超声波声孔效应中气泡动力学的研究

在超声快速制取组织细胞病理切片的过程中，发现激励信号对切片制取效果有明显的影响.为了掌握超声激励信号对组织细胞的影响规律，达到快速制取病理切片的最佳状态，从气泡空化模型入手，通过改变激励信号频率、声压、气泡初始半径和液体黏滞系数等参量，研究了声孔效应中气泡动力学激励机制.数值计算表明：空化泡振动随激励声压增强而升高，随液体黏滞系数增强而减弱；一定频率范围内空化泡振动能保持在膨胀、收缩和振荡的稳定空化状态，存在空化泡稳态振动的最佳激励频率；一定初始半径能保证空化泡产生稳定的振动，存在空化泡稳态振动幅度最大的初始半径.实际操作中，在频率、声压、初始半径和黏滞系数综合作用的若干空化阈内，声孔效应使超声快速法制取细胞组织切片获得最佳效果. 关键词： 声孔效应 超声空化 气泡振动 稳态空化域     

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.     

Structural and permeability sensitivity of cells to low intensity ultrasound: Infrared and fluorescence evidence in vitro

This work is focused on the in vitro study of the effects induced by medical ultrasound (US) in murine fibroblast cells (NIH-3T3) at a low-intensity of exposure (spatial peak temporal average intensity I_ta < 0.1 W cm⁻²). Conventional 1 MHz and 3 MHz US devices of therapeutic relevance were employed with varying intensity and exposure time parameters. In this framework, upon cells exposure to US, structural changes at the molecular level were evaluated by infrared spectroscopy; alterations in plasma membrane permeability were monitored in terms of uptake efficiency of small cell-impermeable model drug molecules, as measured by fluorescence microscopy and flow cytometry. The results were related to the cell viability and combined with the statistical PCA analysis, confirming that NIH-3T3 cells are sensitive to therapeutic US, mainly at 1 MHz, with time-dependent increases in both efficiency of uptake, recovery of wild-type membrane permeability, and the size of molecules entering 3T3. On the contrary, the exposures from US equipment at 3 MHz show uptakes comparable with untreated samples.     

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.     

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