Quantitative measurement of ultrasound disruption of polymer-shelled microbubbles

The goal of this study was to assess the threshold of disruption and subsequent time-course of acoustic response of four experimental nitrogen-filled polymer-shelled microbubbles. Using an in vitro measurement system, a sequence of low-amplitude detection pulses measured the change in echo caused by a high-amplitude disruption pulse on a dilute suspension of bubbles. Detection pulses were transmitted 0.5 ms before disruption and between 1 and 200 ms after disruption. Separate transducers, aligned confocally and orthogonally, were used to transmit and receive bubble echoes. After disruption, all agents demonstrated a transient increase in scattered power. Above the disruption threshold, highly echogenic, nonlinear scatterers were observed. Their echoes slowly disappeared after disruption with median decay times from 7.4 to 13.6 ms, calculated by fitting to a mono-exponential decay. This is consistent with a process wherein the shell is disrupted, releasing the gas and generating free gas bubbles, which cause high-amplitude nonlinear scattering followed by relatively slow diffusion of the gas into solution. This picture has been observed optically with single bubbles and differs from the concept of "stimulated acoustic emission" from disrupted bubbles.     

Targeted tissue transfection with ultrasound destruction of plasmid-bearing cationic microbubbles

The aim of this study was to assess the relative efficacy and mechanism of gene transfection by ultrasound (US) destruction of plasmid-bearing microbubbles. Luciferase reporter plasmid was charge-coupled to cationic lipid microbubbles. Rat hindlimb skeletal muscle was exposed to intermittent high-power US during dose-adjusted intra-arterial (IA) or IV administration of plasmid-bearing microbubbles via the carotid artery or jugular vein, respectively. At 4 days, luciferase activity in US-exposed skeletal muscle was 200-fold greater with IA than with IV administration of plasmid-bearing microbubbles, and was similar to transfection achieved by IM injection of plasmid (positive control). No transfection occurred with US and IA injection of plasmid alone. Intravital microscopy of the cremaster muscle in mice following administration of microbubbles and US exposure demonstrated perivascular deposition of fluorescent plasmid, the extent of which was twofold greater for IA compared to IV injection. Electron microscopy demonstrated a greater extent of myocellular microporations in US-exposed muscle after IA injection of microbubbles. We conclude that muscle transfection by US destruction of plasmid-bearing cationic microbubbles is amplified by IA, rather than IV, injection of microbubbles due to greater extravascular deposition of plasmid and to greater extent of myocellular microporation. (E-mail: jlindner@virginia.edu)     

纳米级微泡:一种新的超声造影剂

随着分子生物学技术的发展和延伸,超声分子成像获得了飞速的发展,但由于常规造影剂微泡不能透过血管壁,超声分子成像的研究仅限于血管内的靶分子.最近纳米级微泡超声造影剂的出现为针对肿瘤细胞的超声分子成像带来了希望.本文对纳米级微泡超声造影剂的研究背景、研究现状及下一步的研究与应用进行了综述.     

超声分子显像与治疗及设备的研究

近年来,随着医学影像技术的发展,超声分子成像成为当前医学影像学研究的热点之一,其中超声分子探针的设计是分子成像研究的重点和先决条件。靶向超声微泡(球)造影剂在超声分子显像及治疗中的研究和应用愈来愈受到人们的关注,而多学科的融合使其具有更大的发展空间。将超声分子成像设备、超声微泡(球)触发装置、超声分子成像监控与超声分子探针有机结合的"低功率超声分子显像与治疗系统",有望实现超声分子显像及精细、适形、高效的药物体内定位递送、定量控释和疗效评价一体化,为疾病的超声分子显像诊断与治疗提供创新的、适合多学科使用的新技术和科研平台。     

超声分子成像的机制及研究现状

随着分子生物学的迅速发展以及疾病分子机制的逐步阐明,医学影像学已深入到细胞及分子水平.超声分子成像技术是医学分子影像学领域中重要组成部分,在当今分子影像学中占据重要地位.本文对其成像机制及研究现状进行综述.     

Temperature-dependent differences in the nonlinear acoustic behavior of ultrasound contrast agents revealed by high-speed imaging and bulk acoustics

Previous work by the authors has established that increasing the temperature of the suspending liquid from 20°C to body temperature has a significant impact on the bulk acoustic properties and stability of an ultrasound contrast agent suspension (SonoVue, Bracco Suisse SA, Manno, Lugano, Switzerland). In this paper the influence of temperature on the nonlinear behavior of microbubbles is investigated, because this is one of the most important parameters in the context of diagnostic imaging. High-speed imaging showed that raising the temperature significantly influences the dynamic behavior of individual microbubbles. At body temperature, microbubbles exhibit greater radial excursion and oscillate less spherically, with a greater incidence of jetting and gas expulsion, and therefore collapse, than they do at room temperature. Bulk acoustics revealed an associated increase in the harmonic content of the scattered signals. These findings emphasize the importance of conducting laboratory studies at body temperature if the results are to be interpreted for in vivo applications.     

Validation of ultrasound contrast destruction imaging for flow quantification

Our purpose was to validate in vitro a kinetic flow model based on microbubble signal decay curve. Using a 3.5 MHz transducer and phase-inversion (1.8 MHz central transmit frequency), a renal dialysis cartridge oriented vertically was imaged in the transverse plane as 1:1000 dilution of AF0150 was infused at 50, 100, 200, 300 and 400 mL/min. Ten gray-scale images were acquired at each infusion rate using 2.5, 5 and 10 frames/s at 100%, 40%, 15% or 1% of maximum transmit power. Video-intensity measured on each 10 images was fit to a kinetic model using Sigma Plot that yielded microbubble concentration, velocity and destruction per frame. These were correlated with the experimental conditions. At 100% power, video-intensity on the first frame (microbubble concentration at equilibrium) was similar for all flow and frame rates. The model fit the experimental data for all flows at 10 frames/s and for flows lower than 400 and 100 mL/min at 5 frames/s and 2.5 frames/s, respectively. The calculated flow was similar to the experimental flow rates, regardless of technique (r² = 0.98). Microbubble fraction destroyed per frame was similar for all flow and frame rates and increased linearly with transmit power (r² > 0.98). These results suggest that using appropriate power and frame rate for a given flow rate, estimates of fractional blood volume, flow and destruction fraction can be calculated from the decay curve using 10 frames that can be acquired in 1 to 4 s. (E-mail: rmattrey@ucsd.edu)     

Dynamics of coated microbubbles adherent to a wall

Molecular imaging with ultrasound is a promising noninvasive technique for disease-specific imaging, enabling for instance, the diagnosis of thrombus and inflammation. Selective imaging is performed by using ultrasound contrast agent microbubbles functionalized with ligands, which bind specifically to the target molecules. Here, we investigate in a model system, the influence of adherence at a wall on the dynamics of the microbubbles, in particular, on the frequency of maximum response, by recording the radial response of individual microbubbles as a function of the applied acoustic pressure and frequency. The frequency of maximum response of adherent microbubbles was found to be over 50% lower than for bubbles in the unbounded fluid and over 30% lower than that of a nonadherent bubble in contact with the wall. The change is caused by adhesion of the bubbles to the wall as no influence was found due solely to the presence of the targeting ligands on the bubble dynamics. The shift in the frequency of maximum response may prove to be important for molecular imaging with ultrasound as this application would benefit from an acoustic imaging method to distinguish adherent microbubbles from freely circulating microbubbles.     

载BDNF脂质体纳米微粒脂膜微泡超声造影剂的制备与初步评价

目的 探索制备一种新型的耦连包载BDNF脂质体纳米颗粒的脂膜微泡超声造影剂(BDNF-UMCA)。方法 以冷冻干燥法在“脂氟显”制备的基础上加入一定比例的含生物素化棕榈酰磷脂酰甘油钠-聚乙二醇2000-生物素[DPPG-PEG(2000)-Biotin]制备含生物素的脂质超声微泡造影剂,通过链亲和素来耦连含生物素化PEG载BDNF脂质体纳米微粒制备BDNF-UMCA,检测其理化性质、载药量、包封率、稳定性和体内声学特性进行检测。结果 BDNF-UMCA平均粒径4.2±0.79 μm,浓度为1.02×10₉/ml;总药物含量为1.18±1.96 mg/mL,包封率为71.6±2.6%;BDNF-UMCA在4℃条件下,平均粒径和包封率均无明显的变化;在24℃条件下,载BDNF脂质体纳米微粒的平均粒径随时间逐渐增大,第1、3、5、7天的粒径与初始粒径比较具有明显的统计学差异(均P<0.05),包封率在24℃下各个时间点无明显的统计学差异(均P>0.05);BDNF-UMCA能显著增强实验动物肝脏显影,平均峰值强度为21.4±0.9 dB,平均达峰时间为4.7±0.4 s。结论 应用生物素-亲和素偶联可成功制备载BDNF脂质体纳米微粒的脂膜超声微泡造影剂,为靶向显影及药物通过血脑屏障释放提供工具。     

多功能超声造影剂体外超声-磁共振双模态显像及增效高强度聚焦超声的实验研究

目的 制备一种新型的多功能超声造影剂,体外观察其超声、磁共振(MR)成像效果及增强高强度聚焦超声(high-intensity focused ultrasound,HIFU)消融效果.方法 采用双乳化法合成载超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)纳米颗粒的高分子微球(s-PLGA),检测其一般性质.制备体外成像模型,应用超声诊断仪及磁共振扫描仪对不同浓度s-PLGA分别行超声及MR成像.另取新鲜离体牛肝,局部注射s-PLGA后,给予不同HIFU辐照参数,通过计算辐照区凝固性坏死体积评价s-PLGA增强HIFU消融效果.结果 制备的s-PLGA呈球形,平均直径为(885.6±133.2)nm.体外超声显像,sPLGA呈高回声,回声强度随浓度及机械指数减小而降低;磁共振T2WI呈负增强显像.注射s-PLGA后行HIFU辐照,辐照区凝固性坏死体积明显增大(P＜0.05).结论 自制的多功能超声造影剂-载超顺磁性氧化铁高分子微球具备超声、MR双模态复合成像与增效HIFU的功能.     

载量子点高分子光-声双模式成像剂制备及显像实验

目的 制备载量子点(QDs,CdTe-MPA)高分子乳酸羟基乙酸共聚物(PLGA)微气泡——荧光-超声双模式成像剂(MBQDs@PLGA).方法 双乳化-冷冻干燥充气法制备双模式成像剂MBQDs@PLGA并评价其显像性能,激光共聚焦显微镜(CLSM)、透射电镜(TEM)技术检测量子点装载及MBQDs@PLGA荧光成像稳定性,荧光分光光度计线性回归法计算量子点的包封率.体外模型成像、在体小动物荧光成像及超声造影增强成像(荷瘤裸鼠)评价MBQDs@PLGA显像.结果 在激发波长272 nm MBQDs@PLGA成像剂荧光发射光谱峰值为549 nm,量子点在微球中的装载率约为54％,平均粒径为(1.7±0.2)μm.体内、外成像结果表明MBQDs@PLGA有超声造影增强显像和荧光成像功能.结论 量子点包埋在高分子微泡内制得MBQDs@PLGA呈显荧光-超声双模式成像性能,为多模式成像剂研发开辟思路.     

The potential of a new stable ultrasound contrast agent for site-specific targeting. An in vitro experiment

Microbubble-based ultrasound contrast agents can be used for specific site targeting, but demonstrate time-limited opacification. We have previously demonstrated the potential of gold-bound microtubules to provide a stable ultrasound contrast effect. Aim of the present study was to test the feasibility of gold-bound microtubules specifically to bind to human thrombi and to inflammatory activated human umbilical vein endothelial cells (HUVEC) in vitro. HUVEC were incubated with tumor necrosis factor, to induce expression of adhesion molecules. Human clots and HUVEC were incubated with biotinylated monoclonal antifibrin and anti-E-selectin antibodies, respectively. Probes were incubated with excess avidin followed by biotinylated gold-bound microtubules and by secondary Cy3-anti-beta-tubulin antibody and processed for immune fluorescence microscopy. Clots were transferred in copolymer foils filled with buffer and were ultrasonographically imaged before and after their treatment with the antifibrin antibody and with biotinylated microtubules, using a broadband harmonic transducer, transmitting and receiving at a mean frequency of 1.7 MHz and 3.2 MHz. The feasibility of specific gold-bound microtubules conjugation to antibody treated clots and HUVEC was confirmed using immune fluorescence analysis. Contrast intensities of the clots significantly increased after their treatment with antifibrin antibody and incubation with gold-bound microtubules (39 +/- 2 dB versus 26 +/- 2 dB, p < 0.001) and remained high after 20 min of ultrasound exposure (37 +/- 2 dB versus 39 +/- 2 dB, p = NS). Thus, gold-bound microtubules can specifically bind to human thrombi and to endothelial cells, providing a significant contrast effect which remains stable in the ultrasound field. This may be a promising approach to target thrombi and inflammatory active atherosclerotic plaques.     

肿瘤分子地址、分子靶确认与超声分子成像

肿瘤分子靶确认是借助于分子影像技术对肿瘤特异性分子地址进行分子成像,为肿瘤的早期发现、治疗方案的制定以及治疗效果的评估提供依据.超声分子成像为肿瘤分子靶确认这一崭新的肿瘤诊治理念的实现带来了巨大机遇,在肿瘤"分子靶确认"领域展示明确的应用前景.     

Leveraging the power of ultrasound for therapeutic design and optimization

Contrast agent-enhanced ultrasound can facilitate personalized therapeutic strategies by providing the technology to measure local blood flow rate, to selectively image receptors on the vascular endothelium, and to enhance localized drug delivery. Ultrasound contrast agents are micron-diameter encapsulated bubbles that circulate within the vascular compartment and can be selectively imaged with ultrasound. Microbubble transport-based estimates of local blood flow can quantify changes resulting from anti-angiogenic therapies and facilitate differentiation of angiogenic mechanisms. Microbubbles that are conjugated with targeting ligands attach to endothelial surface receptors that are upregulated in disease, providing high signal-to-noise ratio images of pathological vasculature. In addition to imaging applications, microbubbles can be used to enhance localized gene and drug delivery, either by changing membrane and vascular permeability or by carrying and locally releasing cargo. Our goal in this review is to provide an overview of the use of contrast-enhanced ultrasound methodologies in the design and evaluation of therapeutic strategies with emphases on quantitative blood flow mapping, molecular imaging, and enhanced drug delivery.     

Critical Appraisal of Targeted Ultrasound Contrast Agents for Molecular Imaging in Large Arteries

Molecular imaging may provide new insights into the early detection and development of atherosclerosis before first symptoms occur. One of the techniques in use employs noninvasive ultrasound. In the past decade, experimental and clinical validation studies showed that for the microcirculation targeted ultrasound contrast agents, such as echogenic liposomes, microbubbles and perfluorocarbon emulsions, do improve visualization of specific structures. For large arteries, however, successful application is less obvious. In this review, we will address the challenges for molecular imaging of large arteries. We will discuss the problems encountered in the use of targeted ultrasound contrast agents presently available, mainly based on data obtained in flow chambers and animal studies because clinical studies are lacking. We conclude that molecular imaging of activated endothelium in large- and middle-sized arteries by site-specific accumulation of contrast material is still difficult to achieve due to wall shear stress conditions in these vessels. (A.Hoeks@bf.unimaas.nl)     

Physicochemical characteristics of Sonazoid, a new contrast agent for ultrasound imaging

The objective of the current work is to describe the physicochemical characteristics of Sonazoid™, a new ultrasound contrast agent for detection and characterisation of focal liver lesions. It has been demonstrated that Sonazoid™ powder for injection consists of microspheres of perfluorobutane (PFB) stabilised by a monomolecular membrane of hydrogenated egg phosphatidyl serine, embedded in an amorphous sucrose structure. Upon reconstitution with sterile water, stabilised microspheres of PFB are released in a predefined amount and size into a low viscosity, isotonic sucrose solution with a neutral pH. Sonazoid™ reconstituted product contains approximately 8 μl microspheres/ml with volume median diameter of approximately 2.6 μm. The product contains approximately 1.2 billion microspheres/ml of which less than 0.1% are larger than 7 μm. The acoustic properties of Sonazoid™ such as attenuation efficacy, fundamental and second harmonic backscatter efficacy are all well correlated to the microsphere volume concentration. The stability of Sonazoid™ after reconstitution is good, with no significant changes in physicochemical properties 2 h after reconstitution. Pressure stress is well tolerated by both concentrated and diluted Sonazoid™ with no permanent effects of pressures up to 300 mm Hg. The level and consistency of the investigated physicochemical properties demonstrate that Sonazoid™ should be well suited as a contrast agent for medical imaging with ultrasound. (E-mail: per.sontum@ge.com)     

Design and characterization of targeted ultrasound microbubbles for diagnostic use

Targeted ultrasound (US) contrast agents represent, because of their size (1 to 5 μm), a unique class of diagnostic imaging agents enabling true vascular imaging of conditions like inflammation and tumor angiogenesis. The objective of this study was to develop technology for preparing targeted microbubbles with binding and acoustic properties compatible with diagnostic use. Phosphatidylcholine (PC) was shown to represent the most favorable wall material. Various thiolated peptide binders were effectively conjugated to PC-based microbubbles containing maleimide functionalized lipids (95:5) without the need for biotin-streptavidin or antibody technology. By optimizing the technology, specific targeting of the inflammatory target E-selectin and the angiogenic target VEGFR2 in the presence of 100% serum was achieved. Increased phospholipid chain length from 18 carbons to 22 carbons improved the stability of the microbubbles during US exposure, without compromising binding or acoustic properties. (E-mail: roald.skurtveit@ge.com)     

Determination of the Interfacial Rheological Properties of a Poly(DL-lactic acid)-Encapsulated Contrast Agent Using In Vitro Attenuation and Scattering

The stabilizing encapsulation of a microbubble-based ultrasound contrast agent (UCA) critically affects its acoustic properties. Polymers, which behave differently from materials commonly used (i.e., lipids or proteins) for monolayer encapsulation, have the potential for better stability and improved control of encapsulation properties. Air-filled microbubbles coated with poly(DL-lactic acid) (PLA) are characterized here using in vitro acoustic experiments and several models of encapsulation. The interfacial rheological properties of the encapsulation are determined according to each model using attenuation of ultrasound through a suspension of microbubbles. Then the model predictions are compared with scattered non-linear (sub- and second harmonic) responses. For this microbubble population (average diameter, 1.9 μm), the peak in attenuation measurement indicates a weighted-average resonance frequency of 2.5–3 MHz, which, in contrast to other encapsulated microbubbles, is lower than the resonance frequency of a free bubble of similar size (diameter, 1.9 μm). This apparently contradictory result stems from the extremely low surface dilational elasticity (around 0.01–0.07 N/m) and the reduced surface tension of the poly(DL-lactic acid) encapsulation, as well as the polydispersity of the bubble population. All models considered here are shown to behave similarly even in the non-linear regime because of the low surface dilational elasticity value. Pressure-dependent scattering measurements at two different excitation frequencies (2.25 and 3 MHz) revealed strongly non-linear behavior with 25–30 dB and 5–20 dB enhancements in fundamental and second-harmonic responses, respectively, for a contrast agent concentration of 1.33 μg/mL in the suspension. Sub-harmonic responses are registered above a relatively low generation threshold of 100–150 kPa, with up to 20 dB enhancement beyond that pressure. Numerical predictions from all models show good agreement with the experimentally measured fundamental response, but not with the experimental second-harmonic response. The characteristic features of sub-harmonic responses and the steady response beyond the threshold are matched well by model predictions. However, prediction of the threshold value depends on estimated properties and size distribution. The variation in size distribution from sample to sample leads to variation in estimates of encapsulation properties: the lowest estimated value for surface dilational viscosity better predicts the sub-harmonic threshold.     

超声新技术对早期前列腺癌的诊断价值及应用进展

近年来,经直肠超声检查技术与超声新技术的联合应用在前列腺癌的临床诊断中发挥了越来越重要的作用,明显提高了对早期前列腺癌的诊断、鉴别诊断和临床分期的准确性及可靠性,为系统评价临床治疗效果提供了必要的参考依据。本文就现阶段医学超声新技术诊断早期前列腺癌的应用现状及进展进行综述。     

留学生超声影像学教学实践及体会

超声影像学教学对医学留学生培养具有重要作用,其全英文教学已引起广泛关注。在教学过程中需要针对超声影像学课程和留学生特点,做好充分教学准备工作,不断改进教学方法,才能提高教学质量。