Microstreaming velocity field and shear stress created by an oscillating encapsulated microbubble near a cell membrane

Sonoporation mediated by microbubbles is being extensively studied as a promising technology to facilitate gene/drug delivery to cells. However, the theoretical study regarding the mechanisms involved in sonoporation is still in its infancy. Microstreaming generated by pulsating microbubble near the cell membrane is regarded as one of the most important mechanisms in the sonoporation process. Here, based on an encapsulated microbubble dynamic model with considering nonlinear rheological effects of both shell elasticity and viscosity, the microstreaming velocity field and shear stress generated by an oscillating microbubble near the cell membrane are theoretically simulated. Some factors that might affect the behaviors of microstreaming are thoroughly investigated, including the distance between the bubble center and cell membrane (d), shell elasticity (χ), and shell viscosity (κ). The results show that (i) the presence of cell membrane can result in asymmetric microstreaming velocity field, while the constrained effect of the membrane wall decays with increasing the bubble-cell distance; (ii) the bubble resonance frequency increases with the increase in d and χ, and the decrease in κ, although it is more dominated by the variation of shell elasticity; and (iii) the maximal microstreaming shear stress on the cell membrane increases rapidly with reducing the d, χ, and κ. The results suggest that microbubbles with softer and less viscous shell materials might be preferred to achieve more efficient sonoporation outcomes, and it is better to have bubbles located in the immediate vicinity of the cell membrane.     

Cavitation microstreaming and stress fields created by microbubbles

Cavitation microstreaming plays a role in the therapeutic action of microbubbles driven by ultrasound, such as the sonoporative and sonothrombolytic phenomena. Microscopic particle-image velocimetry experiments are presented. Results show that many different microstreaming patterns are possible around a microbubble when it is on a surface, albeit for microbubbles much larger than used in clinical practice. Each pattern is associated with a particular oscillation mode of the bubble, and changing between patterns is achieved by changing the sound frequency. Each microstreaming pattern also generates different shear stress and stretch/compression distributions in the vicinity of a bubble on a wall. Analysis of the micro-PIV results also shows that ultrasound-driven microstreaming flows around bubbles are feasible mechanisms for mixing therapeutic agents into the surrounding blood, as well as assisting sonoporative delivery of molecules across cell membranes. Patterns show significant variations around the bubble, suggesting sonoporation may be either enhanced or inhibited in different zones across a cellular surface. Thus, alternating the patterns may result in improved sonoporation and sonothrombolysis. The clear and reproducible delineation of microstreaming patterns based on driving frequency makes frequency-based pattern alternation a feasible alternative to the clinically less desirable practice of increasing sound pressure for equivalent sonoporative or sonothrombolytic effect. Surface divergence is proposed as a measure relevant to sonoporation.     

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.     

微气泡激发的声微流对细胞声孔效应的影响

理论及实验研究了微气泡激发的声微流对声孔效应的影响。实验采用低幅度(0.05~0.3MIPa)连续超声波信号照射MCF-7细胞,PEI:DNA的复合质粒和造影剂气泡的混合溶液,通过扫描电子显微镜测量细胞膜声孔效应。结果表明声孔大小随着激励声压的幅度和照射时间的增加而增大,平均孔径范围为100 nm~1.25μm。基于Marmottant微气泡振动模型的理论计算结果表明微气泡振动所产生的声微流引起的剪切力在低幅度超声引发声致穿孔作用中起着关键作用。      

New insights on the role of ROS in the mechanisms of sonoporation-mediated gene delivery

Reactive oxygen species (ROS) are hypothesized to play a role in the sonoporation mechanisms. Nevertheless, the acoustical phenomenon behind the ROS production as well as the exact mechanisms of ROS action involved in the increased cell membrane permeability are still not fully understood. Therefore, we investigated the key processes occurring at the molecular level in and around microbubbles subjected to ultrasound using computational chemistry methods. To confirm the molecular simulation predictions, we measured the ROS production by exposing SonoVue® microbubbles (MBs) to ultrasound using biological assays. To investigate the role of ROS in cell membrane permeabilization, cells were subjected to ultrasound in presence of MBs and plasmid encoding reporter gene, and the transfection level was assessed using flow cytometry. The molecular simulations showed that under sonoporation conditions, ROS can form inside the MBs. These radicals could easily diffuse through the MB shell toward the surrounding aqueous phase and participate in the permeabilization of nearby cell membranes. Experimental data confirmed that MBs favor spontaneous formation of a host of free radicals where HO was the main ROS species after US exposure. The presence of ROS scavengers/inhibitors during the sonoporation process decreased both the production of ROS and the subsequent transfection level without significant loss of cell viability. In conclusion, the exposure of MBs to ultrasound might be the origin of chemical effects, which play a role in the cell membrane permeabilization and in the in vitro gene delivery when generated in its proximity.     

H-22细胞声孔效应的应力阈值

理论和实验研究了超声空化场中的H-22型肝癌细胞产生可逆声孔效应的剪应力阈值.本文用1.37 MHz的聚焦声场,当超顺磁性纳米氧化铁在细胞悬液中的终浓度为410 μg/mL,换能器负载电功率为2 W,超声辐照60 s,细胞存活率90%以上时,有45.9±13.5%的细胞显示普鲁士蓝染阳性,暗示超声作用下,这些细胞表面曾出现可逆性微孔而使磁性微粒由此进入细胞内.利用无界自由空间微泡运动方程的球对称稳态解对实验条件下细胞膜表面的切变应力进行数值估算,结果表明,使H-22细胞产生可逆性声孔效应的微流剪应力阈值为697 Pa. 关键词： 声孔效应 磁性标记 微流 剪应力     

The behavior of lipid debris left on cell surfaces from microbubble based ultrasound molecular imaging

Lipid monolayer coated microbubbles are currently being developed to identify vascular regions that express certain surface proteins as part of the new technique of ultrasound molecular imaging. The microbubbles are functionalized with targeting ligands which bind to the desired cells holding the microbubbles in place as the remaining unbound microbubbles are eliminated from circulation. Subsequent scanning with ultrasound can detect the highly reflectant microbubbles that are left behind. The ultrasound scanning and detection process results in the destruction of the microbubble, creating lipid fragments from the monolayer. Here we demonstrate that microbubbles targeted to 4T1 murine breast cancer cells and human umbilical cord endothelial cells leave behind adhered fragments of the lipid monolayer after exposure to ultrasound with peak negative pressures of 0.18 and 0.8 MPa. Most of the observed fragments were large enough to be resistant to receptor mediated endocytosis. The fragments were not observed to incorporate into the lipid membrane of the cell over a period of 96 min. They were not observed to break into smaller pieces or significantly change shape but they were observed to undergo translation and rotation across the cell surface as the cells migrated over the substrate. These large fragments will apparently remain on the surface of the targeted cells for significant periods of time and need to be considered for their potential effects on blood flow through the microcapillaries and potential for immune system recognition.     

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.     

Mechanisms underlying sonoporation: Interaction between microbubbles and cells

The past several decades have witnessed great progress in “smart drug delivery”, an advance technology that can deliver genes or drugs into specific locations of patients’ body with enhanced delivery efficiency. Ultrasound-activated mechanical force induced by the interactions between microbubbles and cells, which can stimulate so-called “sonoporation” process, has been regarded as one of the most promising candidates to realize spatiotemporal-controllable drug delivery to selected regions. Both experimental and numerical studies were performed to get in-depth understanding on how the microbubbles interact with cells during sonoporation processes, under different impact parameters. The current work gives an overview of the general mechanism underlying microbubble-mediated sonoporation, and the possible impact factors (e.g., the properties of cavitation agents and cells, acoustical driving parameters and bubble/cell micro-environment) that could affect sonoporation outcomes. Finally, current progress and considerations of sonoporation in clinical applications are reviewed also.     

超声造影剂微泡非线性动力学响应的机理及相关应用

随着生命科学及现代医学的发展, 一体化无创精准诊疗已经日益成为人们关注的焦点问题, 而关于超声造影剂微泡的非线性效应的相关机理、动力学建模及其在超声医学领域中的应用研究也得到了极大的推动. 本文对下列课题进行了总结和讨论, 包括: 1)基于Mie散射技术和流式细胞仪对造影剂微泡参数进行定征的一体化解决方案; 2)通过对微泡包膜的黏弹特性进行非线性修正, 构建新的包膜微泡动力学模型; 3)探索造影剂惯性空化阈值与其包膜参数之间的相关性; 以及4)研究超声联合造影剂微泡促进基因/药物转染效率并有效降低其生物毒性的相关机理.     

Development of a theoretical model describing sonoporation activity of cells exposed to ultrasound in the presence of contrast agents

Sonoporation uses ultrasound, with the aid of ultrasound contrast agents (UCAs), to enhance cell permeabilization, thereby allowing delivery of therapeutic compounds noninvasively into specific target cells. The objective of this study was to determine if a computational model describing shear stress on a cell membrane due to microstreaming would successfully reflect sonoporation activity with respect to the peak rarefactional pressure. The theoretical models were compared to the sonoporation results from Chinese hamster ovary cells using Definity(?) at 0.9, 3.15, and 5.6 MHz and were found to accurately describe the maximum sonoporation activity, the pressure where a decrease in sonoporation activity occurs, and relative differences between maximum activity and the activity after that decrease. Therefore, the model supports the experimental findings that shear stress on cell membranes secondary to oscillating UCAs results in sonoporation.     

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.     

Difference frequency and its harmonic emitted by microbubbles under dual frequency excitation

Vibro-acoustography is an elasticity imaging method that uses two ultrasound beams of slightly different frequency to excite an object and detects the resulting acoustic emission (AE) at the difference frequency. This method is especially sensitive to bubbles due to their nonlinearity. This study explores the harmonic acoustic emission (HAE) at twice the difference frequency emitted from bubbles. A perturbation method based on the dynamic bubble equation is used to derive the AE and HAE from a single bubble excited by dual frequency waves. Simulation shows that HAE is generated only by microbubbles whose resonant frequencies match the incident ultrasound frequencies. In contrast, AE is more sensitive to resonance at the difference frequency, which is relevant to sub-millimeter bubbles. This finding was confirmed by experiments where HAE was produced from Optison microbubbles, but not from larger air bubbles which are off resonance at the incident ultrasound frequency. In conclusion, harmonic acoustic emission is present for microbubbles. It is very sensitive to the size of the bubble and may be used for selective detection of microbubbles.     

Numerical investigation of shock-induced bubble collapse dynamics and fluid–solid interactions during shock-wave lithotripsy

In this paper we investigate the bubble collapse dynamics under shock-induced loading near soft and rigid bio-materials, during shock wave lithotripsy. A novel numerical framework was developed, that employs a Diffuse Interface Method (DIM) accounting for the interaction across fluid–solid-gas interfaces. For the resolution of the extended variety of length scales, due to the dynamic and fine interfacial structures, an Adaptive Mesh Refinement (AMR) framework for unstructured grids was incorporated. This multi-material multi-scale approach aims to reduce the numerical diffusion and preserve sharp interfaces. The presented numerical framework is validated for cases of bubble dynamics, under high and low ambient pressure ratios, shock-induced collapses, and wave transmission problems across a fluid–solid interface, against theoretical and numerical results. Three different configurations of shock-induced collapse applications near a kidney stone and soft tissue have been simulated for different stand-off distances and bubble attachment configurations. The obtained results reveal the detailed collapse dynamics, jet formation, solid deformation, rebound, primary and secondary shock wave emissions, and secondary collapse that govern the near-solid collapse and penetration mechanisms. Significant correlations of the problem configuration to the overall collapse mechanisms were found, stemming from the contact angle/attachment of the bubble and from the properties of solid material. In general, bubbles with their center closer to the kidney stone surface produce more violent collapses. For the soft tissue, the bubble movement prior to the collapse is of great importance as new structures can emerge which can trap the liquid jet into induced crevices. Finally, the tissue penetration is examined for these cases and a novel tension-driven tissue injury mechanism is elucidated, emanating from the complex interaction of the bubble/tissue interaction during the secondary collapse phase of an entrapped bubble in an induced crevice with the liquid jet.     

Translation of cavitation bubble near the different walls

The interaction between spherical cavitation bubble and flat wall is transformed into that between the real bubble and imaging bubble by the method of images. Firstly, we investigate the dynamics of real bubble and matched, inversed or mis-matched imaging bubble driven by a small amplitude ultrasound, revealing the characteristics of the interaction between cavitation bubble and rigid, soft and impedance walls. Then, we emphatically study the dynamics of real bubble and mis-matched imaging bubble driven by a finite amplitude ultrasound, and the interaction characteristics between cavitation bubble and real impedance wall are revealed. The results show that the cavitation bubble is always close to the rigid wall and far away from the soft wall; For the impedance wall, whether the cavitation bubble is far away or close depends on the specific wall parameters. Moreover, the direction and magnitude of bubble's translation velocity can be changed by adjusting the driving parameters. Understanding the interaction between cavitation bubble and impedance wall is of great significance for efficient application of ultrasonic cavitation.     

Synergistic inhibition of malignant melanoma proliferation by melphalan combined with ultrasound and microbubbles

The cavitational effects of ultrasound (US) exposure induce transient pores on the cell membrane (sonoporation). Sonoporation have been applied in the field of cancer therapy by promoting delivery of extracellular molecules such as drugs and genes into cytoplasm. In addition, it is known that using US together with microbubbles (MB) elevates permeability of these agents. In this study, by applying the US-MB strategy for melanoma chemotherapy, we evaluated the antitumor effect of melphalan combined with US-MB on a melanoma cell line (C32) in vitro and in vivo. The in vitro cytotoxic effect of the melphalan with US-MB was greater than that of melphalan alone or melphalan in combination with US. In vivo experiments using xenografts, intratumoral injection of melphalan and MB with US exposure led to a greater degree of tumor regression than did the intratumoral injection of the melphalan alone or melphalan in combination with US. These results suggest that US-MB promotes the antitumor effect of melphalan by increasing delivery of molecules into cells and that this strategy may become an effective method of adjuvant therapy against malignant melanoma.     

Monitoring the effect of sonoporation on the cells using electrochemical approach

Sonoporation is applied to enhance the permeability of the cell to bioactive materials by employing the acoustic cavitation of microbubbles. This phenomena would be helpful in molecular biology, delivery of large molecules into the cells and gene therapy. Many methods have been applied to monitor the biological effects and trace of sonoporation on the cells such as scanning/transmission electron microscopy, confocal imaging and flow cytometry. Here, we monitored the effect of sonoporation on the cells using electrochemical method with an integrated three electrode system. Electrochemical responses of stimulated cells, compared to flow cytometry and electron microscopy results, presented different patterns of sonoporation in the cells detectable by cyclic voltammetry. In addition, confocal microscopy from actin stress fibers and young’s modulus measured by AFM revealed the correlation of cell mechanics and amount of induced sonopores in the cells. This method could be applied as a new trend in cellular mechanochemical studies.     

Limited damage of tissue mimic caused by a collapsing bubble under low-frequency ultrasound exposure

In this study, we investigated the bubble induced serious damage to tissue mimic exposed to 27-kHz ultrasound. The initial bubble radius ranged from 80 to 100 μm, which corresponded approximately to the experimentally-evaluated resonant radius of the given ultrasound frequency. The tissue mimic consisted of 10 wt% gelatine gel covered with cultured canine kidney epithelial cells. The collapsing bubble behaviour during the ultrasound exposure with negative peak pressures of several hundred kPa was captured by a high-speed camera system. After ultrasound exposure, a cell viability test was conducted based on microscopic bright-field images and fluorescence images for living and dead cells. In the viability test, cells played a role in indicating the damaged area. The bubble oscillations killed the cells, and on occasion detached layers of cultured cells from the gel. The damaged area was comparable or slightly larger than the initial bubble size, and smaller than the maximum bubble size. We concluded that only a small area in close proximity to the bubble could be damaged even above transient cavitation threshold.     

Barrier-breaking effects of ultrasonic cavitation for drug delivery and biomarker release

Recently, emerging evidence has demonstrated that cavitation actually creates important bidirectional channels on biological barriers for both intratumoral drug delivery and extratumoral biomarker release. To promote the barrier-breaking effects of cavitation for both therapy and diagnosis, we first reviewed recent technical advances of ultrasound and its contrast agents (microbubbles, nanodroplets, and gas-stabilizing nanoparticles) and then reported the newly-revealed cavitation physical details. In particular, we summarized five types of cellular responses of cavitation in breaking the plasma membrane (membrane retraction, sonoporation, endocytosis/exocytosis, blebbing and apoptosis) and compared the vascular cavitation effects of three different types of ultrasound contrast agents in breaking the blood-tumor barrier and tumor microenvironment. Moreover, we highlighted the current achievements of the barrier-breaking effects of cavitation in mediating drug delivery and biomarker release. We emphasized that the precise induction of a specific cavitation effect for barrier-breaking was still challenged by the complex combination of multiple acoustic and non-acoustic cavitation parameters. Therefore, we provided the cutting-edge in-situ cavitation imaging and feedback control methods and suggested the development of an international cavitation quantification standard for the clinical guidance of cavitation-mediated barrier-breaking effects.     

Fluorescent microscope system to monitor real-time interactions between focused ultrasound,echogenic drug delivery vehicles,and live cell membranes

Rapid development in the field of ultrasound triggered drug delivery has made it essential to study the real-time interaction between the membranes of live cells and the membranes of echogenic delivery vehicles under exposure to focused ultrasound. The objective of this work was to design an analysis system that combined fluorescent imagining, high speed videography, and definable pulse sequences of focused ultrasound to allow for real time observations of both cell and vehicle membranes. Documenting the behavior of the membranes themselves has not previously been possible due to limitations with existing optical systems used to understand the basic physics of microbubble/ultrasound interaction and the basic interaction between microbubbles and cells. The performance of this new system to monitor membrane behavior was demonstrated by documenting the modes of vehicle fragmentation at different ultrasound intensity levels. At 1.5 MPa the membranes were shown to completely fragment while at intensities below 1 MPa the membranes pop open and slowly unfold. The interaction between these vehicles and cell membranes was also documented by the removal of fluorescent particles from the surfaces of live cells out to 20 μm from the microbubble location. The fluid flow created by microstreaming around ensonated microbubbles was documented at video recording speeds from 60 to 18,000 frames per second. This information about membrane behavior allows the chemical and physical properties of the drug delivery vehicle to be designed along with the ultrasound pulse sequence to cause the most efficient drug delivery.