Characterization of Bioeffects on Endothelial Cells under Acoustic Droplet Vaporization

Gas embolotherapy is achieved by locally vaporizing microdroplets through acoustic droplet vaporization, which results in bubbles that are large enough to occlude blood flow directed to tumors. Endothelial cells, lining blood vessels, can be affected by these vaporization events, resulting in cell injury and cell death. An idealized monolayer of endothelial cells was subjected to acoustic droplet vaporization using a 3.5-MHz transducer and dodecafluoropentane droplets. Treatments included insonation pressures that varied from 2 to 8 MPa (rarefactional) and pulse lengths that varied from 4 to 16 input cycles. The bubble cloud generated was directly dependent on pressure, but not on pulse length. Cellular damage increased with increasing bubble cloud size, but was limited to the bubble cloud area. These results suggest that vaporization near the endothelium may impact the vessel wall, an effect that could be either deleterious or beneficial depending on the intended overall therapeutic application.     

Characterization of Acoustic Droplet Vaporization for Control of Bubble Generation Under Flow Conditions

This study investigated the manipulation of bubbles generated by acoustic droplet vaporization (ADV) under clinically relevant flow conditions. Optical microscopy and high-frequency ultrasound imaging were used to observe bubbles generated by 2-MHz ultrasound pulses at different time points after the onset of ADV. The dependence of the bubble population on droplet concentration, flow velocity, fluid viscosity and acoustic parameters, including acoustic pressure, pulse duration and pulse repetition frequency, was investigated. The results indicated that post-ADV bubble growth spontaneously driven by air permeation markedly affected the bubble population after insonation. The bubbles can grow to a stable equilibrium diameter as great as twice the original diameter in 0.5–1 s, as predicted by the theoretical calculation. The growth trend is independent of flow velocity, but dependent on fluid viscosity and droplet concentration, which directly influence the rate of gas uptake by bubbles and the rate of gas exchange across the wall of the semipermeable tube containing the bubbles and, hence, the gas content of the host medium. Varying the acoustic pressure does not markedly change the formation of bubbles as long as the ADV thresholds of most droplets are reached. Varying pulse duration and pulse repetition frequency markedly reduces the number of bubbles. Lengthening pulse duration favors the production of large bubbles, but reduces the total number of bubbles. Increasing the PRF interestingly provides superior performance in bubble disruption. These results also suggest that an ADV bubble population cannot be assessed simply on the basis of initial droplet size or enhancement of imaging contrast by the bubbles. Determining the optimal acoustic parameters requires careful consideration of their impact on the bubble population produced for different application scenarios.     

Bubble dynamics in boiling histotripsy

Boiling histotripsy is a non-invasive, cavitation-based ultrasonic technique which uses a number of millisecond pulses to mechanically fractionate tissue. Though a number of studies have demonstrated the efficacy of boiling histotripsy for fractionation of solid tumours, treatment monitoring by cavitation measurement is not well studied because of the limited understanding of the dynamics of bubbles induced by boiling histotripsy. The main objectives of this work are to (a) extract qualitative and quantitative features of bubbles excited by shockwaves and (b) distinguish between the different types of cavitation activity for either a thermally or a mechanically induced lesion in the liver. A numerical bubble model based on the Gilmore equation accounting for heat and mass transfer (gas and water vapour) was developed to investigate the dynamics of a single bubble in tissue exposed to different High Intensity Focused Ultrasound fields as a function of temperature variation in the fluid. Furthermore, ex vivo liver experiments were performed with a passive cavitation detection system to obtain acoustic emissions. The numerical simulations showed that the asymmetry in a shockwave and water vapour transport are the key parameters which lead the bubble to undergo rectified growth at a boiling temperature of 100°C. The onset of rectified radial bubble motion manifested itself as (a) an increase in the radiated pressure and (b) the sudden appearance of higher order multiple harmonics in the corresponding spectrogram. Examining the frequency spectra produced by the thermal ablation and the boiling histotripsy exposures, it was observed that higher order multiple harmonics as well as higher levels of broadband emissions occurred during the boiling histotripsy insonation. These unique features in the emitted acoustic signals were consistent with the experimental measurements. These features can, therefore, be used to monitor (a) the different types of acoustic cavitation activity for either a thermal ablation or a mechanical fractionation process and (b) the onset of the formation of a boiling bubble at the focus in the course of HIFU exposure.     

Predicting the acoustic response of a microbubble population for contrast imaging in medical ultrasound

Although the behavior of a bubble in an acoustic field has been studied extensively, few theoretical treatments to date have been applied to simulate the acoustic response of a real population of variably sized microbubbles in a finite-width sound beam. In this paper, we present a modified Trilling equation for single bubble dynamics that has been solved numerically for different conditions. Radiated waveforms from a large number of such bubbles are combined, reflecting their size distribution and location and the shape of a real acoustic beam. The resulting time-domain pressure waveforms can be compared with those obtained experimentally. The dependence of second-harmonic radiation on incident focal amplitude at different frequencies is presented. This model is particularly suited to the study of interaction between a medical ultrasound beam and microbubble contrast agents in aqueous media.     

Effects of acoustic insonation parameters on ultrasound contrast agent destruction

Ultrasound contrast agents (UCAs) are used to enhance the acoustic backscattered intensity of blood and thereby assist the assessment of blood perfusion. Characterization of UCA destruction provides important information for the design of contrast-assisted perfusion imaging. High-speed optical observation of single microbubble destruction during acoustic insonation has been performed in previous studies. The results identified that pressure, center frequency and transmission phase have significant effects on the fragmentation threshold. We proposed an acoustic-based experiment method to demonstrate the relationship between different acoustic exposure conditions and the degree of UCA destruction. The method also provides a simple and convenient way to determine the microbubble destruction threshold. The experiments introduced three insonation parameters, including acoustic pressure (0 to 1 MPa), pulse frequency (1, 2.25, 5 and 7.5 MHz) and pulse length (1 to 10 cycles). The term of surviving percentage (SP) was proposed to represent the ratio of UCA backscattered power with and without acoustic insonation. The results showed that the SP decreased with decreasing pulse frequency, but with increasing transmission acoustic pressure and pulse length. In addition, there was an exponential relationship between SP and acoustic pressure, and thus the UCA destruction pressure threshold could be predicted from the fitted exponential curve. The results also show that the degree of UCA destruction was not related to mechanical index (MI). Potential applications of this method include UCA high-resolution destruction/replenishment imaging model, microbubble cavitation, sonoporation in drug delivery and gene therapy.     

Modeling of Microbubble-Enhanced High-Intensity Focused Ultrasound

Heat enhancement at the target in a high intensity focused ultrasound (HIFU) field is investigated by considering the effects of the injection of microbubbles in the vicinity of the tumor to be ablated. The interaction between the bubble cloud and the HIFU field is investigated using a 3-D numerical model. The propagation of non-linear ultrasonic waves in the tissue or in a phantom medium is modeled using the compressible Navier–Stokes equations on a fixed Eulerian grid, while the microbubbles dynamics and motion are modeled as discrete singularities, which are tracked in a Lagrangian framework. These two models are coupled to each other such that both the acoustic field and the bubbles influence each other. The resulting temperature rise in the field is calculated by solving a heat transfer equation applied over a much longer time scale. The compressible continuum part of the model is validated by conducting axisymmetric HIFU simulations without microbubbles and comparing the pressure and temperature fields against available experiments. The coupled Eulerian–Lagrangian approach is then validated against existing experiments conducted with a phantom tissue. The bubbles are distributed randomly in a 3-D fashion inside a cylindrical volume, while the background acoustic field is assumed axisymmetric. The presence of microbubbles modifies the ultrasound field in the focal region and significantly enhances heat deposition. The various mechanisms through which heat deposition is increased are then examined. Among these effects, viscous damping of the bubble oscillations is found to be the main contributor to the enhanced heat deposition. The effects of the initial void fraction in the cloud are then sought by considering the changes in the attenuation of the primary ultrasonic wave and the modifications of the enhanced heat deposition in the focal region. It is observed that although high bubble void fractions lead to increased heat deposition, they also cause significant pre-focal heating because of acoustic shielding. The effects of the microbubble cloud size and its location in the focal region are studied, and the effects of these parameters in altering the temperature rise and the location of the temperature peak are discussed. It is found that concentrating the bubbles adjacent to the focus and farther away from the acoustic source leads to effective heat deposition. Finally, the presence of a shell at the bubble surface, as in contrast agents, is seen to reduce heat deposition by restraining bubble oscillations.     

Over-Pressure Suppresses Ultrasonic-Induced Drug Uptake

Ultrasound (US) is used to enhance and target delivery of drugs and genes to cancer tissues. The present study further examines the role of acoustic cavitation in US-induced permeabilization of cell membranes and subsequent drug or gene uptake by the cell. Rat colon cancer cells were exposed to ultrasound at various static pressures to examine the hypothesis that oscillating bubbles, also known as cavitating bubbles, permeabilize cells. Increasing pressure suppresses bubble cavitation activity; thus, if applied pressure were to reduce drug uptake, cell permeabilization would be strongly linked to bubble cavitation activity. Cells were exposed to 476 kHz pulsed ultrasound at average intensities of 2.75 W/cm² and 5.5 W/cm² at various pressures and times in an isothermal chamber. Cell fractions with reversible membrane damage (calcein uptake) and irreversible damage (propidium iodide uptake) were analyzed by flow cytometry. Pressurization to 3 atm nearly eliminated the biological effect of US in promoting calcein uptake. Data also showed a linear increase in membrane permeability with respect to insonation time and intensity. This research shows that US-mediated cell membrane permeability is likely linked to cavitation bubble activity. (E-mail: pitt@byu.edu)     

Quantification of microbubble destruction of three fluorocarbon-filled ultrasonic contrast agents

The assessment of myocardial blood velocity using ultrasonic contrast agents is based on the premise that the vast majority of contrast microbubbles within a myocardial region can be destroyed by an acoustic pulse of sufficient magnitude. Determination of the period of time after destruction that a region of myocardium needs to reperfuse may be used to assess myocardial blood velocity. In this study, we investigated the acoustic pressure sensitivity of three solutions of intravenous fluorocarbon-filled contrast agents and the magnitude of acoustic pulse required to destroy the contrast agent microbubbles. A novel tissue-mimicking phantom was designed and manufactured to investigate the relationships between mean integrated backscatter, incident acoustic pressure and number of frames of insonation for three fluorocarbon-filled contrast agents (Definity(R), Optison(R), and Sonazoid(R), formerly NC100100). Using a routine clinical ultrasound (US) scanner (Acuson XP-10), modified to allow access to the unprocessed US data, the contrast agents were scanned at the four acoustic output powers. All three agents initially demonstrated a linear relationship between mean integrated backscatter and number of frames of insonation. For all three agents, mean integrated backscatter decreased more rapidly at higher acoustic pressures, suggesting a more rapid destruction of the microbubbles. In spite of the fact that there was no movement of microbubbles into or out of the beam, only the results from Definity(R) suggested that a complete destruction of the contrast agent microbubbles had occurred within the total duration of insonation in this study.     

Coupling agents in therapeutic ultrasound: acoustic and thermal behavior

OBJECTIVE: To compare transmissivity data and thermal behavior of 4 coupling media. DESIGN: Experimental. SETTING: Postgraduate rehabilitation program in Brazil. SPECIMENS: Four coupling media: gel, mineral oil, white petrolatum, and degassed water. INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: The transmission, attenuation, reflection coefficient, and acoustic impedance of gel, mineral oil, white petrolatum, and degassed water were measured with a density measurement cell. The temperature variation in the therapeutic ultrasound transducer was measured with a thermocouple. RESULTS: The transmissivity data showed that the water and gel presented the highest transmission coefficient, the lowest reflection, and an attenuation coefficient and acoustic impedance close to that of the skin. The thermal data revealed the highest heating in the transducer during the insonation with white petrolatum and mineral oil, resulting from the thermal conductivity features of each medium. CONCLUSIONS: Transmissivity data obtained showed that water and gel present the best acoustic features. In ultrasound therapy, with the direct contact technique using thin layers of coupling agents, any product may be used, because the effect of the attenuation coefficient does not play a significant role when layers are as thin as those used in this experiment.     

Contrast agent stability: a continuous B-mode imaging approach.

The stability of contrast agents in suspensions with various dissolved gas levels has not been reported in the literature. An in vitro investigation has been carried out that studied the combined effect of varying the acoustic pressure along with degassing the suspension environment. In this study, the contrast agents were introduced into suspensions with different oxygen concentration levels, and their relative performance was assessed in terms of decay rate of their backscatter echoes. The partial pressures of oxygen in those solutions ranged between 1.5 and 26 kPa. Two IV and one arterial contrast agents were used: Definity, Quantison, and Myomap. It was found that Quantison and Myomap released free bubbles at high acoustic pressure that also dissolved faster in degassed suspensions. The backscatter decay for Definity did not depend on the air content of the suspensions. The destruction of bubbles was dependent on acoustic pressure. Different backscatter performance was observed by different populations of bubbles of the last two agents. The physical quantity of "overall backscatter" (OB) was defined as the integral of the decay rate over time of the backscatter of the contrast suspensions, and improved significantly the understanding of the behaviour of the agents. A quantitative analysis of the backscatter properties of contrast agents using a continuous imaging approach was difficult to achieve. This is due to the fact that the backscatter in the field of view is representative of a bubble population affected by the ultrasound (US) field, but this bubble population is not representative of the contrast suspension in the whole tank. Single frame insonation is suggested to avoid the effects of decay due to the ultrasonic field, and to measure a tank-representative backscatter. The definition of OB was useful, however, in understanding the behaviour of the agents.     

Ultrasonically induced gas bubble production in agar based gels: Part I. Experimental investigation

Macroscopically visible gas bubbles can be produced in an agar based gel by irradiation with either continuous or pulsed ultrasound at frequencies from 0.75 to 3.0 MHz. The variation in the number of bubbles formed with frequency, acoustic pressure, pulse length, duty cycle, and temperature closely resembles that seen in vivo. Furthermore, the acoustic pressure required to initiate bubble formation is also close to that required in vivo. It has been observed that alterations in the concentration and pH of the gels can have a profound effect on the nature and quantity of bubbles. This suggests that not only is this gel model suitable for the representation of the macroscopic features of bubble formation in vivo, but can be used to gain information about the preexisting bubble nuclei. Based on the experimental results obtained it can be suggested that for peak negative acoustic pressures of up 1 MPa (equivalent, for a plane travelling sinusoidal wave, to a time averaged intensity of 30 W/cm2) bubble formation can be avoided by the use of high frequencies, short pulse lengths and long duty cycles.     

Modest enhancement of ultrasound-induced mutations in V79 cells in vitro.

V79 cells were insonated (1 MHz, 35 W/cm2, 2 min continuous wave) under temperature conditions previously reported to cause a large increase in acoustic cavitation. The goal was to increase the ease of detecting ultrasound-induced mutations. The data supported the hypothesis that a 3 degrees C gas-equilibration period followed by insonation at 37 degrees C resulted in a detectable increase in resistance to 6-thioguanine with 6 trials per regimen.     

Temperature Dependent Behavior of Ultrasound Contrast Agents

Recent interest in ultrasound contrast agents (UCAs) as tools for quantitative imaging and therapy has increased the need for accurate characterization. Laboratory investigations are frequently undertaken in a water bath at room temperature; however, implications for in vivo applications are not presented. Acoustic investigation of a bulk suspension of SonoVue (Bracco Research, Geneva, Switzerland) was made in a water bath at temperatures of 20–45 °C. UCA characteristics were significantly affected by temperature, particularly between 20 and 40 °C, leading to an increase in attenuation from 1.7–2.5 dB, respectively (p = 0.002) and a 2-dB increase in scattered signal over the same range (p = 0.05) at an insonation pressure of 100 kPa. Optical data supported the hypothesis that a temperature-mediated increase in diameter was the dominant cause, and revealed a decrease in bubble stability. In conclusion, measurements made at room temperature require careful interpretation with regard to behavior in vivo. (E-mail: h.mulvana@imperial.ac.uk)     

Cavitation induced by asymmetric, distorted pulses of ultrasound: a biological test

Prediction of the response of gaseous microbubbles to ultrasonic waves is complicated by the finite-amplitude distortion associated with large amplitude acoustic fields. Typical finite-amplitude pulses in medical applications consist of a sharp positive spike followed by a smaller, slowly varying negative pressure. In previous theoretical studies it was found that: (a) the peak-positive pressure is a very poor index of bubble response; (b) the peak-negative pressure typically underestimates the bubble response; (c) a better predictor of bubble response is the pressure amplitude of the fundamental in a Fourier series expansion of the distorted pulse. It is reported here that the killing of Drosophila larvae exposed to pulsed, symmetric, sinusoidal fields and to pulsed, asymmetric, distorted fields is consistent with these predictions.     

Multifocus Thermal Strain Imaging Using a Curved Linear Array Transducer for Identification of Lipids in Deep Tissue

Thermal strain imaging (TSI) is an ultrasound-based imaging technique intended primarily for diseases in which lipid accumulation is the main biomarker. The goal of the research described here was to successfully implement TSI on a single, commercially available curved linear array transducer for heating and imaging of organs at a deeper depth. For an effective temperature rise of the tissue over a large area, which is key to TSI performance, an innovative multifocus beamforming approach was applied. This yielded a heating area from 32 to 96 mm in the axial direction and –7 to +7 mm in the lateral direction. The pressure fields generated from simulation were in agreement with pressure fields measured with the hydrophone. TSI with safe acoustic power identified with high contrast a rubber inclusion and liposuction fat tissue embedded in a gelatin block.     

Problems in establishing exposure criteria for medical ultrasound

This paper described the need for adequate quantification of dosage by identifying the most relevant physical factors so that a proper risk assessment can be made for any treatments involving exposure to ultrasound. As a demonstration an acoustic quantity was plotted for a continuous ultrasound field and called Q representing the square of the pressure amplitude. It was shown that this quantity Q would be relevant to biological effects of ultrasound in several ways, such as in the derivation of radiation pressure which has relevance to insonation of cell cultures. Q is also applicable to temperature calculations and to cavitation which would occur most vigorously when the pressure amplitude is greatest. It was pointed out that the suitability of parameters chosen to quantify ultrasound exposure will depend on both the feasibility of measuring it, and its relevance to biological effects and safety.     

Cavitation-enhanced ultrasound thermal therapy by combined low- and high-frequency ultrasound exposure

This paper demonstrates a novel approach for enhancing ultrasound-induced heating by the introduction of acoustic cavitation using simultaneous sonication with low- and high-frequency ultrasound. A spherical focused transducer (566 or 1155 kHz) was used to generate the thermal lesions, and a low-frequency planar transducer (40 or 28 kHz) was used to enhance the bubble activity. Ex vivo fresh porcine muscles were used as the target of ultrasound ablation. The emitted signals and the signals backscattered from the bubble activity were also recorded during the heating process by a PVDF-type needle hydrophone, and thermocouples were inserted to measure temperatures. Compared with the lesions formed by a single focused transducer, the size of the lesions generated by this approach were as much as 140% larger along the axial direction and 200% larger along the radial direction for combined 566- and 40-kHz sonication. They were 47% and 66% larger along the axial and radial directions, respectively, for combined 1155- and 28-kHz sonication. Cavitation activities enhanced by low-frequency ultrasound were confirmed by the presence of subharmonics in the spectrum and temperature increase as a result of increased tissue absorption. These observations imply that cavitation-enhanced lesions can be generated without reducing the penetration ability; they also show the advantage of producing larger and more uniform thermal lesions by multiple sonications. This technique provides an easy and effective way to achieve cavitation-enhanced heating, and may be useful for generating large and deep-seated thermal lesions.     

The experimental investigation of ultrasonic properties for a sonicated contrast agent and its application in biomedicine

The ultrasonic properties of a promising ultrasound (US) contrast agent, named SDA (sonicated dextrose albumin) are reported in this paper. SDA is a suspension of stable microencapsulated gas bubbles with average diameter 2.0 microm prepared from sonicated dextrose albumin. The ultrasonic linear and nonlinear parameters, such as acoustic velocity, sound attenuation and acoustic nonlinearity parameter B/A of SDA, as a function of its bubble concentration from 1.0 x 10(7) to 2.05 x 10(8) microbubbles/mL in the frequency range of 2-6 MHz are measured in vitro. The sound attenuation coefficients over 2-6 MHz are linearly proportional to the bubble concentration and frequency. It is important to point out that the acoustic nonlinearity parameter B/A for SDA has a very large value that nonlinearly increases with the increase of bubble concentration.     

Synthesis and Characterization of Transiently Stable Albumin-Coated Microbubbles via a Flow-Focusing Microfluidic Device

We describe a method for synthesizing albumin-shelled, large-diameter (>10 μm), transiently stable microbubbles using a flow-focusing microfluidic device (FFMD). The microfluidic device enables microbubbles to be produced immediately before insonation, thus relaxing the requirements for stability. Both reconstituted fractionated bovine serum albumin (BSA) and fresh bovine blood plasma were investigated as shell stabilizers. Microbubble coalescence was inhibited by the addition of either dextrose or glycerol and propylene glycol. Microbubbles were observed to have an acoustic half-life of approximately 6 s. Microbubbles generated directly within a vessel phantom containing flowing blood produced a 6.5-dB increase in acoustic signal within the lumen. Microbubbles generated in real time upstream of in vitro rat aortic smooth muscle cells under physiologic flow conditions successfully permeabilized 58% of the cells on insonation at a peak negative pressure of 200 kPa. These results indicate that transiently stable microbubbles produced via flow-focusing microfluidic devices are capable of image enhancement and drug delivery. In addition, successful microbubble production with blood plasma suggests the potential to use blood as a stabilizing shell.