy. Previous studies have shown that 120 kHz ultrasound enhanced thrombolysis and allowed efficient penetration through the temporal bone. The objective of our study was to develop an accurate finite-difference model of acoustic propagation through the skull based on computed tomography (CT) images. The computational approach, which neglected shear waves, was compared with a simple analytical model including shear waves. Acoustic pressure fields from a two-element annular array (120 and 60 kHz) were acquired in vitro in four human skulls. Simulations were performed using registered CT scans and a source term determined by acoustic holography. Mean errors below 14% were found between simulated pressure fields and corresponding measurements. Intracranial peak pressures were systematically underestimated and reflections from the contralateral bone were overestimated. Determination of the acoustic impedance of the bone from the CT images was the likely source of error. High correlation between predictions and measurements (R(2) = 0.93 and R(2) = 0.88 for transmitted and reflected waves amplitude, respectively) demonstrated that this model is suitable for a quantitative estimation of acoustic fields generated during 40-200 kHz ultrasound-enhanced ischemic stroke treatment.

Interstitial ultrasound applicators can be a minimally invasive alternative for treating targets that are unresectable or are inaccessible by extracorporeal methods. Dual-mode transducers for ultrasound imaging and therapy were developed to address the constraints of a miniaturized applicator and real-time treatment monitoring. We propose an original treatment strategy that combines ultrasound imaging and therapy using a dual-mode transducer rotating at 8 revolutions per second. Real-time B-mode imaging was interrupted to emit high-intensity ultrasound over a selected therapy aperture. A full 360 degrees image was taken every 8th rotation to image the therapy aperture. Numerical simulations were performed to study the effect of rotation on tissue heating, and to study the effect of the treatment sequence on transducer temperature. With the time-averaged transducer surface intensity held at 12 W/cm(2) to maintain transducer temperature below 66 degrees C, higher field intensities and deeper lesions were produced by narrower therapy apertures. A prototype system was built and tested using in vitro samples of porcine liver. Lesions up to 8 mm were produced using a time-averaged transducer surface intensity of 12 W/cm(2) applied for a period of 240 s over a therapy aperture of 40 degrees. Apparent strain imaging of the therapy aperture improved the contrast between treated and spared tissues, which could not be differentiated on B-mode images. With appropriate limits on the transducer output, real-time imaging and deep thermal ablation are feasible and sustainable using a rotating dual-mode transducer.

The purpose of this study was to define and validate an inertial cavitation dose (CD) based on the detection of broadband noise, designed to monitor ultrasound-mediated drug release from liposomes. The validation consists of using the terephthalate dosimeter to quantify by fluorescence measurements the extent of hydroxyl radical ( OH) production during inertial cavitation. Sonication of samples was performed using tone bursts (pulse repetition frequency (PRF): 10 Hz −1 kHz, duty cycle (dc): 5–25%, Isppa: 4100–12,200 W/cm 2 ) generated by a 1 MHz focused transducer. Three sets of ultrasound parameters with different PRF and dc were selected to be more precisely compared. Results demonstrated an excellent correlation between OH radical production and CD for each set of parameters, but significant differences in hydroxyl radical levels were observed among the sets of parameters. The results were compared with other studies, and the same tendency of variation with pulse duration was demonstrated. Results also showed that the CD was not distorted by peak intensity variations and was a much more reliable indicator than sonication time. Consequently, one validated parameter was selected to monitor drug release from two liposome formulations, and compare their ultrasound sensitivity.

Medical imaging is a vital component of high intensity focused ultrasound (HIFU) therapy, which is gaining clinical acceptance for tissue ablation and cancer therapy. Imaging is necessary to plan and guide the application of therapeutic ultrasound, and to monitor the effects it induces in tissue. Because they can transmit high intensity continuous wave ultrasound for treatment and pulsed ultrasound for imaging, dual-mode transducers aim to improve the guidance and monitoring stages. Their primary advantage is implicit registration between the imaging and treatment axes, and so they can help ensure before treatment that the therapeutic beam is correctly aligned with the planned treatment volume. During treatment, imaging signals can be processed in real-time to assess acoustic properties of the tissue that are related to thermal ablation. Piezocomposite materials are favorable for dual-mode transducers because of their improved bandwidth, which in turn improves imaging performance while maintaining high efficiency for treatment. Here we present our experiences with three dual-mode transducers for interstitial applications. The first was an 11-MHz monoelement designed for use in the bile duct. It had a 25 × 7.5 mm 2 aperture that was cylindrically focused to 10 mm. The applicator motion was step-wise rotational for imaging and therapy over a 360°, or smaller, sector. The second transducer had 5-elements, each measuring 3.0 × 3.8 mm 2 for a total aperture of 3.0 × 20 mm 2 . It operated at 5.6 MHz, was cylindrically focused to 14 mm, and was integrated with a servo-controlled oscillating probe designed for sector imaging and directive therapy in the liver. The last transducer was a 5-MHz, 64-element linear array designed for beam-formed imaging and therapy. The aperture was 3.0 × 18 mm 2 with a pitch of 0.280 mm. Characterization results included conversion efficiencies above 50%, pulse-echo bandwidths above 50%, surface intensities up to 30 W / cm 2 , and axial imaging resolutions to 0.2 mm. The second transducer was evaluated in vivo using porcine liver, where coagulation necrosis was induced up to a depth of 20 mm in 120 s. B-mode and M-mode images displayed a hypoechoic region that agreed well with lesion depth observed by gross histology. These feasibility studies demonstrate that the dual-mode transducers had imaging performance that was sufficient to aid the guidance and monitoring of treatment, and could sustain high intensities to induce coagulation necrosis in vivo.

Interstitial probes have been shown as effective devices to deliver high-intensity ultrasound therapy. Here, cylindrically-focused dual-mode transducers with either one or 5-elements were characterized, and a monoelement probe was evaluated in vitro. In therapy mode, the transducers were maximally efficient (or =70%) at 5.6 MHz with surface intensities up to 20 W/cm(2). In imaging mode, fractional bandwidths were 46% and 50+/-4% (ave+/-std) for the monoelement and 5-element transducers respectively. Axial and lateral resolutions were 0.5 mm and 1.0 mm, respectively, for both transducers as measured with a point scatterer in the focal plane. After characterization, the oscillating probe was used to image and apply therapy to porcine liver. B-mode images over a 140 degrees sector were formed before and after therapy, which was applied for 90 s at each of 5 angles separated by 20 degrees (e.g. -40 degrees , -20 degrees, 0 degrees, 20 degrees, 40 degrees) to form a composite lesion. Transducer surface intensity was 18 W/cm(2). Therapy was interrupted at 125 ms intervals to collect pulse/echo data along the therapy axes. Data were displayed in real-time as an M-mode image to monitor therapy. B-mode images adequately represented the liver tissue. M-mode image data agreed well with the formation of lesions in the liver.

Deep‐seated tumours can be treated by high intensity interstitial ultrasound using a miniature transducer brought in contact with the target. A dual mode transducer was designed for performing simultaneous imaging. Two therapeutic strategies are envisaged in this study. First, the transducer can be rotated step‐by‐step and therapy interrupted periodically for acquiring echo lines. In a second approach, fast rotation of the transducer allows 2D‐imaging and therapy is applied transiently over a small angular sector. Simultaneous imaging and therapy were performed in vitro with the designed transducer (2.5×7.5mm2, cylindrically focused at 10mm, 11MHz) following these two approaches. Up to 10mm deep lesions were obtained with the immobile transducer emitting an intensity of 17W/cm2 for 120s. M‐mode images were constructed on‐line during treatment. For the second strategy, the transducer rotated at a 7 rotations per second pace. Each turn, imaging was interrupted for ≈0.02s in order to perform therapy at insta...

Although it is possible to both measure temperature and monitor the damage caused by HIFU treatment sessions with magnetic resonance imaging, a number of studies have been carried out proposing an ultrasound solution. Conventional B-mode ultrasound seems to be valid, but cannot be applied to all organs, and the hyperechogenic areas often observed during HIFU treatments do not necessarily and exactly correspond to the thermal damage. The aim of this paper is to summarize recent work in this field. The various techniques have been divided into three main groups, characterizing the lesions by measuring the acoustic properties, mechanical properties or vascularization.

High intensity focused ultrasound has been shown to be very effective for thermally ablating localised tumours through extracorporeal or endocavitary routes. More recently, miniature interstitial ultrasound probes have been investigated for the treatment of deep-seated tumours. All treatments must be performed under imaging guidance in order to bring the probe focus precisely on the targeted site and to monitor the treatment on-line. Imaging objectives and constraints may vary depending on the targeted organs, access routes, and treatment process. MRI provides high contrast images and is presently recognized as the gold standard for thermal mapping. MRI compatible high intensity ultrasound transducers have been designed for this purpose. Conventional B mode imaging is efficient at visualising scattering gas bodies. Many other ultrasound techniques have been tested and are likely to be further developed in order to make use of the large potential of ultrasound for targeting and monitoring the treatments. More recently it has been shown that dual mode transducers can be used for both imaging and therapeutic objectives. This again opens new possibilities for extracorporeal, endo-cavitary and interstitial applications. In all the cases, the therapeutic transducer has to be designed with the chosen imaging modality in mind

It has been shown that a very deep and selective interstitial thermal therapy can be performed using a rotating flat miniature ultrasound transducer. This heating modality would be significantly improved by implementing imaging and monitoring. The space limitations in interstitial applications require combining imaging and therapy on the same transducer. In conventional endoscopic ultrasound imaging, a miniprobe can be positioned inside the lumen of an organ and fast rotation of a single element transducer enables real time peripheral imaging of the lumen. Based on this idea, we proposed an original therapeutic strategy. Imaging can be interrupted for a short period of time corresponding to an angle Deltaalpha in order to apply high intensity ultrasound for thermal ablation. First, theoretical simulations were conducted. A 1D thermal model of the transducer demonstrated that this operating mode is not critical for the transducer. Simulations using the BioHeat Transfer Equation showed that Deltaalpha=pi/4 induced efficient therapy. An applicator was built in order to test this treatment strategy. The active element consisted in a 2.5 times 7.5 mm2 dual mode single element transducer operating at a frequency of 11 MHz. The applicator was driven in rotation at 7-turns per second pace. Experiments were performed in vitro on porcine liver. Ultrasound images were reconstructed and the size of the -6 dB point spread function at focal distance was 0.55 mm times 0.25 mm. The electronics for performing the therapeutic strategy considered above was set up. Up to 8 millimeters-deep thermal lesions were obtained with Deltaalpha=pi/4 and peak intensity of 110 W/cm 2. This work demonstrated that thermal lesions can be performed with a fast-rotating dual mode transducer. This approach enabled real-time image-guided therapy

Echogenic liposomes can be used as a vector for co-encapsulation of the thrombolytic drug rt-PA and microbubbles. These agents can be acoustically activated for localized cavitation-enhanced drug delivery. The objective of our study was to characterize thrombolytic efficacy and sustained cavitation nucleation and activity from rt-PA-loaded echogenic liposomes (t-ELIP). A spectrophotometric method was used to determine the enzymatic activity of rt-PA released from t-ELIP and compared to unencapsulated rt-PA. The thrombolytic efficacy of t-ELIP, rt-PA alone, or rt-PA and the commercial contrast agent Definity® exposed to sub-megahertz ultrasound was determined in an in vitro flow model. Ultraharmonic (UH) emissions from stable cavitation were recorded during insonation. Both UH emissions and thrombolytic efficacy were significantly greater for rt-PA and Definity® over either rt-PA alone or t-ELIP with equivalent rt-PA loading. Furthermore, the enzymatic activity of t-ELIP was significantly lower than free rt-PA. When the dosage of t-ELIP was adjusted to compensate for the lack of enzymatic activity, similar thrombolytic efficacy was found for t-ELIP and Definity® and rt-PA. However, sustained ultraharmonic emissions were not observed for t-ELIP in the flow phantom.

Adjuvant ultrasound exposure improves rtPA thrombolysis in stroke patients. Transmission of 120-kHz ultrasound through the temporal bone is efficient but exhibits skull-dependent distortion and reflection. Numerical models of acoustic propagation through human skull based on computed tomography (CT) data have been developed. The objective of our study was to validate a finite-difference model of transcranial ultrasound quantitatively. The acoustic fields from a two-element annular array (120 kHz and 60 kHz) were acquired in four ex-vivo human skulls with a calibrated hydrophone (10 kHz-800 kHz frequency range). The spatial distributions of the acoustomechanical properties of each skull were obtained from CT scans and used for simulations. Predicted acoustic fields and waveform shapes were compared with corresponding hydrophone measurements and were in good agreement. Transmitted wave amplitudes were systematically underestimated (14%) and reflected wave amplitudes were overestimated (30%). The acoustic im...

1350-4177/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ultsonch.2010.12.007 DOI of original article: 10.1016/j.ultsonch.2010.07.009 ⇑ Corresponding author at: Universite de Lyon, Lyon F-69003, France. E-mail address: lucie.somaglino@yahoo.fr (L. Somaglino). 4th paragraph (line 35 of the Section), the line ‘‘off-time duration induced a decrease of hydroxyl radical production’’ should read as follows

Previous in vitro studies conducted in our group have shown the feasibility of monitoring drug release from liposomes by an inertial acoustic cavitation index. We currently report in vivo experiments utilizing the cavitation index in combined treatment of AT2 Dunning tumor grafted rats with focused ultrasound and liposomal doxorubicin. Sixty‐three rats were allocated into seven groups: control, low level ultrasound treatment, high level ultrasound treatment, free doxorubicin+high level ultrasound treatment, and liposomal doxorubicin, liposomal doxorubicin+low level ultrasound treatment, and liposomal doxorubicin+high level ultrasound treatment. Based on pharmacokinetic studies, it was decided to apply ultrasound to the tumor 48 h after drug injection. An experimental setup was built to perform repeatable and rapid sonications of tumors monitored by the cavitation index. Tumor growth was assessed for a period of 35 days after tumor inoculation. Results showed that liposomal doxorubicin significantly slowed...

Drug release from liposomes can be activated by ultrasound and inertial cavitation is assumed to be the main involved mechanism. Broadband noise is known as a good indicator for such cavitation. The feasibility of assessing the extent of drug release by acoustic noise measurements has been investigated. A 1.17‐MHz focused transducer (50‐mm diameter and 50‐mm focal length) was excited with tone‐bursts (duty cycle: 10%–100%, PRF: 100 Hz–10 kHz, and Ispta < 800 W/cm2). Emitted noise was registered with a broadband needle hydrophone and bandpass filtered between the fundamental and the first harmonic frequency for assessing the instantaneous inertial cavitation activity. A cavitation index (CI) was computed by integrating the filtered signal over time. This index was validated experimentally using terephthalate oxidized to fluorescent hydroxyterephthalate under the action of free hydroxyl radicals generated by inertial cavitation. The hydroxyterephthalate fluorescence and the CI were well correlated (R2≃0.92)...

Miniature dual‐mode transducers can be used for minimally invasive treatment of deep‐seated tumors. While in contact with the tissue, the transducer guides and monitors localized necrosis. Here, an oscillating 5‐element piezo‐composite transducer was characterized, and then evaluated in vitro using porcine liver. Each element was 3.0 x 3.8 mm2 with a geometric cylindrical focus of 14 mm. The transmit frequency was determined by the maximal electro‐acoustic efficiency, 65%, which was found at 5.6 MHz. The transmit‐receive impulse response was 400 ns long at ‐6 dB, and the ‐6 dB fractional bandwidth, centered at 5.6 MHz, was 30%. Axial and lateral resolution measured with a 0.1 mm diameter wire was 0.5 mm and 2.0 mm, respectively. For therapy, all elements radiated simultaneously, and for imaging, independently. Treatment was performed at increments of 20° to form a composite volume of necrosis. At each angle, ultrasound was applied for 60 s at a transducer surface intensity of 15 W/cm2. Pulse‐echo data wer...

Aggressive treatment of localized hepatic metastases, by surgery or other means, was proven to be a viable strategy for improving the prognoses of many patients. In that context, thermal ablation by high intensity ultrasound was proposed and used in clinics. However, for treating deep‐seated tumors and in most cases, radiofrequency and cryotherapy probes are applied interstitially. Interstitial ultrasound applicators were proposed as an intermediate solution. The treatment can be focused, deeper than with other physical agents, and the transducer can eventually both treat and image tissues. In our experience, two approaches were investigated: percutaneous and intratissular, or endo vascular. The active element was a miniature flat transducer operating at a frequency of 5 MHz, for a satisfactory tradeoff between beam penetration and energy absorption. In vivo trials on a porcine model demonstrated that both procedures are minimally invasive and that large thermal lesions, up to 20 mm deep, can be obtained....

Echogenic liposomes (ELIP), that can encapsulate both recombinant tissue-type plasminogen activator (rt-PA) and microbubbles, are under development to improve the treatment of thrombo-occlusive disease. However, the enzymatic activity, thrombolytic efficacy, and stable cavitation activity generated by this agent has yet to be evaluated and compared to another established ultrasound-enhanced thrombolytic scheme. A spectrophotometric method was used to compare the enzymatic activity of the rt-PA incorporated into ELIP (t-ELIP) to that of rt-PA. An in vitro flow model was employed to measure the thrombolytic efficacy and dose of ultraharmonic emissions from stable cavitation for 120-kHz ultrasound exposure of three treatment schemes: rt-PA, rt-PA and the perfluorocarbon-filled microbubble Definity(®), and t-ELIP. The enzymatic activity of rt-PA incorporated into t-ELIP was 28 % that of rt-PA. Thrombolytic efficacy of t-ELIP or rt-PA and Definity(®) was equivalent when the dose of t-ELIP was adjusted to produce comparable enzymatic activity. Sustained bubble activity was nucleated from Definity but not from t-ELIP exposed to 120-kHz ultrasound. These results emphasize the advantages of encapsulating a thrombolytic and the importance of incorporating an insoluble gas required to promote sustained, stable cavitation activity.