Ultrasound Disruption Research Articles

Caveolin Delivered by Ultrasound-Mediated Microbubble Destruction Prevents Endothelial Cell Proliferation.

The nitric oxide synthase (eNOS) is an important regulator of vascular homeostasis. eNOS is modulated by intracellular mechanisms that include protein-protein interaction with Caveolin-1 (Cav). Cav binds to and impairs eNOS activation reducing vascular permeability and angiogenesis. Blocking of eNOS by Cav has been proposed as therapeutic antiangiogenic approach. However, the efficient and controlled delivery of the peptide requires to be solved. The effect of antennapedia (AP)-Cav loaded into microbubbles (MBs) and delivered by ultrasound-mediated microbubble destruction (UMMD) into brain endothelial cells (bEnd.3 cells) was evaluated on NO production using DAF2-DA, cell migration assessed by the wound healing assay, cell proliferation with BrdU, and ex-vivo angiogenesis in rat aortic rings. An enhanced inhibitory effect of AP-Cav was observed on cells treated with UMMD. MBs and ultrasound disruption delivery of AP-Cav increased acetylcholine-induced NO release, wound healing, cell proliferation, and angiogenesis inhibition on bEnd.3 cells, compared to free AP-Cav administration. We demonstrated that the delivery of Cav via AP-Cav-loaded MBs and UMMD may be an administration method for Cav that would increase its therapeutic potential by enhancing efficacy and cellular specificity.

Abstract 195: A focused ultrasound blood brain barrier disruption model to test the influence of tight junction genes to treat brain tumors

Abstract A major hindrance to advances in the care of patients with malignant gliomas is the presence of the blood brain barrier (BBB) and blood-brain tumor barrier (BBTB) that greatly restricts drug access from the plasma to the tumor cells. Bubble-assisted Focused Ultrasound (BAFUS) has proven effective in opening the BBB for treatment of glial tumors in adults and pediatric cases. BAFUS has been previously shown to disrupt noninvasively, selectively, and transiently the BBB in small animals in vivo. However, there is a lack of an in vitro preclinical model suitable for testing the genetic determinants of endothelial cell tight junction integrity and vulnerability to the physical disruption. Our BBB organ-on-chip platform will enable precision medicine of brain cancers through identifying patient-specific parameters by which to open the BBB allowing use of drugs and drug combinations otherwise unsuitable. We intend to sequence these in vitro models to verify that the genotype (alleles/SNPs) of tight junction proteins contribute to BBB structure and integrity. To initiate this effort, we report the development of an ultrasound transparent organ-on-chip model populated by iPSC-derived endothelial cells (iPSC-EC) co-cultured with astrocytes. Western blot, immunocytochemistry, permeability, and transelectrical endothelial resistance (TEER) studies all convey expression of key EC proteins and marked barrier integrity. Further benchmarking of device-ultrasound interactions, successful iPSC differentiation, tight junction formation, and fabrication of nanobubbles and their assistance in ultrasound BBB disruption will be presented. Efforts are underway to analyze nine characteristic BBB tight junction genes from WGS data to determine associations between iPSC-EC genotype and phenotype. Citation Format: Jayashree Iyer, Adam Akkad, Nanyun Tang, Sen Peng, Michael Berens, Frederic Zenhausern, Jian Gu. A focused ultrasound blood brain barrier disruption model to test the influence of tight junction genes to treat brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 195.

EXTH-17. A FOCUSED ULTRASOUND BLOOD BRAIN BARRIER DISRUPTION MODEL TO TEST THE INFLUENCE OF TIGHT JUNCTION GENES TO TREAT BRAIN TUMORS

Abstract Treating primary or metastatic tumors in the brain (glioblastomas, melanoma, lung cancer, breast cancer) proves challenging by virtue of the protective function of the blood brain barrier (BBB). The tight junction proteins (TJPs) binding the specialized endothelial cells of the BBB largely contribute to the limited permeability of cancer-therapeutic drugs. In both preclinical and clinical models, low intensity focused ultrasound (LIFU) coupled with microbubbles has been proven to safely and transiently open the BBB. Despite this method being established, potential genetic influences on the durability and vulnerability of tight junctions to LIFU have not been elucidated, nor have the determinants of tight junction repair post LIFU been thoroughly investigated. We report the development of an ultrasound transparent organ-on-chip model populated by iPSC-derived endothelial cells (iPSC-EC) co-cultured with astrocytes. We aim to probe the contributions of various tight junction genes to barrier integrity along with the subsequent protein topology involved in reassembly post ultrasound. Thus, this model serves to determine parameters for ultrasound disruption for precision opening of the BBB. The BBB-On-Chip was successfully fabricated and assembled with an optimized technique that has an 80% yield of leak-free devices, with stable cavitation post nanobubble injection. Furthermore, Western blots show expression of claudin-5, a key TJP, in our iPSC-ECs. We have also demonstrated by confocal microscopy that another component of the TJP complex, ZO-1, can be visualized at iPSC-derived cell junctions. Further benchmarking of device-ultrasound interactions, successful iPSC differentiation, tight junction formation, and fabrication of nanobubbles and their assistance in ultrasound BBB disruption will be presented. Efforts are underway to characterize the contributions of tight junction genes and their variations to the integrity and disruption of the BBB.

Encapsulated calcium carbonate suspensions: A drug delivery vehicle sensitive to ultrasound disruption

A calcium carbonate suspension, encapsulated within particles of calcium alginate hydrogel, is proposed as a drug delivery device susceptible to ultrasound disruption. Spheres approximately 1mm in diameter were prepared by the coaxial airflow method from mixtures of 1% sodium alginate (m/v) and each of 50%, 75% and 100% calcium carbonate (m/v) in distilled water. This product was subjected to cycles of 85 Watt ultrasound in 1 second on/off bursts via a lab sonication system until fully disintegrated, a process requiring between 8 and 20 minutes depending upon initial calcium carbonate concentrations. The spheres subjected to vortex did not demonstrate any signs of mechanical degeneration after 30 minutes. Before use as a model implant, further work is required to develop a method of drying the particles to make them impermeable to drug diffusion before the time of their disruption with ultrasound.

Corrigendum: Water Science and Technology 81 (6), 1231-1241: Improving biogas production performance of dairy activated sludge via ultrasound disruption prior to microwave disintegration, M. Al Ramahi, G. Keszthelyi-Szabó and S. Beszédes, https://iwaponline.com/wst/article/81/6/1231/74038/Improving-biogas-production-performance-of-dairy?searchresult = 1.

Corrigendum: Water Science and Technology 81 (6), 1231-1241: Improving biogas production performance of dairy activated sludge via ultrasound disruption prior to microwave disintegration, M. Al Ramahi, G. Keszthelyi-Szabó and S. Beszédes, https://iwaponline.com/wst/article/81/6/1231/74038/Improving-biogas-production-performance-of-dairy?searchresult = 1.

Improving biogas production performance of dairy activated sludge via ultrasound disruption prior to microwave disintegration.

In this study, ultrasound disruption was employed to enhance the efficiency of microwave disintegration of dairy sludge. Results revealed that ultrasound specific energy input of 1,500 kJ/kg TS was found to be optimum with limited cell lysis at the end of the disruption phase. Biodegradability study suggested an enhancement in suspended solids reduction (16%) and biogas production (180 mL/gVS) in floc disrupted (deflocculated) samples when compared to sole microwave pretreatment (8.3% and 140 mL/gVS, respectively). Energy assessment to attain the 15% optimum solubilization revealed a positive net production of 26 kWh per kg sludge in deflocculated samples compared to 18 kWh in flocculated (sole microwave) samples. Thus, ultrasound disruption prior to microwave disintegration of dairy sludge was considered to be a feasible pretreatment technique.

Enzyme activity of waste activated sludge extracts.

Wastewater treatment and generated biological sludge provide an alternative source of enzymes to conventional industrial production methods. Here, we present a protocol for extracting enzymes from activated sludge using ultrasonication and surfactant treatment. Under optimum conditions, ultrasound disruption of activated sludge gave recovery rates of protease and cellulase enzymes equivalent to 63.1% and ∼100%, respectively. The extracting of enzymes from activated sludge represents a potentially significant, high-value, resource recovery option for biological sludge generated by municipal wastewater treatment.

Localized difference in blood–brain barrier permeability of rat brain after focused ultrasound induced disruption

Localized difference in blood–brain barrier permeability of rat brain after focused ultrasound induced disruption

Science to Practice: Can Focused Ultrasound Disruption of the Blood-Brain Barrier Improve Malignant Brain Tumor Treatment Outcomes?

A relatively novel combination treatment for malignant brain tumors that includes focused ultrasound has been shown to improve tumor response and treatment outcome in a preclinical mouse model. This method directly addresses the great need for better treatments for this lethal disease. However, there are substantial technologic hurdles that must be addressed before clinical efficacy will be known. Unfortunately, few of these questions can be readily answered outside the clinical environment. Although it would be enormously challenging, a phase I clinical trial seems feasible and needed to determine the clinical value of this technique.

Differences in soluble COD and ammonium when applying ultrasound to primary, secondary and mixed sludge.

Ultrasound treatment is often applied to enhance the anaerobic digestion of sludge. Optimal conditions for organic matter solubilisation of primary, secondary and mixed sludge were assessed by implementing ultrasound disruption at different specific energies (from 3,500 to 21,000 kJ/kgTS). The variation in soluble chemical oxygen demand (sCOD) and ammonium nitrogen (NH4+-N) was monitored following the treatment, and after a subsequent fermentation (24 h, 37 °C). The effect of the treatment was clearly more pronounced in secondary sludge than in the other types of sludge. Relatively minimal values in solubility were found when applying ultrasound at different energies depending on the sludge (3,500-7,000 kJ/kgTS in primary sludge and 10,500-14,000 kJ/kgTS in secondary sludge). This minimal value was not so noticeable in mixed sludge. The addition of inoculum was not required after ultrasound disruption in order to perform the subsequent fermentation. After this final stage, no general pattern in terms of sCOD was observed. Increases and decreases were conditioned by the coverage of the ultrasound irradiation; NH4+-N values increased notably during the fermentation.

Bubble nucleation in lipid bilayers: A mechanism for low frequency ultrasound disruption

Recent experiments have shown that low frequency ultrasound (LFUS) induces leakage from lipid vesicles. However, the mechanism by which LFUS disrupts the lipid bilayer structure is not clear. In this paper we develop a theoretical model to test the possibility that gas molecule partitioning from the aqueous media into the lipid bilayer core can lead to the nucleation of microscale gas bubbles. If those can, indeed, form, then their presence in the lipid bilayer and interactions with an ultrasound field can cause bilayer disruption and leakage. The model derived here for the nucleation of stable bubbles accounts for the ‘surface tension’ that the lipid bilayer exerts on the bubble, a result of the associated disruption of the lipid packing. The model predicts that the probability of bubble nucleation is highly sensitive to the bilayer thickness, and largely insensitive to the bilayer phase. The probability of stable bubble formation is shown to correlate with experimentally measured sensitivity of lipid bilayers to LFUS, suggesting that membrane disruption may be due to embedded bubbles that nucleated in the bilayer.

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB(1-5). The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size(6). FUS induced BBB disruption (BBBD) is temporary and reversible(4) and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB(2,5,7), there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Blood-Brain Barrier: Real-time Feedback-controlled Focused Ultrasound Disruption by Using an Acoustic Emissions–based Controller

To determine if focused ultrasound disruption of the blood-brain barrier (BBB) can be safely controlled by using real-time modulation of treatment pressures on the basis of acoustic emissions from the exposed microbubbles. All experiments were performed with the approval of the institutional animal care committee. Transcranial focused ultrasound (551.5 kHz, 10-msec bursts, 2-Hz pulse repetition frequency, 2 minute sonication) in conjunction with circulating microbubbles was applied in 86 locations in 27 rats to disrupt the BBB. Acoustic emissions captured during each burst by using a wideband polyvinylidene fluoride hydrophone were analyzed for spectral content and used to adjust treatment pressures. Pressures were increased incrementally after each burst until ultraharmonic emissions were detected, at which point the pressure was reduced to a percentage of the pressure required to induce the ultraharmonics and was maintained for the remainder of the sonication. Disruption was evaluated at contrast material-enhanced T1-weighted magnetic resonance (MR) imaging. Mean enhancement was calculated by averaging the signal intensity at the focus over a 3 × 3-pixel region of interest and comparing it with that in nonsonicated tissue. Histologic analysis was performed to determine the extent of damage to the tissue. Statistical analysis was performed by using Student t tests. For sonications resulting in BBB disruption, the mean peak pressure was 0.28 MPa ± 0.05 (standard deviation) (range, 0.18-0.40 MPa). By using the control algorithm, a linear relationship was found between the scaling level and the mean enhancement on T1-weighted MR images after contrast agent injection. At a 50% scaling level, mean enhancement of 19.6% ± 1.7 (standard error of the mean) was achieved without inducing damage. At higher scaling levels, histologic analysis revealed gross tissue damage, while at a 50% scaling level, no damage was observed at high-field-strength MR imaging or histologic examination 8 days after treatment. This study demonstrates that acoustic emissions can be used to actively control focused ultrasound exposures for the safe induction of BBB disruption.

Focused ultrasound disruption of the blood-brain barrier: a new frontier for therapeutic delivery in molecular neurooncology

Recent advances in molecular neurooncology provide unique opportunities for targeted molecular-based therapies. However, the blood-brain barrier (BBB) remains a major limitation to the delivery of tumor-specific therapies directed against aberrant signaling pathways in brain tumors. Given the dismal prognosis of patients with malignant brain tumors, novel strategies that overcome the intrinsic limitations of the BBB are therefore highly desirable. Focused ultrasound BBB disruption is emerging as a novel strategy for enhanced delivery of therapeutic agents into the brain via focal, reversible, and safe BBB disruption. This review examines the potential role and implications of focused ultrasound in molecular neurooncology.

Enzyme Reaction in the Pores of CaCO3 Particles upon Ultrasound Disruption of Attached Substrate‐Filled Liposomes

Enzyme Reaction in the Pores of CaCO₃ Particles upon Ultrasound Disruption of Attached Substrate‐Filled Liposomes

The impact of standing wave effects on transcranial focused ultrasound disruption of the blood–brain barrier in a rat model

Microbubble-mediated disruption of the blood–brain barrier (BBB) for targeted drug delivery using focused ultrasound shows great potential as a therapy for a wide range of brain disorders. This technique is currently at the pre-clinical stage and important work is being conducted in animal models. Measurements of standing waves in ex vivo rat skulls were conducted using an optical hydrophone and a geometry dependence was identified. Standing waves could not be eliminated through the use of swept frequencies, which have been suggested to eliminate standing waves. Definitive standing wave patterns were detected in over 25% of animals used in a single study. Standing waves were successfully eliminated using a wideband composite sharply focused transducer and a reduced duty cycle. The modified pulse parameters were used in vivo to disrupt the BBB in a rat indicating that, unlike some other bioeffects, BBB disruption is not dependent on standing wave conditions. Due to the high variability of standing waves and the inability to correctly estimate in situ pressures given standing wave conditions, attempts to minimize standing waves should be made in all future work in this field to ensure that results are clinically translatable.

Application of High-Intensity Ultrasounds to Control the Size of Whey Proteins Particles

In this paper, we reported a new method to prepare whey protein microparticles via high-intensity ultrasound disruption. Particles morphology was characterized by confocal microscopy, and their size and distribution were analyzed by light scattering technique. Starting whey protein isolate (WPI) exhibited changes in size and distribution according to its concentration. For WPI, 7.5% (w/w) mean size was 0.7 µm, and upon sonication at ambient temperature, the size was reduced up to 0.2 µm showing the particles a rounded morphology. Sonication at room temperature of gelled WPI led to particles with sizes between 0.1 and 10 µm which had a tendency to flocculate. When WPI was submitted to sonication under heating at protein denaturation temperature, different effects were observed according to protein concentration. The particle size was reduced for the lowest WPI concentration (7.5 wt.%), did not change at 9 wt.%, but strongly increased at 12 wt.%, in comparison with the untreated sample. WPI particles of desired size in the micron range may be obtained either by sonication of gelled WPI or by sonication under heating at denaturation temperature by controlling processing variables.

Extraction of isoflavonoids from Pueraria by combining ultrasound with microwave vacuum

This study proposes a new method to quickly extract and dry isoflavonoids from Pueraria Lobata Ohwi (Pueraria) by combining ultrasound and microwave technologies. The time required to extract isoflavonoids at comparable levels of production by ultrasound disruption was 20 times shorter than by conventional reflux extraction. Moreover, test results for drying the extracted substance from Pueraria show that the microwave-vacuum method is 10 times faster than the conventional two-step vacuum approach. Finally, different instrumental analyses of isoflavonoids obtained from Pueraria using the proposed new method show that extraction by ultrasound disruption and microwave-vacuum drying affects neither the structure nor the composition of the extracted substance.

Quantitative Measurement of Ultrasound Disruption of Polymer-Shelled Microbubbles

The goal of this study was to assess the threshold of disruption and subsequent time-course of acoustic response of four experimental nitrogen-filled polymer-shelled microbubbles. Using an in vitro measurement system, a sequence of low-amplitude detection pulses measured the change in echo caused by a high-amplitude disruption pulse on a dilute suspension of bubbles. Detection pulses were transmitted 0.5 ms before disruption and between 1 and 200 ms after disruption. Separate transducers, aligned confocally and orthogonally, were used to transmit and receive bubble echoes. After disruption, all agents demonstrated a transient increase in scattered power. Above the disruption threshold, highly echogenic, nonlinear scatterers were observed. Their echoes slowly disappeared after disruption with median decay times from 7.4 to 13.6 ms, calculated by fitting to a mono-exponential decay. This is consistent with a process wherein the shell is disrupted, releasing the gas and generating free gas bubbles, which cause high-amplitude nonlinear scattering followed by relatively slow diffusion of the gas into solution. This picture has been observed optically with single bubbles and differs from the concept of “stimulated acoustic emission” from disrupted bubbles. (E-mail: burns@sri.utoronto.ca)