Ultrasound-mediated disruption of cell membranes. II. Heterogeneous effects on cells

Ultrasound has been shown to reversibly and irreversibly disrupt membranes of viable cells through a mechanism believed to involve cavitation. Because cavitation is both temporally and spatially heterogeneous, flow cytometry was used to identify and quantify heterogeneity in the effects of ultrasound on molecular uptake and cell viability on a cell-by-cell basis for suspensions of DU145 prostate cancer and aortic smooth muscle cells exposed to varying peak negative acoustic pressures (0.6-3.0 MPa). exposure times (120-2,000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent. Cell-to-cell heterogeneity was observed at all conditions studied and was classified into three subpopulations: nominal uptake (NUP), low uptake (LUP), and high uptake (HUP) populations. The average number of molecules within each subpopulation was generally constant: 10(4)-10(5) molecules/cell in NUP, approximately 10(6) molecules/cell in LUP, and approximately 10(7) molecules/cell in HUP. However, the fraction of cells within each subpopulation showed a strong dependence on both acoustic pressure and exposure time. Varying pulse length produced no significant effect. The distribution of cells among the three subpopulations correlated with acoustic energy exposure, which suggests that energy exposure may govern the ability of ultrasound to induce bioeffects by a nonthermal mechanism.     

Ultrasound-mediated disruption of cell membranes. I. Quantification of molecular uptake and cell viability

Ultrasound-mediated drug delivery is a nonchemical, nonviral, and noninvasive method for targeted transport of drugs and genes into cells. Molecules can be delivered into cells when ultrasound disrupts the cell membrane by a mechanism believed to involve cavitation. This study examined molecular uptake and cell viability in cell suspensions (DU145 prostate cancer and aortic smooth muscle cells) exposed to varying peak negative acoustic pressures (0.6-3.0 MPa), exposure times (120-2000 ms), and pulse lengths (0.02-60 ms) in the presence of Optison (1.7% v/v) contrast agent. With increasing pressure and exposure time, molecular uptake of a marker compound, a calcein, increased and approached equilibrium with the extra cellular solution, while cell viability decreased. Varying pulse length produced no significant effect. All viability and molecular uptake measurements collected over the broad range of ultrasound conditions studied correlated with acoustic energy exposure. This suggests that acoustic energy exposure may be predictive of ultrasound's nonthermal bioeffects.     

Ultrasound-induced cell lysis and sonoporation enhanced by contrast agents.

The enhancement of ultrasound-induced cell destruction, lysis, and sonoporation in low cell concentration suspensions (2 x 10(5)/mL) by the presence of contrast agents (gas bubble to cell ratio = 230) was demonstrated using cervical cancer cells (HeLa S3) suspensions containing micron-size denatured albumin microspheres filled with air (Albunex) or octafluoropropane (Optison). The suspensions were insonificated by 2-MHz continuous or tone burst ultrasound in near field. The spatial peak-pressure amplitude was 0.2 MPa. The enhancement of cell destruction due to Optison was shown to be much higher than that due to Albunex for similar bubble concentration and ultrasound conditions. For tone burst exposures, significant lysis and sonoporation only occurred in the presence of a contrast agent. The majority of the bioeffects observed occurred in the first 5 min of exposure. The relationship between the enhancement of bioeffects and duty cycle of tone burst ultrasound appears to indicate that both stable gas spheres of contrast agents and cavitation nuclei created by the disruption of the gas spheres play a significant role in causing the bioeffects.     

A comparison of the fragmentation thresholds and inertial cavitation doses of different ultrasound contrast agents

Contrast bubble destruction is important in several new diagnostic and therapeutic applications. The pressure threshold of destruction is determined by the shell material, while the propensity for of the bubbles to undergo inertial cavitation (IC) depends both on the gas and shell properties of the ultrasound contrast agent (UCA). The ultrasonic fragmentation thresholds of three specific UCAs (Optison, Sonazoid, and biSpheres), each with different shell and gas properties, were determined under various acoustic conditions. The acoustic emissions generated by the agents, or their derivatives, characteristic of IC after fragmentation, was also compared, using cumulated broadband-noise emissions (IC "dose"). Albumin-shelled Optison and surfactant-shelled Sonazoid had low fragmentation thresholds (mean = 0.13 and 0.15 MPa at 1.1 MHz, 0.48 and 0.58 MPa at 3.5 MHz, respectively), while polymer-shelled biSpheres had a significant higher threshold (mean = 0.19 and 0.23 MPa at 1.1 MHz, 0.73 and 0.96 MPa for thin- and thick-shell biSpheres at 3.5 MHz, respectively, p<0.01). At comparable initial concentrations, surfactant-shelled Sonazoid produced a much larger IC dose after shell destruction than did either biSpheres or Optison (p<0.01). Thick-shelled biSpheres had the highest fragmentation threshold and produced the lowest IC dose. More than two and five acoustic cycles, respectively, were necessary for the thin- and thick-shell biSpheres to reach a steady-state fragmentation threshold.     

Evaluation of the properties of daughter bubbles generated by inertial cavitation of preformed microbubbles

Inertial cavitation (IC) of the preformed microbubbles is being investigated for ultrasound imaging and therapeutic applications. However, microbubbles rupture during IC, creating smaller daughter bubbles (DBs), which may cause undesired bioeffects in the target region. Thus, it is important to determine the properties of DBs to achieve controllable cavitation activity for applications. In this study, we theoretically calculated the dissolution dynamics of sulfur hexafluoride bubbles. Then, we applied a 1-MHz single tone burst with different peak negative pressures (PNPs) and pulse lengths (PLs), and multiple 5-MHz tone bursts with fixed acoustic conditions to elicit IC of the preformed SonoVue microbubbles and scattering of DBs, respectively. After the IC and scattering signals were received by a 7.5-MHz transducer, time- and frequency-domain analysis was performed to obtain the IC dose and scattering intensity curve. The theoretical dissolution curves and measured scattering intensity curves were combined to determine the effect of the incident pulse parameters on the lifetime, mean radius and distribution range of DBs. Increased PNP reduced the lifetime and mean size of the DBs population and narrowed the size distribution. The proportion of small DBs (less than resonance size) increased from 36.83% to 85.98% with an increase in the PNP from 0.6 to 1.6 MPa. Moreover, increased PL caused a shift of the DB population to the smaller bubbles with shorter lifetime and narrower distribution. The proportion of small bubbles increased from 25.74% to 95.08% as the PL was increased from 5 to 100 µs. Finally, increased IC dose caused a smaller mean size, shorter lifetime and narrower distribution in the DB population. These results provide new insight into the relationship between the incident acoustic parameters and the properties of DBs, and a feasible strategy for achieving controllable cavitation activity in applications.     

Energy balance of high-energy stable acoustic cavitation within dual-frequency sonochemical reactor

The acoustic cavitation bubble as an open energetic system is the seat of conversion of various forms of energy accompanying the bubble oscillation. The energy conversion would explain specific dynamical, thermal and kinetical behaviors. In the present paper, the energy balance related to a stable bubble irradiated by dual-frequency field is simulated numerically and interpreted in accordance with the phenomena occurring inside it. The study particularly focuses on the comparison of the energetic behavior of high-energy stable cavitation with bubbles that are non-active in sonochemistry, submitted to couples of 35, 140, 300 and 515 kHz. The simulation results revealed that pressure forces work is the major energetic input during the bubble oscillation lifetime, while the main energetic loss comes from heat transfer by diffusion and enthalpy loss accompanying water condensation. Besides, high rates of condensation of water molecules and low amounts of accumulated energy inside the bubble volume were identified as the key factors preventing the achievement of the sonochemical activity threshold.     

The acoustic emissions of cavitation bubbles in stretched vortices

Pairs of unequal strength, counter-rotating vortices were produced in order to examine the inception, dynamics, and acoustic emission of cavitation bubbles in rapidly stretching vortices. The acoustic signatures of these cavitation bubbles were characterized during their inception, growth, and collapse. Growing and collapsing bubbles often produced a sharp, broadband, pop sound. The spectrum of these bubbles, and the peak resonant frequency can generally be related to quiescent flow bubble dynamics and corresponding resonant frequencies. However, some elongated cavitation bubbles produced a short tonal burst, or chirp, with frequencies on the order of a few kilohertz. Theses frequencies are too low to be related to resonant frequencies of a bubble in a quiescent flow. Instead, the frequency content of the acoustic signal during bubble inception and growth is related to the volumetric oscillations of the bubble while it interacted with vortical flow that surrounds the bubble (i.e., the resonant frequency of the vortex-bubble system). A relationship was determined between the observed peak frequency of the oscillations, the highly stretched vortex properties, and the water nuclei content. It was found that different cavitation spectra could relate to different flow and fluid properties and therefore would not scale in the same manner.     

Nonlinear dynamics and acoustic emissions of interacting cavitation bubbles in viscoelastic tissues

The cavitation-mediated bioeffects are primarily associated with the dynamic behaviors of bubbles in viscoelastic tissues, which involves complex interactions of cavitation bubbles with surrounding bubbles and tissues. The radial and translational motions, as well as the resultant acoustic emissions of two interacting cavitation bubbles in viscoelastic tissues were numerically investigated. Due to the bubble–bubble interactions, a remarkable suppression effect on the small bubble, whereas a slight enhancement effect on the large one were observed within the acoustic exposure parameters and the initial radii of the bubbles examined in this paper. Moreover, as the initial distance between bubbles increases, the strong suppression effect is reduced gradually and it could effectively enhance the nonlinear dynamics of bubbles, exactly as the bifurcation diagrams exhibit a similar mode of successive period doubling to chaos. Correspondingly, the resultant acoustic emissions present a progressive evolution of harmonics, subharmonics, ultraharmonics and broadband components in the frequency spectra. In addition, with the elasticity and/or viscosity of the surrounding medium increasing, both the nonlinear dynamics and translational motions of bubbles were reduced prominently. This study provides a comprehensive insight into the nonlinear behaviors and acoustic emissions of two interacting cavitation bubbles in viscoelastic media, it may contribute to optimizing and monitoring the cavitation-mediated biomedical applications.     

Selective aggregation by ultrasonic standing waves through gas nuclei on the particle surface

Gas nuclei in water are usually too small to be directly observed. They will grow into bubbles under the negative pressure, which is called cavitation (heterogeneous cavitation). In this study, the gas nuclei in the hydrophilic and hydrophobic silica particle suspension were investigated using the transient cavitation threshold measured by a high-intensity focused ultrasound (HIFU). The transient cavitation bubbles were also observed by a high-speed camera. The results showed that the nuclei only exist on the surface of hydrophobic particles. Furthermore, the aggregation experiments revealed that the aggregates were only formed in the hydrophobic silica suspension by ultrasonic standing waves (USW) at 200 kHz. This distinct difference was mainly due to the formation of gas nuclei on hydrophobic silica particles, which grew and coalesced into stable bubbles under the 200 kHz USW. The aggregation process in suspension was observed by a CCD camera. Moreover, the cavitation thresholds and acoustic radiation forces were analyzed to explain the mechanism of the acoustic aggregation. This study showed a very promising acoustic method for the selective aggregation of hydrophobic particles, which might be efficiently used in the mineral separation industry.     

A dual passive cavitation detector for localized detection of lithotripsy-induced cavitation in vitro

A passive cavitation detector (PCD) identifies cavitation events by sensing acoustic emissions generated by the collapse of bubbles. In this work, a dual passive cavitation detector (dual PCD), consisting of a pair of orthogonal confocal receivers, is described for use in shock wave lithotripsy. Cavitation events are detected by both receivers and can be localized to within 5 mm by the nature of the small intersecting volume of the focal areas of the two receivers in association with a coincidence detection algorithm. A calibration technique, based on the impulse response of the transducer, was employed to estimate radiated pressures at collapse near the bubble. Results are presented for the in vitro cavitation fields of both a clinical and a research electrohydraulic lithotripter. The measured lifetime of the primary growth-and-collapse of the cavitation bubbles increased from 180 to 420 microseconds as the power setting was increased from 12 to 24 kV. The measured lifetime compared well with calculations based on the Gilmore-Akulichev formulation for bubble dynamics. The radiated acoustic pressure 10 mm from the collapsing cavitation bubble was measured to vary from 4 to 16 MPa with increasing power setting; although the trends agreed with calculations, the predicted values were four times larger than measured values. The axial length of the cavitation field correlated well with the 6-dB region of the acoustic field. However, the width of the cavitation field (10 mm) was significantly narrower than the acoustic field (25 mm) as bubbles appeared to be drawn to the acoustic axis during the collapse. The dual PCD also detected signals from "rebounds," secondary and tertiary growth-and-collapse cycles. The measured rebound time did not agree with calculations from the single-bubble model. The rebounds could be fitted to a Rayleigh collapse model by considering the entire bubble cloud as an effective single bubble. The results from the dual PCD agreed well with images from high-speed photography. The results indicate that single-bubble theory is sufficient to model lithotripsy cavitation dynamics up to time of the main collapse, but that upon collapse bubble cloud dynamics becomes important.     

超声空化及其声流结构实验研究*

超声空化及其声流效应在医学、化工和能源等领域得到广泛应用。本文采用高速摄像和粒子图像测速系统分别研究了超声场下的空化形态和声流场结构的时空演化规律。实验研究了50W,100W,200W和250W等四种不同输入功率对18kHz的超声变幅杆附近空化及其声流场的影响。研究结果表明：（1）在变幅杆下端面处观察到由大量空化气泡均匀分布组成的倒置锥形空泡结构,并且锥形空泡结构为稳态流动结构。（2）在超声变幅杆附近产生了两种不同的声流形式,第一种是变幅杆底端的射流型声流,第二种是变幅杆两侧的回旋流。此外,通过研究空泡与声流场中最大速度点之间的空间对应关系,发现声流是因为空泡流动带动而产生的。（3）空间位置和输入功率能显著影响射流型声流的流场结构,但是对回旋流的影响十分微弱。     

Effect of polymer surface activity on cavitation nuclei stability against dissolution

The persistence of acoustic cavitation in a pulsed wave ultrasound regime depends upon the ability of cavitation nuclei, i.e., bubbles, to survive the off time between pulses. Due to the dependence of bubble dissolution on surface tension, surface-active agents may affect the stability of bubbles against dissolution. In this study, measurements of bubble dissolution rates in solutions of the surface-active polymer poly(propyl acrylic acid) (PPAA) were conducted to test this premise. The surface activity of PPAA varies with solution pH and concentration of dissolved polymer molecules. The surface tension of PPAA solutions (55-72 dynes/cm) that associated with the polymer surface activity was measured using the Wilhelmy plate technique. Samples of these polymer solutions then were exposed to 1.1 MHz high intensity focused ultrasound, and the dissolution of bubbles created by inertial cavitation was monitored using an active cavitation detection scheme. Analysis of the pulse echo data demonstrated that bubble dissolution time was inversely proportional to the surface tension of the solution. Finally, comparison of the experimental results with dissolution times computed from the Epstein-Plesset equation suggests that the radii of residual bubbles from inertial cavitation increase as the surface tension decreases.     

脉冲超声激励下SonoVue微泡的瞬态空化特性

将SonoVue微泡从临床疾病诊断拓展至治疗引起了诸多研究人员的兴趣。为了平衡治疗效率和生物安全性,深入理解声学参数和SonoVue微泡瞬态空化的关系至关重要。本研究自行制备仿体容器放置SonoVue微泡,使用1 MHz发射换能器激励其产生空化效应,另一个7.5 MHz的聚焦换能器接收声信号,经放大及高速数据采集后送上位机处理。通过深入分析信号的时频域特征,我们提出以宽带信号的能量及其随时间变化曲线的半高宽来表征瞬态空化的剂量(ICD)和相对持续时间(ICP),并确定:瞬态空化的发生和ICD依赖于峰值负声压,但ICP随峰值负声压的增加而减小;脉冲重复频率和脉冲持续时间都和ICD及ICP正相关;且脉冲持续时间的影响较大。这些结果有望为SonoVue微泡的治疗应用提供理论支持。      

Generation and control of acoustic cavitation structure

The generation and control of acoustic cavitation structure are a prerequisite for application of cavitation in the field of ultrasonic sonochemistry and ultrasonic cleaning. The generation and control of several typical acoustic cavitation structures (conical bubble structure, smoker, acoustic Lichtenberg figure, tailing bubble structure, jet-induced bubble structures) in a 20–50 kHz ultrasonic field are investigated. Cavitation bubbles tend to move along the direction of pressure drop in the region in front of radiating surface, which are the premise and the foundation of some strong acoustic cavitation structure formation. The nuclei source of above-mentioned acoustic cavitation structures is analyzed. The relationship and mutual transformation of these acoustic cavitation structures are discussed.     

Thermodynamic and kinetic considerations of nucleation and stabilization of acoustic cavitation bubbles in water

Qualitative explanation for a homogeneous nucleation of acoustic cavitation bubbles in the incompressible liquid water with simple phenomenological approach has been provided via the concept of the desorbtion of the dissolved gas and the vaporization of local liquid molecules. The liquid medium has been viewed as an ensemble of lattice structures. Validity of the lattice structure approach against the Brownian motion of molecules in the liquid state has been discussed. Criterion based on probability for nucleus formation has been defined for the vaporization of local liquid molecules. Energy need for the enthalpy of vaporization has been considered as an energy criterion for the formation of a vaporous nucleus. Sound energy, thermal energy of the liquid bulk (Joule-Thomson effect) and free energy of activation, which is associated with water molecules in the liquid state (Brownian motion) as per the modified Eyring's kinetic theory of liquid are considered as possible sources for the enthalpy of vaporization of water molecules forming a single unit lattice. The classical nucleation theory has then been considered for expressing further growth of the vaporous nucleus against the surface energy barrier. Effect of liquid property (temperature), and effect of an acoustic parameter (frequency) on an acoustic cavitation threshold pressure have been discussed. Kinetics of nucleation has been considered.     

Spatial and temporal observation of phase-shift nano-emulsions assisted cavitation and ablation during focused ultrasound exposure

Background: Phase-shift nano-emulsions (PSNEs) with a small initial diameter in nanoscale have the potential to leak out of the blood vessels and to accumulate at the target point of tissue. At desired location, PSNEs can undergo acoustic droplet vaporization (ADV) process, change into gas bubbles and enhance focused ultrasound efficiency. The threshold of droplet vaporization and influence of acoustic parameters have always been research hotspots in order to spatially control the potential of bioeffects and optimize experimental conditions. However, when the pressure is much higher than PSNEs’ vaporization threshold, there were little reports on their cavitation and thermal effects.Object: In this study, PSNEs induced cavitation and ablation effects during pulsed high-intensity focused ultrasound (HIFU) exposure were investigated, including the spatial and temporal information and the influence of acoustic parameters.Methods: Two kinds of tissue-mimicking phantoms with uniform PSNEs were prepared because of their optical transparency. The Sonoluminescence (SL) method was employed to visualize the cavitation activities. And the ablation process was observed as the heat deposition could produce white lesion.Results: Precisely controlled HIFU cavitation and ablation can be realized at a relatively low input power. But when the input power was high, PSNEs can accelerate cavitation and ablation in pre-focal region. The cavitation happened layer by layer advancing the transducer. While the lesion appeared to be separated into two parts, one in pre-focal region stemmed from one point and grew quickly, the other in focal region grew much more slowly. The influence of duty cycle has also been examined. Longer pulse off time would cause heat transfer to the surrounding media, and generate smaller lesion. On the other hand, this would give outer layer bubbles enough time to dissolve, and inner bubbles can undergo violent collapse and emit bright light.     

Shear strain from irrotational tissue displacements near bubbles

Particle displacements can be much greater near bubbles than they would be in a homogeneous liquid or tissue when exposed to an acoustic wave. In a plane wave, shear and bulk strains are of the same order of magnitude. In contrast, for a bubble oscillating close to its resonance frequency, the shear strain in the medium near the bubble is roughly four orders of magnitude greater than the bulk strain. This can lead to shear strains of a few percent even with acoustic excitation pressures far below the pressure thresholds required to cause inertial cavitation. High shear strains near oscillating bubbles could potentially be the cause of bioeffects. After acoustic exposures at audio frequencies, hemorrhages in tissues as diverse as lung, liver, and kidney have been observed at shear strains on the order of 1%.     

Stability of alteplase in presence of cavitation

Several experimental studies have demonstrated that ultrasound (US) can accelerate enzymatic fibrinolysis and this effect is further enhanced in the presence of ultrasound contrast agents (UCA). Although UCA have been shown to be safe when administered to ischemic stroke patients, safety information of these agents in the thrombolysis setting is limited. Therefore, in this study we investigated potential adverse effects of acoustic cavitation generated by UCA on alteplase (t-PA), the drug used for treatment of ischemic stroke patients. A volume of 0.9 mL of alteplase was dispensed into a custom-made polyester sample tube. For treatments in the presence or absence of cavitation either 0.1 mL Optison or phosphate buffer saline was combined with alteplase. Three independent samples of each treatment group were exposed to ultrasound of 2 MHz frequency at three different peak negative acoustic pressures of 0.5, 1.7, and 3.5 MPa for a duration of 60 min. All treatments were carried out in a cavitation detection system which was used to insonify the samples and record acoustic emissions generated within the sample. After ultrasound exposure, the treated samples and three untreated drug samples were tested for their enzymatic activity using a chromogenic substrate. The insonified samples containing Optison demonstrated cavitational activity proportional to acoustic pressure. No significant cavitation activity was observed in the absence of Optison. Enzymatic activity of alteplase in both insonified groups was comparable to that in the control group. These tests demonstrated that exposure of alteplase to 60 min of 2 MHz ultrasound at acoustic pressures ranging from 0.5 MPa to 3.5 MPa, in the presence or absence of Optison had no adverse effects on the stability of this therapeutic compound.     

Radial oscillation and translational motion of a gas bubble in a micro-cavity

According to classical nucleation theory, a gas nucleus can grow into a cavitation bubble when the ambient pressure is negative. Here, the growth process of a gas nucleus in a micro-cavity was simplified to two “events”, and the full confinement effect of the surrounding medium of the cavity was considered by including the bulk modulus in the equation of state. The Rayleigh–Plesset-like equation of the cavitation bubble in the cavity was derived to model the radial oscillation and translational motion of the cavitation bubble in the local acoustic field. The numerical results show that the nucleation time of the cavitation bubble is sensitive to the initial position of the gas nucleus. The cavity size affects the duration of the radial oscillation of the cavitation bubble, where the duration is shorter for smaller cavities. The equilibrium radius of a cavitation bubble grown from a gas nucleus increases with increasing size of the cavity. There are two possible types of translational motion: reciprocal motion around the center of the cavity and motion toward the cavity wall. The growth process of gas nuclei into cavitation bubbles is also dependent on the compressibility of the surrounding medium and the magnitude of the negative pressure. Therefore, gas nuclei in a liquid cavity can be excited by acoustic waves to form cavitation bubbles, and the translational motion of the cavitation bubbles can be easily observed owing to the confining influence of the medium outside the cavity.     

Boiling-up of liquid argon at high superheatings under the impact of weak ultrasonic fields

The technique of lifetime measurement for metastable liquid and continuous depressurization technique were applied for study of kinetics of spontaneous cavitation in superheated liquid argon under impact of weak ultrasonic fields. It was demonstrated that acoustic cavitation may occur through the mechanism of homogeneous generation of vapor phase or through “swinging” of vapor bubbles generated by high-energy particles and/or other kinds of initiative factors. The thresholds of acoustic cavitation are described by the theory of homogeneous nucleation.