The Effect of Binding on the Subharmonic Emissions from Individual Lipid-Encapsulated Microbubbles at Transmit Frequencies of 11 and 25 MHz

Targeted microbubble imaging at ultrasound frequencies above 5 MHz has applications in both a preclinical context for a range of disease processes and clinically for the assessment of atherosclerosis and superficial tumors. Although the feasibility of ultrasound molecular imaging has been well demonstrated for a range of target molecules, little work has examined the effects of binding on microbubble oscillations, which is of potential relevance to improving the sensitivity, specificity, and quantification of bound-bubble detection. In this study we investigated the influence of binding on the subharmonic response of bubbles at transmit frequencies of 11 and 25 MHz. Individual bubbles were situated adjacent to a boundary in either a bound or an unbound state, optically sized and acoustically interrogated with pressures ranging from 0.02 to 1.2 MPa. At 11 MHz, unbound bubbles (n = 53) were found to have strong subharmonic activity for sizes between 2.4 and 2.6 μm, whereas bound bubbles (n = 50) were most active from 2.6 to 3.0 μm. Destruction thresholds were found to be lower for bound bubbles. At 25 MHz, bound-bubble (n = 57) activity was found to peak at 1.9 μm as compared to 2.1 μm in the unbound cases (n = 53), with a 20% increase in amplitude. Comparison with simulations indicates that both unbound and bound bubbles undergo compression-only behavior at 11 MHz, and expansion-dominated behavior at 25 MHz. Subharmonic emissions elicited from 0 radian transmit pulses were found to be π/2 radians out of phase with those elicited from a π radian transmit pulse, suggesting inefficient subharmonic preservation from pulse inversion schemes. With the appropriate postprocessed phase correction, an increase in the subharmonic amplitude of up to 60% was shown, depending on the bubble size and transmit frequency.     

Characterization of the Dynamic Activities of a Population of Microbubbles Driven by Pulsed Ultrasound Exposure in Sonoporation

Ultrasound-driven microbubble activities have been exploited to transiently disrupt the cell membrane (sonoporation) for non-viral intracellular drug delivery and gene transfection both in vivo and in vitro. In this study, we investigated the dynamic behaviors of a population of microbubbles exposed to pulsed ultrasound and their impact on adherent cells in terms of intracellular delivery and cell viability. By systematically analyzing the bubble activities at time scales relevant to pulsed ultrasound exposure, we identified two quantification parameters that categorize the diverse bubble activities subjected to various ultrasound conditions into three characteristic behaviors: stable cavitation/aggregation (type I), growth/coalescence and translation (type II) and localized inertial cavitation/collapse (type III). Correlation of the bubble activities with sonoporation outcome suggested that type III behavior resulted in intracellular delivery, whereas type II behavior caused the death of a large number of cells. These results provide useful insights for rational selection of ultrasound parameters to optimize outcomes of sonoporation and other applications that exploit the use of ultrasound-driven bubble activities.     

Characterizing the subharmonic response of phospholipid-coated microbubbles for carotid imaging

The subharmonic vibration of BR14 (Bracco Research S.A., Geneva, Switzerland) contrast agent microbubbles is investigated within the preferable frequency range for carotid ultrasound imaging (8-12 MHz). The response of the bubbles was recorded optically with an ultra-fast recording camera (Brandaris 128) at three acoustic pressures (50, 100 and 120 kPa). The vibration of the microbubbles was measured as a function of the excitation frequency and its frequency content was determined. Among 390 recordings, 40% showed subharmonic oscillations. It was observed that for smaller microbubbles (diameter < 3 μm) the frequency of the maximum subharmonic response increases for increasing pressures (shell hardening) opposite to what has been reported for larger microbubbles (3 μm < diameter < 15 μm). These findings are well predicted by the model proposed by Marmottant et al. (2005) after including the dilatational shell viscosity of the microbubbles measured by Van der Meer et al. (2007), which indicates a marked shear-thinning behavior of the phospholipid shell.     

Quantitative Guidelines for the Prediction of Ultrasound Contrast Agent Destruction During Injection

Experiments and theory were undertaken on the destruction of ultrasound contrast agent microbubbles on needle injection, with the aim of predicting agent loss during in vivo studies. Agents were expelled through a variety of syringe and needle combinations, subjecting the microbubbles to a range of pressure drops. Imaging of the bubbles identified cases where bubbles were destroyed and the extent of destruction. Fluid-dynamic calculations determined the pressure drop for each syringe and needle combination. It was found that agent destruction occurred at a critical pressure drop that depended only on the type of microbubble. Protein-shelled microbubbles (sonicated bovine serum albumin) were virtually all destroyed above their critical pressure drop of 109 ± 7 kPa Two types of lipid-shelled microbubbles were found to have a pressure drop threshold above which more than 50% of the microbubbles were destroyed. The commercial lipid-shelled agent Definity was found to have a critical pressure drop for destruction of 230 ± 10 kPa; for a previously published lipid-shelled agent, this value was 150 ± 40 kPa. It is recommended that attention to the predictions of a simple formula could preclude unnecessary destruction of microbubble contrast agent during in vivo injections. This approach may also preclude undesirable release of drug or gene payloads in targeted microbubble therapies. Example values of appropriate injection rates for various agents and conditions are given.     

Impact of Filling Gas on Subharmonic Emissions of Phospholipid Ultrasound Contrast Agents

Subharmonic signals backscattered from gas-filled lipid-shelled microbubbles have generated significant research interest because they can improve the detection and sensitivity of contrast-enhanced ultrasound imaging. However, the emission of subharmonic signals is strongly characterized by a temporal dependence, the origins of which have not been sufficiently elucidated. The features that influence subharmonic emissions need to be identified not only to better develop next-generation microbubble contrast agents, but also to develop more efficient subharmonic imaging (SHI) modes and therapeutic strategies. We examined the effect of microbubble filling gas on subharmonic emissions. Phospholipid shelled-microbubbles with different gaseous compositions such as sulfur hexafluoride (SF₆), octafluoropropane (C₃F₈) or decafluorobutane (C₄F₁₀), nitrogen (N₂)/C₄F₁₀ or air were insonated using a driving frequency of 10 MHz and peak negative pressure of 450 kPa, and their acoustic responses were tracked by monitoring both second harmonic and subharmonic emissions. Microbubbles were first acoustically characterized with their original gas and then re-characterized after substitution of the original gas with air, SF₆ or C₄F₁₀. A measureable change in intensity of the subharmonic emissions with a 20- to 40-min delayed onset and increasing subharmonic emissions of the order 12–18 dB was recorded for microbubbles filled with C₄F₁₀. Substitution of C₄F₁₀ with air eliminated the earlier observed delay in subharmonic emissions. Significantly, substitution of SF₆ for C₄F₁₀ successfully triggered a delay in the subharmonic emissions of the resultant agents, whereas substitution of C₄F₁₀ for SF₆ eliminated the earlier observed suppression of subharmonic emissions, clearly suggesting that the type of filling gas contained in the microbubble agent influences subharmonic emissions in a time-dependent manner. Because our agents were dispersed in air-stabilized phosphate-buffered saline, these results suggest that the diffusivity of the gas from the agent to the surrounding medium is correlated with the time-dependent evolution of subharmonic emissions.     

Single Bubble Acoustic Characterization and Stability Measurement of Adherent Microbubbles

This article examines how the acoustic and stability characteristics of single lipid-shelled microbubbles (MBs) change as a result of adherence to a target surface. For individual adherent and non-adherent MBs, the backscattered echo from a narrowband 2-MHz, 90-kPa peak negative pressure interrogation pulse was obtained. These measurements were made in conjunction with an increasing amplitude broadband disruption pulse. It was found that, for the given driving frequency, adherence had little effect on the fundamental response of an MB. Examination of the second harmonic response indicated an increase of the resonance frequency for an adherent MB: resonance radius increasing of 0.3 ± 0.1 μm for an adherent MB. MB stability was seen to be closely related to MB resonance and gave further evidence of a change in the resonance frequency due to adherence.     

The Effect of Short Duration Ultrasound Pulses on the Interaction Between Individual Microbubbles and Fibrin Clots

In previous work, we examined microscale interactions between microbubbles and fibrin clots under exposure to 1 ms ultrasound pulses. This provided direct evidence that microbubbles were capable of deforming clot boundaries and penetrating into clots, while also affecting fluid uptake and inducing fibrin network damage. Here, we investigate the effect of short duration (15 μs) pulses on microscale bubble-clot interactions as function of bubble diameter (3–9 μm) and pressure. Individual microbubbles (n = 45) were placed at the clot boundary with optical tweezers and exposed to 1 MHz ultrasound. High-speed (10 kfps) imaging and 2-photon microscopy were performed during and after exposure, respectively. While broadly similar phenomena were observed as in the 1 ms pulse case (i.e., bubble penetration, network damage and fluid uptake), substantial quantitative differences were present. The pressure threshold for bubble penetration was increased from 0.39 MPa to 0.6 MPa, and those bubbles that did enter clots had reduced penetration depths and were associated with less fibrin network damage and nanobead uptake. This appeared to be due in large part to increased bubble shrinkage relative to the 1 ms pulse case. Stroboscopic imaging was performed on a subset of bubbles (n = 11) and indicated that complex bubble oscillations can occur during this process.     

Frequency and pressure dependent attenuation and scattering by microbubbles

The aim of this study was to evaluate experimentally the degree of pressure dependence of attenuation and scattering by microbubbles at low acoustic pressures with an empirical nonlinear model. In addition, the pressure dependency over a range of frequencies (1 to 5 MHz) has been studied. A series of transmission and scattering measurements were made with the microbubble SonoVue, using an automated system. Results show that, within the pressure range studied, attenuation as a result of the microbubble is pressure-dependent, whereas no such dependence of scattering was detectable. The pressure dependence of attenuation for SonoVue was found to be most significant at 1.5 MHz. The scattering is shown to be the highest at the lowest insonation frequency, around 1 approximately 1.25 MHz, and then decreases with frequency.     

亚微米级超声微泡造影剂的制备及体外基因转染效率的实验研究

目的 探索制备亚微米级超声微泡造影剂的方法 ,以GFP作为目的 基因验证其作为一种新型基因载体的可行性.方法 以高剪切分散法制备超声微泡造影剂,透射电镜及激光粒度分析仪检测其形态及粒径;将超声微泡造影剂与不同剂量的绿色荧光蛋白质粒PShuttle-IRES-hrGFP-1结合后转染HepG2细胞,利用荧光显微镜观察并检测其基因转染效率.结果 自制超声微泡造影剂为均匀分散的圆泡,粒径分布在282.2～415.7 nm之间,平均值为(335±5)nm,达到亚微米级;该微泡能将GFP基因成功转运到HepG2细胞内并高效表达,转染效率达32.61%±3.42%.结论 自制亚微米级超声微泡造影剂粒径小、分散均匀,并能成功转运外源DNA进入细胞内,可作为一种新型基因载体.     

Generation of Reactive Oxygen Species in Heterogeneously Sonoporated Cells by Microbubbles with Single-Pulse Ultrasound

To develop and realize sonoporation-based macromolecule delivery, it is important to understand the underlying cellular bioeffects involved. It is known that an appropriate level of reactive oxygen species (ROS) is necessary to maintain normal physiologic function, but excessive ROS triggers adverse downstream bioeffects. However, it is still unclear whether a relationship exists between intracellular ROS levels and sonoporation. Using a customized platform for 1.5-MHz ultrasound exposure (13.33 µs duration and 0.70?MPa peak negative pressure) and imaging the dynamics of sonoporation and intracellular ROS at the single-cell level, we quantified the exogenous molecular uptake and the concentration of intracellular ROS indicator to evaluate the extent of sonoporation and ROS change, respectively. Our results revealed that the intracellular ROS level was correlated with the degree of the sonoporation. (i) Within ~120?s of the onset of ultrasound, during which membrane perforation and complete membrane resealing occurred, intracellular ROS rapidly decreased because of extracellular diffusion of dichlorofluorescein through the perforated membrane and positively correlated with the degree of the sonoporation. (ii) In the following 270?s (120–390?s post-exposure), ROS generation in reversibly sonoporated cells gradually increased and was positively correlated with the degree of the sonoporation. (iii) The ROS level in irreversibly sonoporated cells reduced to depletion during this time interval. It is possible that ROS generation in reversibly sonoporated cells can impact their long-term fate. These results thus provide new insight into the biological response to sonoporation.     

Examining and analyzing subcellular morphology of renal tissue treated by histotripsy

Our recent studies have shown that high-intensity pulsed ultrasound can achieve mechanical tissue fragmentation, a process we call histotripsy. Histotripsy has many medical applications where noninvasive tissue removal or significant tissue disruption is needed (e.g., cancer therapy). The primary aim of this study is to investigate tissue regions treated by histotripsy and to characterize the boundary between the treated and untreated zones using transmission electron microscopy (TEM). The nature of the tissue disruption suggests many clinical applications and provides insights on the physical mechanism of histotripsy. Fresh ex vivo porcine kidney tissues were treated using histotripsy. A 1 MHz 100 mm diameter focused transducer was used to deliver 15 cycle histotripsy pulses at a peak negative pressure of 17 MPa and a pulse repetition frequency (PRF) of 100 Hz. Each lesion was produced by a 3 × 3 (lateral) × 4 (axial) grid with 2 mm between adjacent lateral and 3 mm between axial exposure points using mechanical scanning. Two thousand pulses were applied to each exposure point to achieve tissue fragmentation. After treatment, the tissue was processed and examined using TEM. Extensive fragmentation of the tissues treated with histotripsy was achieved. TEM micrographs of the tissue treated by histotripsy, showing no recognizable cellular features and little recognizable subcellular structures, demonstrates the efficacy of this technique in ablating the targeted tissue regions. A boundary, or transition zone, of a few microns separated the affected and unaffected areas, demonstrating the precision of histotripsy tissue targeting. TEM micrographs of the tissue treated by histotripsy showed no discernable cellular structure within the treated region. Histotripsy can minimize fragmentation of the adjoining nontargeted tissues because, as a nonlinear threshold phenomenon, damage can be highly localized. The potential for high lesion precision is evident in the TEM micrographs. (E-mail: fwinterr@umich.edu)     

超声联合微泡对卡铂化疗A549细胞凋亡影响的实验研究

目的 探讨超声联合微泡是否协同卡铂诱导人肺癌A549细胞凋亡。方法 设置对照组、US组、USMB组、CBP组、CBP+US组、CBP+USMB组,其中卡铂采用最适宜剂量50μg/ml,超声辐照参数采用最适宜声强0.6W/cm_²,辐照时间60s。处理后培养24小时,每组分别消化、离心、收集3个独立样本,加入FITC标记的Annexin-V室温避光30 min,再加入PI避光反应5 min,应用流式细胞仪检测各组A549细胞凋亡率。应用SPSS 26.0分析各组凋亡率。结果 A549细胞总凋亡率由高到低依次为CBP+USMB组、CBP+US组、CBP组、USMB组、US组。CBP+US组与CBP组比较,早期凋亡率差异无统计学意义(P=0.964)。CBP+USMB组与CBP组、CBP+US组比较,早期凋亡率差异均有统计学意义(均P<0.01)。CBP+US组、CBP+USMB组与CBP组两两比较,晚期、总凋亡率差异均有统计学意义(均P<0.01)。 结论 US、USMB联合卡铂化疗可以协同诱导A549细胞凋亡。     

超声辐照和SonoVue微泡在介导hAng-1基因体外转染时的作用和量效关系探讨

目的 探讨超声辐照和SonoVue微泡分别使用和联用在介导hAng-1基因体外转染过程中的作用以及辐照强度和微泡浓度对转染效率和细胞活性的影响.方法 实验分四组A组:单纯超声辐照+质粒组;B组:微泡+质粒组;C组:超声辐照+微泡+质粒组和空白对照组D组. C组内转染参数分别设置为超声照射强度0.5、1.0 、1.5和2.0 W/cm~2,微泡浓度5%、10%、20%、30%和40%.将连接有eGFP-C_3-hAng-1质粒的SonoVue微泡对293T细胞进行转染,48 h后检测各组基因转染效率和细胞存活率. 结果转染48 h后C组转染效率最高,荧光阳性细胞数最多,强度最大;A组转染效率很低,见少量荧光表达;B、D组无明显基因转染发生.随着超声照射强度和微泡浓度的增加,基因转染效率会逐步升高,具有统计学意义.微泡浓度大于20%、超声照射强度超过1.5 W/cm~2后基因转染效率不再升高甚至降低,细胞死亡率显著增高(P<0.01).结论 SonoVue微泡介导外源基因转染必须联合超声辐照才能获得较好的转染效率.对于hAng-1基因和SonoVue微泡,选择声强1.5 W/cm~2,微泡浓度20%是相对最佳转染条件.     

Evaluation of the Specific Adsorption of Biotinylated Microbubbles Using a Quartz Crystal Microbalance

Specific adsorption of biotinylated microbubbles to streptavidin was evaluated by measuring the resonant frequency of an AT-cut quartz crystal microbalance (QCM). Streptavidin was fixed via self-assembled monolayers coated onto the QCM electrode. The resonant frequency of the QCM decreased as a result of specific adsorption of the biotinylated microbubbles, compared with the results for microbubbles containing no biotin. Additionally, there was significant evidence indicating that the frequency shift was caused by the internal gas of the microbubble, as well as the mass of the outer-shell material surrounding the gas. These results suggest that the QCM measurement system can be used effectively to evaluate the specific adsorption of targeted microbubbles.     

A Novel Framework for Segmentation of Secretory Granules in Electron Micrographs

It is still a standard practice for biologists to manually analyze transmission electron microscopy images. This is not only time consuming but also not reproducible and prone to induce subjective bias. For large-scale studies of insulin granules inside beta cells of the islet of Langerhans, an automated method for analysis is essential. Due to the complex structure of the images, standard microscopy segmentation techniques cannot be applied. We present a new approach to segment and measure transmission electron microscopy images of insulin granule cores and membranes from beta cells of rat islets of Langerhans. The algorithm is separated into two broad components, core segmentation and membrane segmentation. Core segmentation proceeds through three steps: pre-segmentation using a novel level-set active contour, morphological cleaning and a refining segmentation on each granule using a novel dual level-set active contour. Membrane segmentation is achieved in four steps: morphological cleaning, membrane sampling and scaling, vector field convolution for gap filling and membrane verification using a novel convergence filter. We show results from our algorithm alongside popular microscopy segmentation methods; the advantages of our method are demonstrated. Our algorithm is validated by comparing automated results to a manually defined ground truth. When the number of granules detected is compared to the number of granules in the ground truth a precision of 91% and recall of 87% is observed. The average granule areas differ by 13.35% and 6.08% for core and membranes respectively, when compared to the average areas of the ground truth. These results compare favorably to previously published data.     

牙髓成牙本质细胞层毛细淋巴管的透射电镜研究

目的研究牙髓成牙本质细胞层中有无毛细淋巴管及其超微结构特点。方法取牙根发育完成的健康恒前磨牙15颗,纵向劈开后在2．5％戊二醛中4℃固定1h,完整地取出牙髓,切成截面积约为1mm^2,长约3mm的小长条,再在同一固定液中固定2h,后固定,包埋,半薄切片定位,选取5例牙髓进一步作超薄切片,铀铅染色,用透射电镜观察。结果在成牙本质细胞层中观察到具有典型毛细淋巴管超微结构特点的结构。结论在牙根发育完成的年轻恒前磨牙成牙本质细胞层中有毛细淋巴管,其具有与其他组织毛细淋巴管基本相似的超微结构特征。     

多组织块集中树脂包埋法在透射电镜基础制样中的应用

目的 探讨多组织块集中树脂包埋法在透射电镜基础制样中的应用效果.方法 选取我院2011年6月-2012年5月临床穿刺活检标本30份,其中肝脏标本、肾脏标本各15份,常规电镜取材.分为3组,实验1组采用24h多组织块集中树脂包埋微波快速制样,实验2组采用24h分别（单一）树脂包埋微波快速制样,对照组采用72 h常规分别（单一）树脂包埋方法制样.将同一标本切成5块约1 mm3组织块,将3块平行排列放入实验1组胶囊中,另2块分别放入实验2组、对照组胶囊中,依次进行修块、切片、捞片、铀铅染色后上镜观察.结果 实验1组、实验2组与对照组比较,切片超微结构均保存较好,且染色对比反差度好;染色后电镜对比观察实验1组、实验2组细胞器保存良好,胞膜、核膜、微绒毛等细胞膜结构连结清晰,切片连续成带,延展性能好.实验1组观察范围较实验2组、对照组更为广泛,切面更完全.结论 多组织块集中树脂包埋法用于透射电镜基础制样切片连续性好,制样时间明显缩短.     

Effects of Focused Ultrasound and Microbubbles on the Vascular Permeability of Nanoparticles Delivered into Mouse Tumors

Ultrasound sonication with microbubbles (MBs) was evaluated for enhancement of the release of nanoparticles from vasculature to tumor tissues. In this study, tumor-bearing Balb/c mice were insonicated with focused ultrasound (FUS) in the tumors after the injection of MBs (SonoVue^®) and then lipid-coated quantum dot (LQD) nanoparticles (130 ± 25 nm) were injected through the tail vein. We studied the effects of the injected MB dose (0–300 μL/kg), sonication duration (0–300 s) and treatment-procedure sequence on the accumulation of nanoparticles in the tumors 24 h after the treatment and the time response of the accumulation (0.5–24 h). After the treatment, the mice were sacrificed and perfused and then the tumor tissues were harvested for quantifying the amount of nanoparticles using graphite furnace atomic absorption spectrometry (GF-AAS). The results showed that pulsed-FUS sonication with MBs can effectively enhance the vascular permeability for LQD nanoparticle delivery into the sonicated tumors. It indicates that this technique is promising for a better nanodrug delivery for tumor chemotherapy. (E-mail: winli@ntu.edu.tw)     

超声介导微气泡造影剂促反义寡核苷酸转染的有效性研究

目的探讨超声介导脂质体微气泡转染反义寡核苷酸HA-2741的有效性。方法12孔板培养人乳腺癌细胞,分组为:(1)单纯超声照射;(2)单纯造影剂;(3)单纯HA-2741;(4)超声照射 HA-2741;(5)造影剂 HA-2741;(6)造影剂 HA-2741 超声照射;(7)脂质体 HA-2741;(8)脂质体 HA-2741 超声照射。造影剂浓度2%,超声波发射频率1.3MHz,强度-3dB,照射时间30s,予与实验刺激后,流式细胞仪定量分析HA-2741的转染率,RT-PCR的检测乳腺癌细胞的HER-2mRNA水平,免疫细胞化学检测HER-2蛋白的表达。结果造影剂 HA-2741 超声照射组HA-2741的转染率显著高于其余各组,约94.6%,造影剂 HA-2741 超声照射组乳腺癌细胞的HER-2mRNA水平显著降低,HER-2蛋白表达明显减低,其程度与HA-2741转染率呈正相关。结论超声介导微气泡造影剂显著增加基因的转染效率,方法简便,安全,具有良好的靶向性。