三种肿瘤细胞对超声结合血卟啉敏感性的实验研究

实验采用频率为1．6MHz，强度为1W／cm^2、3W／cm^2、5W／cm^2、7W／cm^2的聚焦超声结合血卟啉分别对腹水型小鼠S180细胞（Sarcomal80，S180）、艾氏腹水瘤细胞（Ehrlich Ascites Tumor，EAT）及H-22肝癌细胞进行杀伤效应研究，利用台盼蓝拒染法检测处理后细胞存活率的变化。实验结果表明，不同类型的细胞对超声结合血卟啉的敏感性不同，声照条件相同时，三种细胞对1．6MHz频率超声的敏感性依次为：S18〉EAT〉H-22，且对超声结合血卟啉敏感性明显大于单纯超声。无论是单纯超声组还是超声结合血卟啉组，三种细胞的存活率均随超声强度的增加而下降。     

脉冲占空比对磁性微泡介导的聚焦超声温升效应的影响

集合多种诊断和治疗功能的声/磁造影剂微泡的研究与开发已经成为当前医学超声、生物医学工程及临床应用领域共同关注的热点问题.超顺磁氧化铁纳米颗粒具有独特的磁性特征和良好的生物相容性,可被用作核磁共振造影剂来提升影像对比度、空间分辨率及临床诊断准确性.我们的前期工作表明,通过将超顺磁氧化铁纳米颗粒挂载于常规超声造影剂微泡表面,可以成功构建多模态诊断及治疗介质,显著改变超声造影剂微泡的尺度分布及包膜粘弹系数等物理特性,进而影响微泡造影剂的声散射特性及其声空化效应和热效应.然而,此前的研究仅考虑了声场强度和微泡浓度等影响因素,对于脉冲超声时间特性对磁性微泡造影剂动力学响应的影响的相关研究仍有所欠缺.本文通过热电偶对凝胶仿体血管模型中流动的双模态磁性微泡在不同占空比超声脉冲信号作用下,产生温升效应开展了系统的实验测量,并基于有限元模型对实验结果进行了仿真验证.结果显示,脉冲信号占空比的提升是增强血管中磁性微泡在聚焦超声作用下温升效果的关键性时间影响因素.本文的研究成果将有助于更好地理解不同超声作用参数对双模态磁性微泡的热效应的影响机制,对保障双模态磁性微泡在临床热疗应用中的安全性和有效性具有重要的...     

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。      

基于受激发光实验的生物光尾流特性

 基于生物受激发光与流场机械刺激间的相关性,构建了库埃特流场刺激下的生物受激发光实验平台,分析了影响生物发光的水动力因素剪应力。实验结果显示：引起多边膝沟藻受激发光的剪应力阈值为0.1 N/m²;当流场中的剪应力大于发光阈值后,生物发光强度随着剪应力的增大而增强。采用FLUENT软件模拟计算了不同航速下某潜艇尾流场中的剪应力,计算结果表明：剪应力随着尾流长度的增加而减小,在近场区域剪应力出现了类似正弦波振荡的减小,且航速越大减幅越大,减速越快;当潜艇航速大于4.1 m/s航行时, 2 000 m处尾流的剪应力仍然大于引起生物发光的剪应力阈值。     

超声滚压中的空化现象*

在超声滚压加工中引入切削液后可能会产生空化现象,由此产生的微射流和冲击波对超声表面强化将有积极作用。为研究超声滚压加工中空化现象是否存在及空化效应在超声滚压中的作用,本文首先分析了超声滚压中的空化阈值,然后进行了染色法试验和超声滚压后试样氧元素能谱分析,最后通过超声滚压加工对比试验研究了空化效应对加工后材料表面粗糙度和显微硬度的影响。研究发现,超声滚压加工中的声压幅值远大于空化阈值,满足空化存在的必要条件;超声滚压中发生了明显的卡纸染色现象,引入切削液后工件超声滚压加工表面氧元素含量显著提高,表明超声滚压中发生了空化现象。超声滚压加工中的空化效应能进一步降低工件表面粗糙度和提高表面显微硬度,有利于提高工件表面强化质量。本研究为空化效应在超声滚压中的积极利用提供了依据。     

超声场中空化泡对弹性粒子微流的影响

从声散射基本理论出发,考虑弹性粒子与空化泡之间耦合作用,结合边界条件,推导了弹性粒子外部声流分布,得到了声微流的n=0和n=1模式近似表达式和粒子表面应力分布函数.数值分析结果表明:气泡和弹性粒子之间的耦合作用增加了粒子周围的微流分布与剪应力场分布,特别是微流速度的切向分量.随着两者间距与相对位置的距离增大,粒子与气泡之间相互作用减弱,粒子周围微流幅值减小;当气泡处于共振状态时,粒子周围的微流分布显著增强.粒子表面剪应力场受粒子半径与声场频率影响,当粒子半径与声场频率越大,外部散射声强越强,粒子表面剪应力幅值越大.     

混响场中超声化学效应的研究

本文借助荧光光谱分析法,使用815kHz超声波对小尺度混响场中声化学产额进行了研究。研究发现混响场中的声化学效应有两个特点:一是声空化的阈值声强下降到约0.3W/cm~2(行波场为0.7W/cm~2);二是声强大于阈值时,声化学产额随声强而增加,随后(声强约为1.69—2.13w/cm~2)出现陡然上升的拐点(行波场则趋向饱和)。理论分析表明,阈值下降是由于混响场的声能密度增大;声化学产额随声强变化的拐点则来自于声波辐射压力对液体表面的干扰.为此,本文从实验与理论上证明,为使声化学获得尽可能高的产额应在声化学反应器内建立混响声场。     

基于蚯蚓背孔射流的仿生射流表面减阻性能研究

为了减小流体对固体壁面的阻力, 基于蚯蚓生物学特征, 对蚯蚓背孔射流特性进行分析, 建立仿蚯蚓背孔射流的仿生射流表面计算模型, 采用SST k-ω 湍流模型对仿生射流表面的减阻特性进行数值模拟, 同时对数值模拟结果进行实验验证, 并以此研究了仿蚯蚓背孔射流表面的减阻机理.结果表明, 在一定条件下, 仿蚯蚓背孔射流的仿生射流表面具有较好的减阻效果; 在同一射流方向角下, 随着射流速度的增加, 减阻率逐渐增大; 在同一射流速度下, 随着射流方向角的增加, 减阻率呈先减小后增大的变化趋势; 数值模拟与实验均在射流速度为1 m·s^-1、射流方向角为-30°时达到最大, 分别为8.69%, 7.86%; 射流表面改变了原有光滑壁面的边界层结构, 对壁面边界层进行了有效的控制, 减小了壁面的剪应力, 降低了壁面边界层的速度.     

生物受激发光的管流实验研究

构建了管流场刺激下的生物受激发光实验平台,分析了管流场中的水动力特性和影响生物受激发光的水动力因素。采用光子计数器测量了3种腰鞭毛虫在管流刺激下的受激发光特性。实验结果表明:海洋生物体受激发光的水动力影响因素是流场剪应力,引起生物体发光的剪应力存在剪应力阈值,只有当剪应力大于阈值,生物体才能被激发发光。根据生物体种类的不同,刺激生物受激发光的剪应力阈值为0.05~0.29N/m2。通过对实验数据的分析得出了生物受激发光的发光强度与剪应力的关系。     

高能超声制备碳纳米管增强AZ91D复合材料的声场模拟

建立了高能超声制备碳纳米管增强AZ91D复合材料的声场计算模型, 并采用有限元方法计算了20 kHz超声直接作用下AZ91D熔体的声场分布, 熔体声场呈辐射状分布, 距离声源越远, 声压幅值越低. 采用超声作用下单一气泡变化模型描述超声作用下AZ91D 熔体中的空化效应, 通过对Rayleigh-Plesset方程的求解, 得到了不同声压作用下气泡的变化规律, 获得了声压幅值与熔体空化效应的关系, 声压幅值越大, 气泡溃灭半径阈值越小, 熔体发生空化效应越容易. 计算了固定坩埚尺寸、不同超声探头没入熔体深度情况下的声场, 得到了超声探头最优没入深度为30 mm左右. 将声场计算结果以及AZ91D熔体中空化效应的发生规律进行综合分析, 得到了超声功率对有效空化区域的影响规律, 超声功率较大时, 有效空化区域体积随超声功率近似成线性增大. 最后, 通过甘油水溶液超声处理实验, 验证了模拟计算的准确性.     

Instant magnetic labeling of tumor cells by ultrasound in vitro

Magnetic labeling of living cells creates opportunities for numerous biomedical applications. Here we describe an instantly cell magnetic labeling method based on ultrasound. We present a detailed study on the ultrasound performance of a simple and efficient labeling protocol for H-22 cells in vitro. High frequency focus ultrasound was investigated as an alternative method to achieve instant cell labeling with the magnetic particles without the need for adjunct agents or initiating cell cultures. Mean diameter of 168 nm dextran-T40 coated superparamagnetic iron oxide (SPIO) nanoparticles were prepared by means of classical coprecipitation in solution in our laboratory. H-22 tumor cells suspended in phosphate-buffered saline (PBS, pH=7.2) were exposed to ultrasound at 1.37 MHz for up to 120 s in the presence of SPIOs. The cellular uptake of iron oxide nanoparticles was detected by prussion blue staining. The viability of cells was determined by a trypan blue exclusion test. At 2 W power and 60 s ultrasound exposure in presence of 410 μg/ml SPIOs, H-22 cell labeling efficiency reached 69.4±6.3% and the labeled cells exhibited an iron content of 10.38±2.43 pg per cell. Furthermore, 95.2±3.2% cells remained viable. The results indicated that the ultrasound protocol could be potentially applied to label cells with large-sized magnetic particles. We also calculated the shear stress at the 2 W power and 1.37 MHz used in experiments. The results showed that the shear stress threshold for ultrasonically induced H-22 cell reparable sonoporation was 697 Pa. These findings provide a quantitative guidance in designing ultrasound protocols for cell labeling.     

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.     

Reparable sonoporation generated by microstreaming

Reparable sonoporation was observed in Jurkat lymphocytes in suspension exposed to a vibrating Mason horn tuned to 21.4 KHz. The diameter of the horn tip was 400 microm and its transverse displacement amplitude was 7.8 microm. It was found that the shear stress associated with microstreaming surrounding the Mason-horn tip was the primary reason for the cell reparable sonoporation. The threshold shear stress was determined to be 12 +/- 4 Pa for exposure time up to 7 min. It was also found that the shorter the exposure time, the greater the threshold.     

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.     

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.     

Sonodynamic therapy--a review of the synergistic effects of drugs and ultrasound

Sonodynamic therapy, the ultrasound dependent enhancement of cytotoxic activities of certain compounds (sonosensitizers) in studies with cells in vitro and in tumor bearing animals, is reviewed. The attractive features of this modality for cancer treatment emerges from the ability to focus the ultrasound energy on malignancy sites buried deep in tissues and to locally activate a preloaded sonosensitizer. Possible mechanisms of sonodynamic therapy include generation of sonosensitizer derived radicals which initiate chain peroxidation of membrane lipids via peroxyl and/or alkoxyl radicals, the physical destabilization of the cell membrane by the sonosensitizer thereby rendering the cell more susceptible to shear forces or ultrasound enhanced drug transport across the cell membrane (sonoporation). Evidence against the role of singlet oxygen in sonodynamic therapy is discussed. The mechanism of sonodynamic therapy is probably not governed by a universal mechanism, but may be influenced by multiple factors including the nature of the biological model, the sonosensitizer and the ultrasound parameters. The current review emphasizes the effect of ultrasound induced free radicals in sonodynamic therapy.     

Effects of extracellular matrix rigidity on sonoporation facilitated by targeted microbubbles: Bubble attachment,bubble dynamics,and cell membrane permeabilization

In this study, we investigated the effects of extracellular matrix rigidity, an important physical property of microenvironments regulating cell morphology and functions, on sonoporation facilitated by targeted microbubbles, highlighting the role of microbubbles. We conducted mechanistic studies at the cellular level on physiologically relevant soft and rigid substrates. By developing a unique imaging strategy, we first resolved details of the 3D attachment configurations between targeted microbubbles and cell membrane. High-speed video microscopy then unveiled bubble dynamics driven by a single ultrasound pulse. Finally, we evaluated the cell membrane permeabilization using a small molecule model drug. Our results demonstrate that: (1) stronger targeted microbubble attachment was formed for cells cultured on the rigid substrate, while six different attachment configurations were revealed in total; (2) more violent bubble oscillation was observed for cells cultured on the rigid substrate, while one third of bubbles attached to cells on the soft substrate exhibited deformation shortly after ultrasound was turned off; (3) higher acoustic pressure was needed to permeabilize the cell membrane for cells on the soft substrate, while under the same ultrasound condition, acoustically-activated microbubbles generated larger pores as compared to cells cultured on the soft substrate. The current findings provide new insights to understand the underlying mechanisms of sonoporation in a physiologically relevant context and may be useful for the clinical translation of sonoporation.     

Stabilizing in vitro ultrasound-mediated gene transfection by regulating cavitation

It is well known that acoustic cavitation can facilitate the inward transport of genetic materials across cell membranes (sonoporation). However, partially due to the unstationary behavior of the initiation and leveling of cavitation, the sonoporation effect is usually unstable, especially in low intensity conditions. A system which is able to regulate the cavitation level during sonication by modulating the applied acoustic intensity with a feedback loop is implemented and its effect on in vitro gene transfection is tested. The regulated system provided better time stability and reproducibility of the cavitation levels than the unregulated conditions. Cultured hepatoma cells (BNL) mixed with 10 μg luciferase plasmids are exposed to 1-MHz pulsed ultrasound with or without cavitation regulation, and the gene transfection efficiency and cell viability are subsequently assessed. Experimental results show that for all exposure intensities (low, medium, and high), stable and intensity dependent, although not higher, gene expression could be achieved in the regulated cavitation system than the unregulated conditions. The cavitation regulation system provides a better control of cavitation and its bioeffect which are crucial important for clinical applications of ultrasound-mediated gene transfection.