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.     

多层膜磁性微泡的非线性声振动特性

超顺磁性氧化铁纳米粒子与造影剂微泡结合形成磁性微泡,用于产生多模态造影剂,以增强医学超声和磁共振成像.将装载有纳米磁性颗粒的微泡包膜层看作由磁流体膜与磷脂膜组合而成的双层膜结构,同时考虑磁性纳米颗粒体积分数a对膜密度及黏度的影响,从气泡动力学基本理论出发,构建多层膜结构磁性微泡非线性动力学方程.数值分析了驱动声压和频率等声场参数、颗粒体积分数、膜层厚度以及表面张力等膜壳参数对微泡声动力学行为的影响.结果表明,当磁性颗粒体积分数较小且a≤0.1时,磁性微泡声响应特性与普通包膜微泡相似,微泡的声频响应与其初始尺寸和驱动压有关;当驱动声场频率f为磁性微泡共振频率f0的2倍(f=2f0)时,微泡振动失稳临界声压最低;磁性颗粒的存在抑制了泡的膨胀和收缩但抑制效果非常有限;磁性微泡外膜层材料的表面张力参数K及膜层厚度d也会影响微泡的振动,当表面张力参数及膜厚取值分别为0.2—0.4 N/m及50—150 nm时,可观察到气泡存在不稳定振动响应区.     

Sonoluminescence characterization of inertial cavitation inside a BSA phantom treated by pulsed HIFU

The aim of this study was to investigate the inertial cavitation inside a phantom treated by pulsed HIFU (pHIFU). Basic bovine serum albumin (BSA) phantoms without any inherent ultrasound contrast agents (UCAs) or phase-shift nano-emulsions (PSNEs) were used. During the treatment, sonoluminescence (SL) recordings were performed to characterize the spatial distribution of inertial cavitation adjacent to the focal region. High-speed photographs and thermal coagulations, comparing with the SL results, were also recorded and presented. A series of pulse parameters (pulse duration (PD) was between 1 and 23 cycles and pulse repetition frequency (PRF) was between 0.5 kHz and 100 kHz) were performed to make a systematic investigation under certain acoustic power (APW). Continuous HIFU (cHIFU) investigation was also performed to serve as control group. It was found that, when APW was 19.5 W, pHIFU with short PD was much easier to form SL adjacent to the focal region inside the phantom, while it was difficult for cHIFU to generate cavitation bubbles. With appropriate PD and PRF, the residual bubbles of the previous pulses could be stimulated by the incident pulses to oscillate in a higher level and even violently collapse, resulting to enhanced physical thermogenesis. The experimental results showed that the most violent inertial cavitation occurs when PD was set to 6 cycles (5 μs) and PRF to 10 kHz, while the highest level of thermal coagulation was observed when PD was set to 10 cycles. The cavitational and thermal characteristics were in good correspondence, exhibiting significant potentiality regarding to inject-free cavitation bubble enhanced thermal ablation under lower APW, compared to the conventional thermotherapy.     

Effect of gold nanoparticle size on acoustic cavitation using chemical dosimetry method

When a liquid is irradiated with high intensities of ultrasound irradiation, acoustic cavitation occurs. Acoustic cavitation generates free radicals from the breakdown of water and other molecules. Cavitation can be fatal to cells and is utilized to destroy cancer tumors. The existence of particles in liquid provides nucleation sites for cavitation bubbles and leads to decrease the ultrasonic intensity threshold needed for cavitation onset. In the present investigation, the effect of gold nanoparticles with appropriate amount and size on the acoustic cavitation activity has been shown by determining hydroxyl radicals in terephthalic acid solutions containing 15, 20, 28 and 35 nm gold nanoparticles sizes by using 1 MHz low level ultrasound. The effect of sonication intensity in hydroxyl radical production was considered.The recorded fluorescence signal in terephthalic acid solutions containing gold nanoparticles was considerably higher than the terephthalic acid solutions without gold nanoparticles at different intensities of ultrasound irradiation. Also, the results showed that the recorded fluorescence signal intensity in terephthalic acid solution containing finer size of gold nanoparticles was lower than the terephthalic acid solutions containing larger size of gold nanoparticles. Acoustic cavitation in the presence of gold nanoparticles can be used as a way for improving therapeutic effects on the tumors.     

流动微泡群瞬态空化强度时域分布的比例反馈调节*

声波、血流环境及流动微泡群的稳定性都会影响焦区内瞬态空化强度(ICI)在超声作用时间内的分布,从而影响基于瞬态空化的治疗效率和生物安全性。本文在搭建仿体中流动微泡群瞬态空化发生和实时测量系统的基础上,设计了基于LabView FPGA的比例反馈控制器,在保持脉冲重复频率和脉冲长度不变的条件下,通过选择适当的比例系数,依据当前周期的声波激励下实时测量的ICI,实时调节下一周期声波信号的峰值负压,以调控ICI在时间上的分布。研究表明,在最优比例系数(1 × 107)下,和开环系统相比,ICI的稳定率提升~2.31 倍,ICI的时域下降速率减小~94.41%;在超声作用时间内总ICI也基本达到期望水平。这些结果表明该比例反馈控制器在调控脉冲超声激励下流动微泡群ICI时域分布的有效性,有望改进瞬态空化在相关疾病治疗中的效率和安全性。     

Wavelength-dependent Faraday–Tyndall effect on laser-induced microbubble in gold colloid

The cavitation microbubbles in dilute gold colloids of different concentrations (2–10 ppm) induced by a focused nanosecond-pulsed laser beam were measured and characterized at different wavelengths by using the passive and active ultrasound measurements. Three colloids with gold nanoparticles (GNPs) of different sizes (10, 45, and 75 nm) were used for experiment. The results show that the lifespan of the microbubble is reduced as the concentration of GNP increases, particularly at the wavelength of 532 nm, the surface plasmon resonance (SPR) of GNP. In contrast, at the off-resonant wavelength (e.g. 700 nm), the lifespan reduction is relatively small. This wavelength-dependent cavitation is attributed to the Faraday–Tyndall effect, a strong light scattering by GNPs. A slight defocusing of the Gaussian beam in gold colloid was proposed. Hence, the waist of the focused beam increases to reduce the optical breakdown in gold colloid. For simplicity, a linear relation between the incremental waist radius of Gaussian beam and the concentration of GNP was assumed. According to this formulation, the theoretical results are consistent with the experimental ones. In addition, the dynamics of the microbubble in gold colloid measured by the active ultrasound method agree with the Rayleigh–Plesset model.     

Sonochemiluminescence observation and acoustic detection of cavitation induced by pulsed HIFU at a tissue–fluid interface

The aim of this study is to investigate the mechanism of the erosion process induced by 1.2 MHz pulsed high-intensity focused ultrasound (pulsed HIFU). By using Sonochemiluminescence (SCL) photograph, the initiation and maintenance of active cavitation were observed. In order to understand the role of both inertial cavitation and stable cavitation, a passive cavitation detection (PCD) transducer was used. Since the exposure variables of HIFU are important in the controlled ultrasound tissue erosion, the influence of pulse length (PL) and duty cycle (DC, T_on:T_off) has been examined. The results of tissue hole, SCL observation and acoustic detection revealed that the erosion was highly efficient for shorter PL. For higher DCs, the area of SCL increased with increasing PL. For lower DCs, the area of SCL increased with increasing PL from 10 to 20 μs and then kept constant. For all PLs, the intensity of SCL decreased with lower DC. For all DCs, the intensity of SCL per unit area (the ratio of SCL intensity to SCL area) also decreased with increasing PL from 10 to 80 μs, which suggested that the higher the intensity of SCL is, the higher the efficiency of tissue erosion is. At DC of 1:10, the position of the maximum pixel in SCL pictures was distant from the tissue–fluid interface with the increasing PL because of shielding effect. By the comparison of inertial cavitation dose (ICD) and the stable cavitation dose (SCD), the mechanisms associated with inertial cavitation are very likely to be the key factor of the erosion process.     

Dual frequency cavitation event sensor with iodide dosimeter

The inertial cavitation activity depends on the sonication parameters. The purpose of this work is development of dual frequency inertial cavitation meter for therapeutic applications of ultrasound waves. In this study, the chemical effects of sonication parameters in dual frequency sonication (40 kHz and 1 MHz) were investigated in the progressive wave mode using iodide dosimetry. For this purpose, efficacy of different exposure parameters such as intensity, sonication duration, sonication mode, duty factor and net ultrasound energy on the inertial cavitation activity have been studied. To quantify cavitational effects, the KI dosimeter solution was sonicated and its absorbance at a wavelength of 350 nm was measured. The absorbance values in continuous sonication mode was significantly higher than the absorbance corresponding to the pulsed mode having duty factors of 20–80% (p < 0.05). Among different combination modes (1 MHz_100% + 40 kHz_100%, 1 MHz_100% + 40 kHz_80%, 1 MHz_80% + 40 kHz_100%, 1 MHz_80% + 40 kHz_80%), the continuous mode for dual frequency sonication is more effective than other combinations (p < 0.05). The absorbance for this combined dual frequency mode was about 1.8 times higher than that obtained from the algebraic summation of single frequency sonications. It is believed that the optimization of dual frequency sonication parameters at low-level intensity (<3 W/cm²) by optically assisted cavitation event sensor can be useful for ultrasonic treatments.     

Modeling photothermal and acoustical induced microbubble generation and growth

Previous experimental studies showed that powerful heating of nanoparticles by a laser pulse using energy density greater than 100 mJ/cm², could induce vaporization and generate microbubbles. When ultrasound is introduced at the same time as the laser pulse, much less laser power is required. For therapeutic applications, generation of microbubbles on demand at target locations, e.g. cells or bacteria can be used to induce hyperthermia or to facilitate drug delivery. The objective of this work is to develop a method capable of predicting photothermal and acoustic parameters in terms of laser power and acoustic pressure amplitude that are needed to produce stable microbubbles; and investigate the influence of bubble coalescence on the thresholds when the microbubbles are generated around nanoparticles that appear in clusters.

We develop and solve here a combined problem of momentum, heat and mass transfer which is associated with generation and growth of a microbubble, filled with a mixture of non-vaporized gas (air) and water vapor. The microbubble’s size and gas content vary as a result of three mechanisms: gas expansion or compression, evaporation or condensation on the bubble boundary, and diffusion of dissolved air in the surrounding water. The simulations predict that when ultrasound is applied relatively low threshold values of laser and ultrasound power are required to obtain a stable microbubble from a single nanoparticle. Even lower power is required when microbubbles are formed by coalescence around a cluster of 10 nanoparticles. Laser pulse energy density of 21 mJ/cm² is predicted for instance together with acoustic pressure of 0.1 MPa for a cluster of 10 or 62 mJ/cm² for a single nanoparticle. Those values are well within the safety limits, and as such are most appealing for targeted therapeutic purposes.     

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.     

Correlation between microbubble-induced acoustic cavitation and hemolysis in vitro

Microbubbles promise to enhance the efficiency of ultrasound-mediated drug delivery and gene therapy by taking advantage of artificial cavitation nuclei.The purpose of this study is to examine the ultrasound-induced hemolysis in the application of drug delivery in the presence of microbubbles.To achieve this goal,human red blood cells mixed with microbubbles were exposed to 1-MHz pulsed ultrasound.The hemolysis level was measured by a flow cytometry,and the cavitation dose was detected by a passive cavitation detecting system.The results demonstrate that larger cavitation dose would be generated with the increase of acoustic pressure,which might give rise to the enhancement of hemolysis.Besides the experimental observations,the acoustic pressure dependence of the radial oscillation of microbubble was theoretically estimated.The comparison between the experimental and calculation results indicates that the hemolysis should be highly correlated to the acoustic cavitation.     

Monitoring of transient cavitation induced by ultrasound and intense pulsed light in presence of gold nanoparticles

One of the most important challenges in medical treatment is invention of a minimally invasive approach in order to induce lethal damages to cancer cells. Application of high intensity focused ultrasound can be beneficial to achieve this goal via the cavitation process. Existence of the particles and vapor in a liquid decreases the ultrasonic intensity threshold required for cavitation onset. In this study, synergism of intense pulsed light (IPL) and gold nanoparticles (GNPs) has been investigated as a means of providing nucleation sites for acoustic cavitation. Several approaches have been reported with the aim of cavitation monitoring. We conducted the experiments on the basis of sonochemiluminescence (SCL) and chemical dosimetric methods. The acoustic cavitation activity was investigated by determining the integrated SCL signal acquired over polyacrylamide gel phantoms containing luminol in the presence and absence of GNPs in the wavelength range of 400–500 nm using a spectrometer equipped with cooled charged coupled devices (CCD) during irradiation by different intensities of 1 MHz ultrasound and IPL pulses. In order to confirm these results, the terephthalic acid chemical dosimeter was utilized as well. The SCL signal recorded in the gel phantoms containing GNPs at different intensities of ultrasound in the presence of intense pulsed light was higher than the gel phantoms without GNPs. These results have been confirmed by the obtained data from the chemical dosimetry method. Acoustic cavitation in the presence of GNPs and intense pulsed light has been suggested as a new approach designed for decreasing threshold intensity of acoustic cavitation and improving targeted therapeutic effects.     

Precise spatial control of cavitation erosion in a vessel phantom by using an ultrasonic standing wave

In atherosclerotic inducement in animal models, the conventionally used balloon injury is invasive, produces excessive vessel injuries at unpredictable locations and is inconvenient in arterioles. Fortunately, cavitation erosion, which plays an important role in therapeutic ultrasound in blood vessels, has the potential to induce atherosclerosis noninvasively at predictable sites. In this study, precise spatial control of cavitation erosion for superficial lesions in a vessel phantom was realised by using an ultrasonic standing wave (USW) with the participation of cavitation nuclei and medium-intensity ultrasound pulses. The superficial vessel erosions were restricted between adjacent pressure nodes, which were 0.87 mm apart in the USW field of 1 MHz. The erosion positions could be shifted along the vessel by nodal modulation under a submillimetre-scale accuracy without moving the ultrasound transducers. Moreover, the cavitation erosion of the proximal or distal wall could be determined by the types of cavitation nuclei and their corresponding cavitation pulses, i.e., phase-change microbubbles with cavitation pulses of 5 MHz and SonoVue microbubbles with cavitation pulses of 1 MHz. Effects of acoustic parameters of the cavitation pulses on the cavitation erosions were investigated. The flow conditions in the experiments were considered and discussed. Compared to only using travelling waves, the proposed method in this paper improves the controllability of the cavitation erosion and reduces the erosion depth, providing a more suitable approach for vessel endothelial injury while avoiding haemorrhage.     

Superhydrophobic silica nanoparticles as ultrasound contrast agents

Microbubbles have been widely studied as ultrasound contrast agents for diagnosis and as drug/gene carriers for therapy. However, their size and stability (lifetime of 5–12 min) limited their applications. The development of stable nanoscale ultrasound contrast agents would therefore benefit both. Generating bubbles persistently in situ would be one of the promising solutions to the problem of short lifetime. We hypothesized that bubbles could be generated in situ by providing stable air nuclei since it has been found that the interfacial nanobubbles on a hydrophobic surface have a much longer lifetime (orders of days). Mesoporous silica nanoparticles (MSNs) with large surface areas and different levels of hydrophobicity were prepared to test our hypothesis. It is clear that the superhydrophobic and porous nanoparticles exhibited a significant and strong contrast intensity compared with other nanoparticles. The bubbles generated from superhydrophobic nanoparticles sustained for at least 30 min at a MI of 1.0, while lipid microbubble lasted for about 5 min at the same settings. In summary MSNs have been transformed into reliable bubble precursors by making simple superhydrophobic modification, and made into a promising contrast agent with the potentials to serve as theranostic agents that are sensitive to ultrasound stimulation.     

Dependence of cavitation,chemical effect,and mechanical effect thresholds on ultrasonic frequency

Cavitation, chemical effect, and mechanical effect thresholds were investigated in wide frequency ranges from 22 to 4880 kHz. Each threshold was measured in terms of sound pressure at fundamental frequency. Broadband noise emitted from acoustic cavitation bubbles was detected by a hydrophone to determine the cavitation threshold. Potassium iodide oxidation caused by acoustic cavitation was used to quantify the chemical effect threshold. The ultrasonic erosion of aluminum foil was conducted to estimate the mechanical effect threshold. The cavitation, chemical effect, and mechanical effect thresholds increased with increasing frequency. The chemical effect threshold was close to the cavitation threshold for all frequencies. At low frequency below 98 kHz, the mechanical effect threshold was nearly equal to the cavitation threshold. However, the mechanical effect threshold was greatly higher than the cavitation threshold at high frequency. In addition, the thresholds of the second harmonic and the first ultraharmonic signals were measured to detect bubble occurrence. The threshold of the second harmonic approximated to the cavitation threshold below 1000 kHz. On the other hand, the threshold of the first ultraharmonic was higher than the cavitation threshold below 98 kHz and near to the cavitation threshold at high frequency.     

Phospholipid-stabilized microbubbles: Influence of shell chemistry on cavitation threshold and binding to giant uni-lamellar vesicles

We demonstrate the feasibility of covalently linking a single microbubble to a single, giant uni-lamellar vesicle (GUV). Such a combination of GUV plus microbubble might prove useful as a new drug delivery vehicle involving microbubble cavitation-induced sonoporation of the vesicle bilayer as a release mechanism. We therefore applied the well known methodology of passive cavitation detection to measure the influence of lipid shell chemistry on inertial cavitation thresholds for externally added microbubbles. We find that cavitation threshold changes significantly with changes in either molecular weight or mole fraction of poly(ethylene glycol), historically used to impede gas dissolution and microbubble coalescence. We attribute changes in cavitation threshold to changes in microbubble resonance frequency resulting from changes in microbubble shell bending elasticity. To further demonstrate the influence of shell chemistry on microbubble behavior, we describe how several common bubble phenomena - and some new - respond to changes in lipid chain length.     

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.     

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。      

Targeted microbubbles for ultrasound mediated gene transfection and apoptosis induction in ovarian cancer cells

Ultrasound-targeted microbubble destruction (UTMD) technique can be potentially used for non-viral delivery of gene therapy. Targeting wild-type p53 (wtp53) tumor suppressor gene may provide a clinically promising treatment for patients with ovarian cancer. However, UTMD mediated gene therapy typically uses non-targeted microbubbles with suboptimal gene transfection efficiency. We synthesized a targeted microbubble agent for UTMD mediated wtp53 gene therapy in ovarian cancer cells. Lipid microbubbles were conjugated with a Luteinizing Hormone–Releasing Hormone analog (LHRHa) via an avidin–biotin linkage to target the ovarian cancer A2780/DDP cells that express LHRH receptors. The microbubbles were mixed with the pEGFP-N1-wtp53 plasmid. Upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm²) for 30 s, the wtp53 gene was transfected to the ovarian cancer cells. The transfection efficiency was (43.90 ± 6.19)%. The expression of wtp53 mRNA after transfection was (97.08 ± 12.18)%. The cell apoptosis rate after gene therapy was (39.67 ± 5.95)%. In comparison with the other treatment groups, ultrasound mediation of targeted microbubbles yielded higher transfection efficiency and higher cell apoptosis rate (p < 0.05). Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles will enhance the gene transfection efficiency in ovarian cancer cells.     

Effect of process intensifying parameters on the hydrodynamic cavitation based degradation of commercial pesticide (methomyl) in the aqueous solution

Methomyl, a carbamate pesticide, is classified as a pesticide of category-1 toxicity and hence shows harmful effects on both human and aquatic life. In the present work, the degradation of methomyl has been studied by using hydrodynamic cavitation reactor (HC) and its combination with intensifying agents such as H₂O₂, fenton reagent and ozone (hybrid processes). Initially, the optimization of operating parameters such pH and inlet pressure to the cavitating device (circular venturi) has been carried out for maximizing the efficacy of hydrodynamic cavitation. Further degradation study of methomyl by the application of hybrid processes was carried out at an optimal pH of 2.5 and the optimal inlet pressure of 5 bar. Significant synergetic effect has been observed in case of all the hybrid processes studied. Synergetic coefficient of 5.8, 13.41 and 47.6 has been obtained by combining hydrodynamic cavitation with H₂O₂, fenton process and ozone respectively. Efficacy of individual and hybrid processes has also been obtained in terms of energy efficiency and extent of mineralization. HC + Ozone process has proved to be the most effective process having highest synergetic coefficient, energy efficiency and the extent of mineralization. The study has also encompassed the identification of intermediate by-products generated during the degradation and has proposed the probable degradation pathway. It has been conclusively established that hydrodynamic cavitation in the presence of intensifying agents can effectively be used for complete degradation of methomyl.