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.     

高强度聚焦超声经颅脑肿瘤治疗焦域的仿真研究*

为了研究脑组织和脑肿瘤组织在HIFU治疗时形成焦域的特性及坏死肿瘤组织、不同治疗剂量参数以及多次治疗时焦点间距和时间间隔对HIFU形成焦域的影响,本文以脑胶质瘤患者为例,利用患者头颅CT图像数据建立HIFU经颅治疗的仿真模型,基于Westervelt声波非线性传播方程和Pennes生物热传导方程进行HIFU经颅治疗的仿真研究。结果表明,脑组织和脑肿瘤组织内形成HIFU焦域的差异较小,坏死肿瘤组织对HIFU温度场分布有较小影响;辐照声强越大,焦点温升达到同一温度所用时间越短,焦域长短轴越短,颅骨处温升越低;当焦点间距在一定范围内时,第一次辐照形成的温度场分布对第二个焦点温升达到同一温度所需时间影响较大;两次辐照时间间隔对颅骨处温升和两次聚焦形成60?C以上温度分布影响较小。     

Comparisons among sensitivities of different tumor cells to focused ultrasound in vitro

This study is to test the sensitivities of different tumor cells to ultrasound irradiation at the frequency of 2.2 MHz for 60 s duration, and investigate the potential mechanism underlying different sensitivities. Three murine tumor models with distinct aggressiveness (S180, H-22 and EAC) were exposed to ultrasound to evaluate their sonodynamic efficiencies, and several biological parameters such as cell membrane permeability, lipid peroxidation (LPO), ultra-structure observation, intracellular reactive oxygen species (ROS) and mitochondria membrane potential (MMP) were analyzed after exposures. The results showed that cellular responses of different cells were distinct, of interest to note, the aggressive S180 cells were much more sensitive than others, whereas EAC cells were relatively more resistant to ultrasound irradiation. The direct comparisons among different types of cells indicate that the sono-sensitization seems to depend on the physiological and chemical properties of tumor cells. Perhaps sections of cell membrane became destabilized following the initial radical attack and LPO reaction, which caused S180 cells more susceptible to mechanical stresses during sonolysis. This study provides important implications for cancer therapy.     

The promise of low-intensity ultrasound: A review on sonosensitizers and sonocatalysts by ultrasonic activation for bacterial killing

Antimicrobial resistance has become one of the main public health issues in modern society. Ultrasonic antimicrobial treatment (UAT) is expected to solve the problem of antimicrobial resistance since ultrasonic treatment does not cause drug resistance during inactivation. However, the ultrasonic application is hindered due to the high energy cost. To cast more lights on the ultrasound in tandem with catalysts as a superior strategy for bacterial inactivation, the present review focuses on the UAT with the assistant of continuous development of organic sonosensitizer and inorganic sonocatalyst. With the application of these nanomaterials, the ultrasonic parameters changed from low-frequency and high-power ultrasound to high-frequency and low-power ultrasound. The review also presents the composition of sonosensitizers/sonocatalysts including organic and inorganic nanoparticles and discusses the ultrasonic activation mechanisms triggered by these catalysts. Based on the synergistic effect of ultrasound and catalysts, we discuss the importance of extracellular oxidation and intracellular oxidation in the process of bacterial inactivation. Overall, UAT combined with catalysts appears to be an effective treatment strategy that can be successfully applied in the field of medicine, environmental treatment, and food industry.     

High intensity focused ultrasound-induced gene activation in sublethally injured tumor cells in vitro

Cultured human cervical cancer (HeLa) and rat mammary carcinoma (R3230Ac) cells were transfected with vectors encoding green fluorescent protein (GFP) under the control of hsp70B promoter. Aliquots of 10-microl transfected cells (5 x 10(7) cells/ml) were placed in 0.2-ml thin-wall polymerase chain reaction tubes and exposed to 1.1-MHz high intensity focused ultrasound (HIFU) at a peak negative pressure P- = 2.68 MPa. By adjusting the duty cycle of the HIFU transducer, the cell suspensions were heated to a peak temperature from 50 to 70 degrees C in 1-10 s. Exposure dependent cell viability and gene activation were evaluated. For a 5-s HIFU exposure, cell viability dropped from 95% at 50 degrees C to 13% at 70 degrees C. Concomitantly, gene activation in sublethally injured tumor cells increased from 4% at 50 degrees C to 41% at 70 degrees C. A similar trend was observed at 60 degrees C peak temperature as the exposure time increased from 1 to 5 s. Further increase of exposure duration to 10 s led to significantly reduced cell viability and lower overall gene activation in exposed cells. Altogether, maximum HIFU-induced gene activation was achieved at 60 degrees C in 5 s. Under these experimental conditions, HIFU-induced gene activation was found to be produced primarily by thermal rather than mechanical stresses.     

超声波与电化学协同作用降解硝基苯溶液的实验研究

本文以硝基苯模拟废水为研究对象，探讨了超声波与电化学协同作用降解硝基苯的实验情况。考察了处理时间、处理温度、硝基苯初始浓度、pH值、电解电压等因素对硝基苯降解率的影响。实验结果表明：随着作用时间增加，硝基苯的降解率升高；温度高于40℃时，硝基苯的降解率随温度的升高而降低，低于40℃时，硝基苯的降解率随温度的升高而增大；硝基苯的降解率随电压的升高而增大；酸性条件有利于硝基苯的降解；硝基苯初始浓度越大降解率越高。     

Study of cell killing effect on S180 by ultrasound activating protoporphyrin IX

The present study was initiated to investigate the potential biological mechanism of cell killing effect on isolate sarcoma 180 (S180) cells induced by ultrasound activating protoporphyrin IX (PPIX). S180 cells were exposed to ultrasound for 30 s duration, at a frequency of 2.2 MHz and an acoustic power of 3 W/cm² in the presence of 120 μM PPIX. The viability of cells was evaluated using trypan blue staining. The generation of oxygen free radicals in cell suspensions was detected immediately after treatment using a reactive oxygen detection kit. A copper reagent colorimetry method was used to measure the level of FFAs released into cell suspensions by the process of cell damage induced by ultrasound and PPIX treatment. Oxidative stress was assessed by measuring the activities of key antioxidant enzymes (i.e., SOD, CAT, GSH-PX) in S180 tumor cells. Treatment with ultrasound and PPIX together increased the cell damage rate to 50.91%, while treatment with ultrasound alone gave a cell damage rate to 24.24%, and PPIX alone kept this rate unchanged. Colorimetry and enzymatic chemical methods showed that the level of FFAs in cell suspension increased significantly after the treatment, while the activity of all the above enzymes decreased in tumor cells at different levels, and were associated with the generation of oxygen free radicals in cell suspension after treatment. The results indicate that oxygen free radicals may play an important role in improving the membrane lipid peroxidation, degrading membrane phospholipids to release FFAs, and decreasing the activities of the key antioxidant enzymes in cells. This biological mechanism might be involved in mediating the effects on S180 cells and resulting in the cell damage seen with SDT.     

纹影法测量远场焦斑实验研究

 报导了用纹影法测量远场焦斑的实验结果。远场焦斑经成像透镜放大成像后,由12bit,1 024×1 024面阵科学级CCD相机采集。将一定大小的小球放置在焦斑位置,用以遮挡光斑中心,控制CCD前的衰减可得旁瓣信息,将其与无遮挡光斑拼接,得到焦斑相对强度的完整分布。该方法扩展了焦斑光强测试的动态范围,可探测焦斑约4个量级的光能密度分布,提高了焦斑的测量精度。     

纹影法测量远场焦斑实验研究

报导了用纹影法测量远场焦斑的实验结果。远场焦斑经成像透镜放大成像后,由12bit,1 024×1 024面阵科学级CCD相机采集。将一定大小的小球放置在焦斑位置,用以遮挡光斑中心,控制CCD前的衰减可得旁瓣信息,将其与无遮挡光斑拼接,得到焦斑相对强度的完整分布。该方法扩展了焦斑光强测试的动态范围,可探测焦斑约4个量级的光能密度分布,提高了焦斑的测量精度。     

Experimental study of the ultrasound attenuation in chemical reactors

Ultrasound is used in different domains, and in sonochemistry particularly, for different purposes and in various flow configurations: monophasic, two-phase and polyphasic reactors. In order to optimize and to design sonochemical reactors, it is important to describe the ultrasonic intensity space and time distribution. In addition, it is important to study the different parameters influencing the intensity profiles of the ultrasonic wave. In this work, a thermoelectric probe has been used to measure the ultrasonic intensity. This procedure has shown that the ultrasound propagation is influenced by the presence of cavitation bubbles, the flow regime and the presence of solid particles.     

The activation mechanism of peroxidase by ultrasound

The activation mechanism of peroxidase by ultrasound was investigated. The catalysis performance of peroxidase with ultrasound treatment was prior to the controls determined by UV–visible spectra and Fourier transform infrared spectra. The transformation of tryptophan residues in peroxidase led to the increase of a-helix and anti-parallel content in the secondary structure, and the content of p-sheet, p-turn and random coil in the secondary structure. In addition, under the atomic force microscope, under ultrasonic treatment, the large molecular clusters of tyrosinase are broken down into small molecular clusters. The current results showed that the activity of peroxidase is activated under ultrasonic treatment, which is mainly caused by ultrasound without conformational change, the catalytic center is exposed, and the affinity with the substrate is stronger.     

Membrane fluidity altering and enzyme inactivating in sarcoma 180 cells post the exposure to sonoactivated hematoporphyrin in vitro

Sonodynamic therapy (SDT) is a novel tumor therapy method. We investigated membrane fluidity, activity of the enzymes and membrane morphology in vitro post hematoporphyrin-SDT treatment. Furthermore, the potential mechanisms behind the changes in membrane fluidity and enzymic activity were discussed. Tumor cells were exposed to ultrasound at 1.75 MHz for up to 3 min in the presence and absence of hematoporphyrin. Fluorescence polarization, contents of Malonaldehyde, and levels of free fatty acid were assessed. Activity of enzymes was checked by the plumbic nitrate detection method. For the morphologic study, a scanning electron microscope was used to observe the cellular surface. Ultrasonically induced cell damage increased in the presence of HPD (from 15% to 24%). Compared with ultrasound treatment alone, the fluidity decreased from 5.037 to 3.908, malonaldehyde content and free fatty acid level increased from 0.743 nmol/mL to 0.979 nmol/mL and from 237.180 μmol/L to 730.769 μmol/L, respectively, post ultrasound combined with HPD treatment. Inactivity of adenylate cyclase and guanylate cyclase and significant deformation of the cellular surface were also observed post SDT treatment. Our results suggested that alterations in membrane modality and lipid composition played important roles in SDT-mediated inhibition of tumor growth, even inducing tumor cell death, which might be attributed to a sono-chemical activation mechanism.     

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。     

Experimental study on the activation process of GaAs spin—polarized electron source

GaAs spin-polarized electron source is a new kind of electron source, where the GaAs semiconductor crystal is used as a photocathode under the irradiation of helicity light. In this paper the activation process of the GaAs spin-polarized electron source is unvestigated experimentally in detail, during which the negative electron affinity of the photo cathode should be achieved more carefully by absorbing the caesium and oxygen on the surface of the GaAs crystal under ultrahigh vacuum conditions. Besides the different activation processes, the important physical parameters are studied to achieve the optimum activation results. At the same time the stability and lifetime of the polarized electron beam are explored for future experiments. Some important experimental data have been acquired.     

复频聚焦超声换能器水中焦区商场特性的研究

本文对新研制的一种复频聚焦超声换能器的辐射声场进行了实验研究,测定了辐射压在轴向上的分布曲线,进而确定了焦区位置,这与理论所得结果符合较好,测定了辐射声压在焦平面上的分布曲线,并对焦区声场进行了定位,最后对换能器在水中焦区辐射声的频谱进行了研究,不仅观测到了两个源波,而且还观测到了和频波,差频波以及倍频波,证实了声散射声效应的存在。     

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。     

Basic study on apoptosis induction into cancer cells U-937 and EL-4 by ultrasound exposure

Recently, the low invasive cancer treatments with small aftereffects have been considered. We are studying on the suppression methods of cancer cell proliferation with ultrasound. Cancer cells of mouse T lymphoma (EL-4) have been used in our study. The human histitocytic lymphoma cells (U-937) was used in this time. The cancer cells were cultured in a culture medium of RPMI1640. The standing wave acoustic field was formed in a water tank of our ultrasound exposure system by a vibrating plate driven with a Langevine type transducer. The U-937 and EL-4 were exposed to ultrasound in the acoustic field with spatial average acoustic intensity of 350 mW/cm(2) at 150 kHz. The viable rate of EL-4 decreased with the lapse of culture time after ultrasound exposure. U-937 did not show the remarkable decrease tendency. The proliferation of U-937 which exposed to ultrasound with 700 mW/cm(2) was suppressed. It can be thought that apoptosis was induced in the cancer cells in this condition. We observed the morphological change on the U-937 exposed to ultrasound with this condition. The morphological changes by apoptosis like the shrink of cells, formation of apoptotic bodies etc. can be observed with an optical microscope and a phase contrast microscope.     

Treatable focal region modulated by double excitation signal superimposition to realize platform temperature distribution during transcranial brain tumor therapy with high-intensity focused ultrasound

Recently, the phase compensation technique has allowed the ultrasound to propagate through the skull and focus into the brain. However, the temperature evolution during treatment is hard to control to achieve effective treatment and avoid over-high temperature. Proposed in this paper is a method to modulate the temperature distribution in the focal region.It superimposes two signals which focus on two preset different targets with a certain distance. Then the temperature distribution is modulated by changing triggering time delay and amplitudes of the two signals. The simulation model is established based on an 82-element transducer and computed tomography(CT) data of a volunteer's head. A finitedifference time-domain(FDTD) method is used to calculate the temperature distributions. The results show that when the distances between the two targets respectively are 7.5–12.5 mm on the acoustic axis and 2.0–3.0 mm in the direction perpendicular to the acoustic axis, a focal region with a uniform temperature distribution(64–65℃) can be created.Moreover, the volume of the focal region formed by one irradiation can be adjusted(26.8–266.7 mm~3) along with the uniform temperature distribution. This method may ensure the safety and efficacy of HIFU brain tumor therapy.     

Experimental study of ultrasound-modulated scattering light using different frequencies ultrasound probes

The focused ultrasound plays a role in localization and modulating the scattering light in ultrasound- modulated optical tomography （UOT）. Both the modulation efficiency of the scattering light and the spatial resolution of UOT are determined by ultrasound. The effects of repetition frequency and pulse energy of impulse ultrasound on the modulated scattering light are derived through experiment in this letter. Purthermore, we compare the imaging sensitivity with 1, 2.25, 5, and 10 MHz center frequencies of impulse ultrasound. Experimental results demonstrate that better signal-to-noise ratios and higher sensitivities can be gained by use of more intense ultrasound and lower ultrasound frequencies.