Platelet-targeted microbubbles inhibit re-occlusion after thrombolysis with transcutaneous ultrasound and microbubbles

Microbubbles (MBs) can augment the acoustic cavitation’ (US), thereby facilitating the thrombolysis of external ultrasound. But we observed re-thrombosis after successful thrombolysis by MBs and transcutaneous ultrasound in an endothelium injury model. This study was designed to explore whether platelet-targeted MBs can prevent the reformation of thrombi. Arterial injury was induced in canine femoral arteries with balloon, and the arteries were completely thrombotically occluded. The arteries were treated with intra-arterial MBs or platelet-targeted MBs (TMB) and transcutaneous low frequency ultrasound (LFUS) to achieve complete thrombolysis. The arterial flow was monitored with angiogram for 4 h following treatment. Results showed that both MBs and TMBs produced successful dissolution of clots in the presence of ultrasound. The re-occlusion began to occur 1 h after thrombolysis in MB/LFUS treatment, and 7 of 8 arteries were re-occluded within 3 h. Most of the arteries (7 of 8) in the TMB/LFUS group remained patent for 4 h following treatment. The flow tended to decrease after thrombolysis in MB/LFUS treatment. These results indicated that platelet-targeted microbubbles were beneficial in preventing re-thrombosis in vivo and microbubbles served as good carrier of thrombolytic and anticoagulation drugs.     

超声联合微泡增强细胞内药物递送的研究进展

超声联合微泡介导的细胞内药物递送是通过微泡的声空化与细胞的相互作用而实现的.作为一种非侵入式的、非病毒的、具有靶向性的、可在成像技术引导下的药物递送技术,超声联合微泡在临床应用上具有独特的优势.该文围绕超声联合微泡实现药物递送的发生机理,从微泡的声学动态响应、细胞响应、细胞外物质进入细胞的动态过程及临床试验进展4方面对...     

Enhanced sonodynamic therapy by carbon dots-shelled microbubbles with focused ultrasound

Sonodynamic therapy involving the non-invasive and local generation of lethal reactive oxygen species (ROS) via ultrasound (US) with sonosensitizers has been proposed as an emerging tumor therapy strategy. However, such therapy is usually associated with inertial cavitation and unnecessary damage to healthy tissue because current sonosensitizers have insufficient sensitivity to US. Here, we report the use of a new proposed sonosensitizer, carbon dots (C-dots), to assemble microbubbles with a gas core (C-dots MBs). As the C-dots were directly integrated into the MB shell, they could effectively absorb the energy of inertial cavitation and transfer it to ROS. Our results revealed the appearance of ¹O₂, •OH, and H₂O₂ after US irradiation of C-dots MBs. In in vitro experiments, treatment with C-dots MBs plus US induced lipid peroxidation, elevation of intracellular ROS, and apoptosis in 32.5%, 45.3%, and 50.1% of cells respectively. In an animal solid tumor model, treatment with C-dots MBs plus US resulted in a 3-fold and 2.5-fold increase in the proportion of ROS-damaged cells and apoptotic cells, respectively, compared to C-dots MBs alone. These results will pave the way for the design of novel multifunctional sonosensitizers for SDT tumor therapy.     

Radionuclide tumour therapy with ultrasound contrast microbubbles

Radionuclides have shown to be effective in tumour therapy. However, the side effects determine the maximum deliverable dose. Recently, it has been demonstrated that cells can be permeabilised through sonoporation using ultrasound and contrast microbubbles. The use of sonoporation in treatment of tumours may increase the anti-tumour efficacy of radionuclide treatment. The mechanisms as well as the effects sonoporation in tumour treatment strategies are still not understood. The purpose of this study is to determine the effects of ultrasound and contrast microbubbles on the internalisation of the radionuclide (111)In-DOTA-Tyr(3)-octreotate in tumour cells. To optimize ultrasound settings for ultrasound adjunctive tumour therapy we incubated rat pancreatic CA20948 tumour cells with two dyes (MW 40 and 70 kDa). The uptake levels were compared with cells treated with ultrasound and contrast microbubbles for different ultrasound settings. The highest molecular uptake was found with addition of contrast microbubbles (ratio of 10 bubbles to 1 cell) and with the ultrasound setting: duty cycle 0.013%, mechanical index (MI) 0.42, and treatment times of 30 and 60 min. These settings were used to enhance the internalisation of (111)In-DOTA-Tyr(3)-octreotate. We found a 160% higher internalisation of (111)In-DOTA-Tyr(3)-octreotate by tumour cells adjunctively treated with ultrasound and contrast microbubbles compared to untreated cells. These results show that adjunctive tumour treatment with the radionuclide (111)In-DOTA-Tyr(3)-octreotate and ultrasound contrast microbubbles may be feasible. When using adjunctive ultrasound contrast microbubble treatment, a lower radionuclide doses are required to reach the same anti-tumour effect.     

Interaction of microbubbles with high intensity pulsed ultrasound

High intensity pulsed ultrasound, interacting with microbubble contrast agents, is potentially useful for drug delivery, cancer treatment, and tissue ablation, among other applications. To establish the fundamental understanding on the interaction of a microbubble (in an infinite volume of water) with an ultrasound pressure field, a numerical study is performed using the boundary element method. The response of the bubble, in terms of its shape at different times, the maximum bubble radius obtained, the oscillation time, the jet velocity, and its translational movement, is studied. The effect of ultrasound intensity and initial bubble size is examined as well. One important outcome is the determination of the conditions under which a clear jet will be formed in a microbubble in its interaction with a specific sound wave. The high speed jet is crucial for the aforementioned intended applications.     

Attenuation of ultrasound in porous media with dispersed microbubbles

The dispersion relation for a granular bed with a small amount of fine bubbles is formulated and analyzed. It is assumed that the grain size is much larger than the bubble's radius and that their volume concentration is small. The study is motivated by the problem of acoustic diagnostics of fixed bed chemical reactors operating in multiphase flow regime.     

Nonlinear response to ultrasound of encapsulated microbubbles

The acoustic backscatter of encapsulated gas-filled microbubbles immersed in a weak compressible liquid and irradiated by ultrasound fields of moderate to high pressure amplitudes is investigated theoretically. The problem is formulated by considering, for the viscoelastic shell of finite thickness, an isotropic hyperelastic neo-Hookean model for the elastic contribution in addition to a Newtonian viscous component. First and second harmonic scattering cross-sections have been evaluated and the quantitative influence of the driving pressure amplitude on the harmonic resonance frequencies for different initial equilibrium bubble sizes and for different encapsulating physical properties has been determined. Conditions for optimal second harmonic imaging have been also investigated and some regions in the parameters space where the second harmonic intensity is dominant over the fundamental have been identified. Results have been obtained for albumin, lipid and polymer encapsulating shells, respectively.     

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。     

Effect of non-acoustic parameters on heterogeneous sonoporation mediated by single-pulse ultrasound and microbubbles

Sonoporation—transient plasma membrane perforation elicited by the interaction of ultrasound waves with microbubbles—has shown great potential for drug delivery and gene therapy. However, the heterogeneity of sonoporation introduces complexities and challenges in the realization of controllable and predictable drug delivery. The aim of this investigation was to understand how non-acoustic parameters (bubble related and bubble-cell interaction parameters) affect sonoporation. Using a customized ultrasound-exposure and fluorescence-imaging platform, we observed sonoporation dynamics at the single-cell level and quantified exogenous molecular uptake levels to characterize the degree of sonoporation. Sonovue microbubbles were introduced to passively regulate microbubble-to-cell distance and number, and bubble size. 1 MHz ultrasound with 10-cycle pulse duration and 0.6 MPa peak negative pressure were applied to trigger the inertial collapse of microbubbles. Our data revealed the impact of non-acoustic parameters on the heterogeneity of sonoporation. (i) The localized collapse of relatively small bubbles (diameter, D < 5.5 μm) led to predictable sonoporation, the degree of which depended on the bubble-to-cell distance (d). No sonoporation was observed when d/D > 1, whereas reversible sonoporation occurred when d/D < 1. (ii) Large bubbles (D > 5.5 μm) exhibited translational movement over large distances, resulting in unpredictable sonoporation. Translation towards the cell surface led to variable reversible sonoporation or irreversible sonoporation, and translation away from the cell caused either no or reversible sonoporation. (iii) The number of bubbles correlated positively with the degree of sonoporation when D < 5.5 μm and d/D < 1. Localized collapse of two to three bubbles mainly resulted in reversible sonoporation, whereas irreversible sonoporation was more likely following the collapse of four or more bubbles. These findings offer useful insight into the relationship between non-acoustic parameters and the degree of sonoporation.     

Prediction of vascular injury by cavitation microbubbles in a focused ultrasound field

Many studies have shown that microbubble cavitation is one mechanism for vascular injury under ultrasonic excitation. Previous work has attributed vascular damage to vessel expansions and invaginations due to the expansion and contraction of microbubbles. However, the mechanisms of vascular damage are not fully understood. In this paper, we investigate, theoretically and experimentally, the vessel injury due to stress induced by ultrasound-induced cavitation (UIC). A bubble-fluid-vessel coupling model is constructed to investigate the interactions of the coupling system. The dynamics process of vessel damage due to UIC is theoretically simulated with a finite element method, and a focused ultrasound (FU) setup is carried out and used to assess the vessel damage. The results show that shear stress contributes to vessel injury by cell detachment while normal stress mainly causes distention injury. Similar changes in cell detachment in a vessel over time can be observed with the experimental setup. The severity of vascular injury is correlated to acoustic parameters, bubble-wall distance, and microbubble sizes, and the duration of insonation..     

Subharmonic behavior of phospholipid-coated ultrasound contrast agent microbubbles

Coated microbubbles, unlike tissue are able to scatter sound subharmonically. Therefore, the subharmonic behavior of coated microbubbles can be used to enhance the contrast in ultrasound contrast imaging. Theoretically, a threshold amplitude of the driving pressure can be calculated above which subharmonic oscillations of microbubbles are initiated. Interestingly, earlier experimental studies on coated microbubbles demonstrated that the threshold for these bubbles is much lower than predicted by the traditional linear viscoelastic shell models. This paper presents an experimental study on the subharmonic behavior of differently sized individual phospholipid coated microbubbles. The radial subharmonic response of the microbubbles was recorded with the Brandaris ultra high-speed camera as a function of both the amplitude and the frequency of the driving pulse. Threshold pressures for subharmonic generation as low as 5 kPa were found near a driving frequency equal to twice the resonance frequency of the bubble. An explanation for this low threshold pressure is provided by the shell buckling model proposed by Marmottant et al. [J. Acoust. Soc. Am. 118, 3499-3505 (2005)]. It is shown that the change in the elasticity of the bubble shell as a function of bubble radius as proposed in this model, enhances the subharmonic behavior of the microbubbles.     

Scaling of the viscoelastic shell properties of phospholipid encapsulated microbubbles with ultrasound frequency

Phospholipid encapsulated microbubbles are widely employed as clinical diagnostic ultrasound contrast agents in the 1–5 MHz range, and are increasingly employed at higher ultrasound transmit frequencies. The stiffness and viscosity of the encapsulating “shells” have been shown to play a central role in determining both the linear and nonlinear response of microbubbles to ultrasound. At lower frequencies, recent studies have suggested that shell properties can be frequency dependent. At present, there is only limited knowledge of how the viscoelastic properties of phospholipid shells scale at higher frequencies. In this study, four batches of in-house phospholipid encapsulated microbubbles were fabricated with decreasing volume-weighted mean diameters of 3.20, 2.07, 1.82 and 1.61 μm. Attenuation experiments were conducted in order to assess the frequency-dependent response of each batch, resulting in resonant peaks in response at 4.2, 8.9, 12.6 and 19.5 MHz, respectively. With knowledge of the size measurements, the attenuation spectra were then fitted with a standard linearized bubble model in order to estimate the microbubble shell stiffness S_p and shell viscosity S_f, resulting in a slight increase in S_p (1.53–1.76 N/m) and a substantial decrease in S_f (0.29 × 10⁻⁶–0.08 × 10⁻⁶ kg/s) with increasing frequency. These results performed on a single phospholipid agent show that frequency dependent shell properties persist at high frequencies (up to 19.5 MHz).     

Synergistic bactericidal effect of ultrasound combined with citral nanoemulsion on Salmonella and its application in the preservation of purple kale

In this study, a novel citral nanoemulsion (CLNE) was prepared by ultrasonic emulsification. The synergistic antibacterial mechanism of ultrasound combined with CLNE against Salmonella Typhimurium and the effect on the physicochemical properties of purple kale were investigated. The results showed that the combined treatment showed obviously inactivate effect of S. Typhimurium. Treatment with 0.3 mg/mL CLNE combined with US (20 kHz, 253 W/cm²) for 8 min reduced S. Typhimurium populations in phosphate-buffered saline (PBS) by 9.05 log CFU/mL. Confocal laser scanning microscopy (CLSM), flow cytometry (FCM), protein and nucleic acid release assays showed that the US combination CLNE disrupt the integrity of S. Typhimurium membranes. Reactive oxygen species (ROS) and malondialdehyde (MDA) detection indicated that US+CLNE exacerbated oxidative stress and lipid peroxidation in cell membranes. The morphological changes of cells after different treatments by field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) illustrated that the synergistic effect of US+CLNE treatment changed the morphology and internal microstructure of the bacteriophage cells. Application of US+CLNE on purple kale leaves for 6 min significantly (P < 0.05) reduced the number of S. Typhimurium, but no changes in the physicochemical properties of the leaves were detected. This study elucidates the synergistic antibacterial mechanism of ultrasound combined with CLNE and provides a theoretical basis for its application in food sterilization.     

超声造影剂微气泡的包膜黏弹特性的定量表征研究

包膜黏弹特性显著影响微气泡超声造影剂的诊断及治疗应用效果. 本文结合原子力显微镜技术及声衰减特性测量提出了一种对微气泡造影剂包膜黏弹特性定量表征的新方法. 首先采用原子力显微镜技术进行机械特性分析得到包膜微气泡的有效硬度及体弹性模量; 然后测量声衰减特性, 基于微气泡动力学理论, 计算包膜微气泡的体黏度系数. 为验证方法的有效性, 实验制备了直径为1-5 μm的白蛋白包膜微气泡造影剂, 原子力显微镜测量的有效硬度和体弹性模量分别为0.149±0.012 N/m和8.31±0.667 MPa, 并与粒径无关. 声衰减特性测量和动力学理论拟合的包膜微气泡的体黏度系数为0.374±0.003 Pa·s. 该方法可推广至其他种类包膜微气泡的黏弹特性表征, 对超声造影剂的制备及其诊断和治疗应用有积极意义.     

Activation of microbubbles by low-intensity pulsed ultrasound enhances the cytotoxicity of curcumin involving apoptosis induction and cell motility inhibition in human breast cancer MDA-MB-231 cells

Ultrasound and microbubbles-mediated drug delivery has become a promising strategy to promote drug delivery and its therapeutic efficacy. The aim of this research was to assess the effects of microbubbles (MBs)-combined low-intensity pulsed ultrasound (LPUS) on the delivery and cytotoxicity of curcumin (Cur) to human breast cancer MDA-MB-231 cells. Under the experimental condition, MBs raised the level of acoustic cavitation and enhanced plasma membrane permeability; and cellular uptake of Cur was notably improved by LPUS–MBs treatment, aggravating Cur-induced MDA-MB-231 cells death. The combined treatment markedly caused more obvious changes of cell morphology, F-actin cytoskeleton damage and cell migration inhibition. Our results demonstrated that combination of MBs and LPUS may be an efficient strategy for improving anti-tumor effect of Cur, suggesting a potential effective method for antineoplastic therapy.     

Effect of surfactant addition on removal of microbubbles using ultrasound

It is difficult to control the bubble in a liquid by the external operation, because the behavior of the bubble is controlled in buoyancy and flow of liquid. On the other hand, microbubbles, whose diameter is several decades μm, stably disperse in static liquid because of their small buoyancy and electrical repulsion. When an ultrasound, whose frequency was 2.4 MHz, was irradiated, the milky white microbubbles suspended solution became rapidly clear. In this study, the effects of surfactant addition on the removal of microbubbles from a liquid in an ultrasonic field were investigated. The efficiency of removal of microbubbles decreased with surfactant addition. Surfactant type influenced the size of agglomerated microbubbles, and the efficiency of removal of microbubbles changed. The surface of microbubble was modified by surfactant adsorption, and the steric inhibition influenced the removal of microbubbles.     

Quantitative evaluation of the use of microbubbles with transcranial focused ultrasound on blood-brain-barrier disruption

It has been shown that focused ultrasound (FUS) can disrupt the blood-brain barrier (BBB) noninvasively and reversibly at target locations when applied in the presence of ultrasound contrast agent (UCA). In this study, the dose-dependent effects of UCA on BBB disruption were investigated in the brains of 16 male Wistar rats sonicated by 1.0-MHz transcranial FUS, with the UCA present at four doses. The BBB disruption was evaluated quantitatively based on the extravasation of Evans blue (EB). The amount of EB extravasation in the brain increased with the quantity of UCA injected into the femoral vein prior to sonication. Moreover, the use of a suitable dose of UCA resulted in the BBB disruption being concentrated in the focal region instead of the entire brain. Our results indicate that injecting an appropriate quantity of UCA effectively increases and localizes the BBB disruption induced by transcranial FUS sonications.     

Minimizing the thermal losses from perfusion during focused ultrasound exposures with flowing microbubbles

This paper demonstrated the use of flowing microbubbles (MBs) to minimize thermal losses from perfusion during focused ultrasound exposures due to acoustic cavitation. Temperature and cavitation were simultaneously investigated as MBs flowing through a wall-less flow phantom with varying flow velocities (2-55 cm/s) and concentrations (0%-0.1%) when exposed at different acoustic power levels (5-120 W). The peak temperature at the end of ultrasonic exposures in the flow and in the outer of the vessel as well as the cavitation were higher than those pure controls measured at the same exposure parameters and flow velocities but without MBs. All the peak temperatures initially increased with increasing flow velocities of MBs, followed by a decrease of the peak temperatures with increasing flow velocities when the velocity was higher than the inflection velocity. Meanwhile, cavitation showed a trend of increases with increasing flow velocity. The inflection velocity and cavitation increased with increasing acoustic power and MBs concentration. Thermal lesion appeared around the vessel as MBs flow through the vessel, at which lesion was not observed originally without MBs. These results suggested that this may provide an effective way to minimize thermal losses from perfusion during focused ultrasound exposures.     

Evaluation of ultrasound and microbubbles effect on pork meat during brining process

In this study combined effect of ultrasound-induced acoustic cavitation and microbubbles during meat brining on pork loin (Longissimus dorsi) was evaluated. Cylindrical shape (diameter 15 mm, height 80 mm) pork loin samples were cut and immersed in 200 g L^-1 NaCl brine and treated with the following brining methods for 180 min: static brining (SB), ultrasound assisted brining (US) and ultrasound combined with microbubbles in brine (USMB). Ultrasound was generated with 20 kHz frequency, 5,09 W/cm² maximum intensity and 100 W maximum power. Microbubbles in brine were produced by a gas–liquid mixing pump. Effect of ultrasound and microbbubles on NaCl content and diffusion in pork loin, mass balance, water binding capacity (WBC), protein denaturation and meat tissue microstructure were evaluated. The US and USMB brinings enhanced the NaCl diffusion into meat compared to meat brined under static conditions. The constant diffusion coefficient (D) model precisely described the NaCl diffusion kinetics during brinings. The ultrasound and microbbubles resulted in microscopic pores on the surface of myofibers. Decreasing WBC was observed for all brining methods. Myosin was not detectable in any of the brining methods. Denaturation temperature of actin showed a decreasing tendency with increasing brining time independently the brining methods.     

微泡对高强度聚焦超声焦域影响的研究

微泡对高强度聚焦超声(HIFU)治疗具有增效作用,而HIFU治疗中不同声学条件下微泡对HIFU治疗焦域的影响尚不清楚。本文基于声传播方程、Yang-Church气泡运动方程、生物热传导方程、时域有限差分法(FDTD)、龙格-库塔(RK)法数值仿真研究输入功率、激励频率和气泡初始半径对HIFU在含气泡体模中形成焦域的影响,并利用含Sono Vue造影剂的仿组织体模研究进行实验验证。结果表明,增大输入功率、气泡初始半径和升高激励频率均可增大焦域,随着输入功率的增大,焦域形状可能发生变化,而随着激励频率升高和气泡初始半径的增大,焦域会向远离换能器的方向移动。