Ultrasound Contrast Agent Microbubbles Research Articles

A sensitive TLRH targeted imaging technique for ultrasonic molecular imaging

The primary goals of ultrasound molecular imaging are the detection and imaging of ultrasound contrast agents (microbubbles), which are bound to specific vascular surface receptors. Imaging methods that can sensitively and selectively detect and distinguish bound microbubbles from freely circulating microbubbles (free microbubbles) and surrounding tissue are critically important for the practical application of ultrasound contrast molecular imaging. Microbubbles excited by low-frequency acoustic pulses emit wide-band echoes with a bandwidth extending beyond 20 MHz; we refer to this technique as transmission at a low frequency and reception at a high frequency (TLRH). Using this wideband, transient echo, we have developed and implemented a targeted imaging technique incorporating a multifrequency colinear array and the Siemens Antares imaging system. The multifrequency colinear array integrates a center 5.4-MHz array, used to receive echoes and produce radiation force, and 2 outer 1.5-MHz arrays used to transmit low-frequency incident pulses. The targeted imaging technique makes use of an acoustic radiation force subsequence to enhance accumulation and a TLRH imaging subsequence to detect bound microbubbles. The radiofrequency (RF) data obtained from the TLRH imaging subsequence are processed to separate echo signatures between tissue, free microbubbles, and bound microbubbles. By imaging biotin-coated microbubbles targeted to avidin-coated cellulose tubes, we demonstrate that the proposed method has a high contrast-to-tissue ratio (up to 34 dB) and a high sensitivity to bound microbubbles (with the ratio of echoes from bound microbubbles versus free microbubbles extending up to 23 dB). The effects of the imaging pulse acoustic pressure, the radiation force subsequence, and the use of various slow-time filters on the targeted imaging quality are studied. The TLRH targeted imaging method is demonstrated in this study to provide sensitive and selective detection of bound microbubbles for ultrasound molecularly targeted imaging.

Cavitation and contrast: The use of bubbles in ultrasound imaging and therapy

Microbubbles and cavitation are playing an increasingly significant role in both diagnostic and therapeutic applications of ultrasound. Microbubble ultrasound contrast agents have been in clinical use now for more than two decades, stimulating the development of a range of new contrast-specific imaging techniques which offer substantial benefits in echocardiography, microcirculatory imaging, and more recently, quantitative and molecular imaging. In drug delivery and gene therapy, microbubbles are being investigated/developed as vehicles which can be loaded with the required therapeutic agent, traced to the target site using diagnostic ultrasound, and then destroyed with ultrasound of higher intensity energy burst to release the material locally, thus avoiding side effects associated with systemic administration, e.g. of toxic chemotherapy. It has moreover been shown that the motion of the microbubbles increases the permeability of both individual cell membranes and the endothelium, thus enhancing therapeutic uptake, and can locally increase the activity of drugs by enhancing their transport across biologically inaccessible interfaces such as blood clots or solid tumours. In high-intensity focused ultrasound (HIFU) surgery and lithotripsy, controlled cavitation is being investigated as a means of increasing the speed and efficacy of the treatment. The aim of this paper is both to describe the key features of the physical behaviour of acoustically driven bubbles which underlie their effectiveness in biomedical applications and to review the current state of the art.

Imaging of the acute scrotum: is there a place for contrast-enhanced ultrasonography?

To present and evaluate the findings of contrast-enhanced ultrasonography (CEUS) in typical cases of acute painful scrotum. Nineteen patients aged from 19 to 61 years old were included in the study. All patients underwent grey-scale and color Doppler ultrasonography (US) of the scrotum, followed by imaging after i.v. administration of 2.4 mL of a second generation ultrasound contrast agent (microbubbles of sulphur hexafluoride). ? dedicated, contrast-sensitive technique was used (Contrast Tissue Imaging - CnTI). The diagnosis was confirmed surgically in 6 cases while in the remaining 8 cases it was based on the combination of clinical, imaging and laboratory findings. The final diagnosis was testicular torsion (n = 4), epididymitis (n = 2, one of the cases complicated by abscess), testicular abscess (n = 1), scrotal abscess (n = 1), testicular trauma of varying severity (n = 6). Five out of 19 cases were true negatives: neither clinical examination nor laboratory tests revealed any pathology. CEUS showed complete lack of enhancement in all cases of torsion, permitting a rapid and definitive diagnosis. In the cases of infection complicated by abscesses, CEUS delineated the lesions much better than the combination of B-mode/Color Doppler US. The severely traumatized testicles showed minimal, inhomogeneous or patchy enhancement, while cases of minor trauma showed no significant enhancement defects. Hematomas were presented as non-enhancing lesions. Generally, there was no advantage over Doppler US as has been previously shown. However, CEUS can be used supplementary to traditional Doppler US in the investigation of blunt testicular trauma especially when there is uncertainty in diagnosis after appropriate clinical and radiographic evaluations occurs. Further studies are required to clearly define the indications of this method.

Ablation of benign prostatic hyperplasia using microbubble-mediated ultrasound cavitation

Benign prostatic hyperplasia (BPH) is a world-wide common disease in elderly male patients. A number of invasive physiotherapies have been used to replace prostatectomy. In this article we report our hypothesis of using microbubbles-mediated ultrasound cavitation effects to ablate prostatic tissues. Microbubble ultrasound contrast agent is widely used contrast media in ultrasonography, yet it is also found to act as cavitation nuclei or enhancer. Once excited by a high peak pressure ultrasound pulse, the mechanical effects, like shock wave and microstream, released from cavitation could produce a series of bioeffects, contributing to sonoporation, microvascular rupture and hematoma. BPH is known to have hyperplastic neovasculature and this make it possible to be disrupted by the physical effects of cavitation under existing microbubbles in circulation. Mechanical ablation of prostatic capillary or small vessels could result in pathological alterations such as thrombosis, micro-circulation blockage, prostatic necrosis and atrophia. Thereupon it could effectively treat BPH by nontraumatic ways.

Morphological study of acoustic liposomes using transmission electron microscopy

Sonoporation is achieved by ultrasound-mediated destruction of ultrasound contrast agents (UCA) microbubbles. For this, UCAs must be tissue specific and have good echogenicity and also function as drug carriers. Previous studies have developed acoustic liposomes (ALs), liposomes that encapsulate phosphate buffer solution and perfluoropropane (C(3)F(8)) gas and function as both UCAs and drug carriers. Few studies have examined the co-existence of gas and liquid in ALs. This study aims to elucidate AL structure using TEM. The size, zeta potential and structure of ALs were compared with those of two other UCAs, human albumin shell bubbles (ABs; Optison) and lipid bubbles (LBs). ABs and LBs encapsulate the C(3)F(8) gas. Particle size was measured by dynamic light scattering. The zeta potential was determined by the Smoluchowski equation. UCA structure was investigated by TEM. ALs were approximately 200 nm in size, smaller than LBs and ABs. ALs and LBs had almost neutral zeta potentials whereas AB values were strongly negative. The negative or double staining TEM images revealed that approximately 20% of ALs contained both liquid and gas, while approximately 80% contained liquid alone (i.e. nonacoustic). Negative staining AB images indicated electron beam scattering near the shell surface, and albumin was detected in filament form. These findings suggest that AL is capable of carrying drugs and high-molecular-weight, low-solubility gases.

Developments in musculoskeletal ultrasound and clinical applications

Ultrasound (US) technologies are rapidly advancing, offering several refined transducer technologies as well as soft and hardware facilities. The aim of this article is to outline US developments, from B-mode technologies over Doppler advances to more sophisticated technologies, and their potential clinical impact in the field of musculoskeletal (MSK) imaging. When using B-mode ultrasound, compound imaging and beam-steering are of help to decrease anisotropy in tendons and ligaments, that are less well depicted due to their oblique course. Doppler imaging has become sensitive in the detection of flow in small vessels, which is of particular value in rheumatologic conditions, tumour and overuse assessment. The use of US microbubble contrast agents improves detection of low-volume blood flow in smaller vessels by increasing the signal-to-noise ratio and thereby facilitating detection of angiogenetic vessels in inflammatory conditions or tumours. The use of US blood pool contrast agents enables molecular imaging in real-time, and thus the diagnostic potential of US is expanded, opening up a new field of US applications. Objective quantification of altered tissue (e.g., synovial proliferation, tumours) is still demanding and might be improved by the use of three-dimensional imaging and software tools as parametric evaluation. Real-time sonoelastography (EUS) is a new development for visualization of tissue elasticity by measurement of tissue displacement in terms of tissue stiffness changes, promising new insights into tendon disorders. Image fusion is an exciting development that enables superimposition of CT/MRI data sets on real-time US scanning. This technique might be helpful in guiding injections under real-time conditions even in regions less easily accessible by US as, for instance, the axial skeleton, and can additionally provide an interesting tool for teaching MSK imaging and ways to guide interventions. In summary, exciting developments are expanding the applications of US in the MSK field, offering the advantages of real-time performance, high tissue resolution and relative speed at a reasonable cost.

In vitro Sonothrombolysis with Duplex Ultrasound: First Results Using a Simplified Model

Background: The main aim was to study the effects of ultrasound (US) alone, in combination with an US contrast agent (UCA), tissue plasminogen activator (tPA), or the combination of both upon blood clots. Methods: In order to learn about sonothrombolysis with diagnostic duplex US, a simplified in vitro test model, using human whole blood clots in Petri dishes, was established. Results: A total of 286 blood clots were analyzed. Improved sonothrombolysis due to insonation with diagnostic duplex US could be achieved, whether it was used alone or in combination with tPA. Although already described, a beneficial effect of UCA microbubbles on sonothrombolysis could not be confirmed due to the study design. Conclusion: Diagnostic duplex US improves thrombolysis significantly, even when it is used without tPA. To study the effect of UCA microbubbles on sonothrombolysis appropriately, any experimental design should provide continuous replenishment of microbubbles at the target site.

Special issue on microbubbles: from contrast enhancement to cancer therapy

The clinical use of microbubbles originated with the fortuitous discovery by Joyner that intravenous injection of certain liquids produced contrast enhancement in ultrasound images [3]. Subsequent investigation revealed that it was not the liquids themselves which were responsible for the observed effect, but rather the presence of gas microbubbles either suspended in the liquid or formed at the tip of the catheter through which it was being injected. Since then, the use of microbubbles in diagnostic and, more recently, therapeutic applications has become a rapidly expanding field of research. The aim of this special issue is to provide an up-to-date overview of the developments in the physics, engineering and clinical applications of microbubbles. Microbubble ultrasound contrast agents have now been in clinical use for more than two decades and their evolving applications are discussed in detail in the comprehensive review by Cosgrove and Harvey [1] who have extensive clinical experience in this area. The primary application of microbubbles is in echocardiography, for ventricular opacification and delineation of endocardial borders. They have also been used successfully for the assessment of systolic function and left ventricular volume, and for identifying myocardial infarction and coronary artery stenoses. As explained in the paper, they are moreover becoming rapidly accepted for the detection, characterisation and image guided treatment of focal liver lesions. They have also been used for functional investigations in other organs including the kidneys, spleen and pancreas and for assessment of disease in the bowel, prostate, breast and lymph nodes as well as non-vascular applications, such as assessment of fallopian tube patency and detection of ureteric reflux. It is the highly non-linear response of microbubbles to ultrasound that has facilitated the development of a range of contrast-specific imaging protocols which have greatly increased the diagnostic potential of ultrasound imaging. For example, in combination with advances in threedimensional visualisation techniques, harmonic imaging with microbubbles has enabled mapping of the microcirculation for the characterisation of tumour vascularity, and

Ultrasound microbubble contrast agents: Application to therapy for peripheral vascular disease

Ultrasound contrast agents are not only effective in ultrasonic imaging but are also important tools for drug or gene delivery. Ultrasound beams can disrupt microbubbles and cell membranes, offering the opportunity to locally deliver drugs or genes. Liposome-shelled microbubbles have many advantages and are widely used in many applications, while Lipofectamine (Invitrogen, Life Technologies, Carlsbad, CA, USA), as a material of microbubble membranes, has been used to enhance the effects of gene delivery. Ultrasound contrast agents that have therapeutic effects can be used for treating peripheral vascular diseases, particularly in thrombotic and angiogenic diseases. A combination of targeted contrast agent and drug-carrying contrast agent may be safer and more effective in treating thrombosis. Vascular endothelial growth factor-loaded microbubbles are expected to treat a variety of neovascular diseases such as severe limb ischemia and other diseases. Although there are several limitations in the application of therapeutic ultrasound microbubble contrast agents, it will offer a new hope for the treatment of peripheral vascular disease.

Influence of finite wall impedance on contrast agent bubble behavior near a membrane.

Experiments investigating the radial dynamics of ultrasound contrast agent (UCA) microbubbles in the vicinity of an optically transparent membrane show that bubble oscillation amplitude and frequency of peak response decrease as bubbles move closer to the membrane (Overvelde et al. Proc. 19th Intl. Congr. Acoust., 2007). However, treating the membrane as a rigid wall predicts that the peak oscillation amplitude should increase at small standoff distances, contrary to experimental observations. Here we present a model describing UCA bubble dynamics near a locally reacting wall having finite acoustic impedance. If finite wall impedance is included in the model, the predicted bubble behavior is in good agreement with observations. The hybrid time‐frequency domain model is based on a linear frequency domain solution [Ingard, J. Acoust. Soc. Am. 23, 329 (1951)] which has been adapted to account for weakly nonlinear bubble oscillations and UCA shell dynamics. Wall impedance parameters are derived from independent experimental measurements. Comparisons between the model and data from microbubble spectroscopy experiments will be presented. [Work supported by the ASA Hunt Fellowship and NIH DK070618.]

VEGFR‐2 Targeted Microbubble Contrast Agents for Ultrasound Molecular Imaging of Tumor Angiogenesis

Ultrasound molecular imaging is an emerging technique for detecting intravascular targets. We have developed a novel microbubble (MB) ultrasound contrast agent covalently coupled to a recombinant single‐chain VEGF construct (scVEGF), which binds VEGFR‐2 (KDR) on the angiogenic endothelium. Ligand conjugation was first validated with a fluorophore (BODIPY‐cystine), and covalently bound dye was detected by fluorometry and flow cytometry. MB were subsequently conjugated to scVEGF, which bound at ~100,000 molecules/MB (ELISA). Targeted adhesion of scVEGF‐MB was demonstrated with in vitro flow assays. At 0.5 dynes/cm2, scVEGF‐MB exhibited 5‐fold higher adhesion to both recombinant murine VEGFR‐2 and porcine aortic endothelial cells transfected with VEGFR‐2 (PAE/KDR) compared to non‐targeted control MB. Additionally, scVEGF‐MB targeted to immobilized VEGFR‐2 in an ultrasound flow phantom showed an 8‐fold increase in mean acoustic signal relative to casein‐coated control channels. A murine model of colon adenocarcinoma was used to assess targeted adhesion in vivo, and scVEGF MB, but not control MB, showed significantly higher ultrasound contrast signal enhancement in tumors. These results demonstrate the functionality of a novel scVEGF‐bearing microbubble contrast agent, which could be a valuable research tool for molecular imaging of VEGFR‐2. Supported by NIH 1R43EB007857.

Shell buckling enhances subharmonic behavior of phospholipid coated ultrasound contrast agent microbubbles.

Subharmonic behavior of coated microbubbles can greatly enhance the contrast in ultrasound imaging. The threshold driving pressure above which subharmonic oscillations are initiated can be calculated from a linearized Rayleigh‐Plesset‐type equation. Earlier experimental studies on a suspension of phospholipid‐coated microbubbles showed a lower threshold than predicted from traditional elastic shell models. Here we present an experimental study of the subharmonic behavior of individual BR‐14 microbubbles (Bracco Research) with initial radii between 1.6 and 4.8 μm. The subharmonic behavior was studied as a function of the amplitude and the frequency of the driving pressure pulse. The radial response of the microbubbles was recorded with the Brandaris ultrahigh‐speed camera, while the resulting acoustic response was measured with a calibrated transducer. It is shown that the threshold pressure is minimum near a driving frequency equal to half the resonance frequency of the bubble, as expected. We found a threshold pressure as low as 10 kPa for certain bubble sizes, which can be explained by the shell buckling model proposed by [Marmottant et al., JASA (2005)]. We show that the origin of subharmonic behavior is a result of the discontinuous transition within the bubble shell from the elastic state to the tensionless buckling state.

Experimental and theoretical investigations of ultrasound contrast agent dynamics between parallel plates and within rectangular channels.

The behavior of ultrasound contrast agent (UCA) microbubbles in confining environments is relevant for applications including ultrasound imaging and targeted microbubbles. In this presentation, measurements from experiments investigating the radial dynamics of UCA microbubbles positioned between parallel plates and within rectangular microchannels will be compared with model simulations. The radial motion of several UCA bubbles was recorded with high‐speed imaging at 15 million frames per second over a range of insonation frequencies at various offset distances from the plates or channel walls. The behavior of each individual bubble was observed in both parallel plate and microchannel environments, thereby isolating effects due purely to the confinement. Several plastics and polymers with well‐defined material properties were used to construct the plates and channels, making the dataset suitable for model validation. Measurements show that the oscillation amplitude and natural frequency of the bubbles dep...

Complex formation and other phase transformations mapped in saturated phosphatidylcholine/DSPE-PEG2000 monolayers

Mixtures containing a saturated phosphatidylcholine (PC)-lipid and polyethylene glycol (PEG)-lipid have been widely used in lipid bilayer and monolayer systems, in particular for forming drug delivery stealth liposomes and for stabilizing medical microbubble ultrasound contrast agents. The miscibility and phase behavior of these systems have been incompletely explored and controversial. Here, using complementary experimental methods and analysis, we examine the phase behavior and construct complete surface pressure (π)–composition phase diagrams for three binary Langmuir monolayer systems of increasing saturated PC-lipid chain-length from n = 14 to n = 18. Specifically, for DMPC, DPPC, and DSPC mixed with DSPE-PEG2000, we examine π–area per molecule isotherms, π–isothermal surface compressibility plots, molecular area–composition plots, and microstructure in fluorescent monolayers. We find that all three of these systems resembled different classical binary phase diagrams at 298K with the following notable phase behavior over wide ranges of composition and π: (1) DMPC/DSPE-PEG2000 forms a single condensed (C) phase containing a stoichiometric complex, DMPC3DSPE-PEG2000, in addition to a eutectic at XDSPE-PEG2000 ≈ 0.66; (2) DPPC/DSPE-PEG2000 forms a single expanded (E) or C phase in addition to a peritectic at XDSPE-PEG2000 ≈ 0.55; and, (3) DSPC/DSPE-PEG2000 displays two-phase coexistence where the two components are almost completely immiscible. The phase diagrams are used to elucidate previously unexplained or conflicting results in the literature and design principles of monolayers and bilayers for medical microbubbles and stealth liposomes. In addition, we suggest that these systems may be ideal models for testing mechanisms of lipid condensation by both experiment and theory.

Performance de l’échographie de contraste en pathologie hépatique : synthèse de la littérature: Contrast-enhanced Ultrasound and liver imaging: review of the literature

The advent of second-generation microbubble ultrasound contrast agents and the development of contrast specific ultrasound techniques improved the ability of contrast enhanced ultrasound (CEUS) in detecting and characterizing focal liver lesions, opening new prospects in liver imaging. A Medline search in June 2008 identified 72 published studies that used CEUS in focal liver lesion detection, characterization, and follow-up to monitor tumor ablation procedures and antiangiogenic treatment. The purpose of this paper, based on literature review, is to describe the technical recommendations when using CEUS for liver imaging and to define the different vascular patterns of the most relevant benign and malignant lesions. Diagnostic performance of CEUS and the important clinical indications are also presented and discussed. CEUS is increasingly accepted in clinical use for diagnostic imaging and post-interventional workup liver imaging. It may replace many computed tomography and magnetic resonance imaging examinations in the near future, according to the European Federation of Societies for Ultrasound in Medicine and Biology guidelines. L’utilisation de produits de contraste ultrasonores de seconde génération et le développement de techniques spécifiques d’échographie de contraste ultrasonore (ECUS) ont permis d’améliorer la capacité de l’ECUS à détecter et caractériser les lésions hépatiques, ouvrant la voie à de nouvelles perspectives en imagerie hépatique. Une recherche Medline réalisée en juin 2008 a permis de sélectionner 72 études publiées ayant utilisé l’ECUS pour la détection de lésions focales hépatiques, leur caractérisation, la surveillance de patients après une procédure de thermoablation par radiofréquence ou sous traitement antiangiogénique. L’objectif de cet article, basé sur une revue de la littérature, est de décrire pour l’ECUS les recommandations techniques en pathologie hépatique et de définir les différents aspects observés dans les lésions hépatiques bénignes et malignes les plus fréquentes. Les performances diagnostiques de l’ECUS ainsi que les principales indications cliniques sont également présentées et discutées. L’ECUS est une technique de plus en plus acceptée et utilisée en routine clinique pour le diagnostic, la caractérisation et la surveillance per et post-thérapeutique des lésions en imagerie hépatique. Dans un futur proche, cette technique se substituera vraisemblablement à de nombreux examens scanner et IRM, en accord avec les recommandations de la Fédération Européenne des Sociétés d’Ultrasons en Médecine et Biologie.

Acoustic characterization of single ultrasound contrast agent microbubbles

Individual ultrasound contrast agent microbubbles (BR14) were characterized acoustically. The bubbles were excited at a frequency of 2 MHz and at peak-negative pressure amplitudes of 60 and 100 kPa. By measuring the transmit and receive transfer functions of both the transmit and receive transducers, echoes of individual bubbles were recorded quantitatively and compared to simulated data. At 100 kPa driving pressure, a second harmonic response was observed for bubbles with a size close to their resonance size. Power spectra were derived from the echo waveforms of bubbles of different sizes. These spectra were in good agreement with those calculated from a Rayleigh-Plesset-type model, incorporating the viscoelastic properties of the phospholipid shell. Small bubbles excited below their resonance frequency have a response dominated by the characteristics of their phospholipid shell, whereas larger bubbles, excited above resonance, have a response identical to those of uncoated bubbles of similar size.

Superparamagnetic nanoparticle-inclusion microbubbles for ultrasound contrast agents

We have developed a new type of ultrasound (US) contrast agent, consisting of a gas core, a layer of superparamagnetic iron oxide Fe3O4 nanoparticles (SPIO) and an oil in water outermost layer. The newly developed US contrast agent microbubbles have a mean diameter of 760 nm with a polydisperity index (PI) of 0.699. Our in vitro and in vivo experiments have shown that they have the following advantages compared to gas-encapsulated microbbubbles without SPIO inclusion: (1) they provide better contrast for US images; (2) the SPIO-inclusion microbubbles generate a higher backscattering signal; the mean grey scale is 97.9, which is 38.6 higher than that of microbubbles without SPIO; and (3) since SPIO can also serve as a contrast agent of magnetic resonance images (MRI) in vitro, they can be potentially used as contrast agents for double-modality (MRI and US) clinical studies.

Use of Contrast-Enhanced Ultrasound Imaging to Detect the First Draining Lymph Node (FDLN) in a Swine Model

The purpose of this study was to investigate the use of contrast-enhanced ultrasound imaging (CEUS) to detect the first draining lymph node (FDLN) in a swine model and to determine whether the distance from the contrast agent injection site to the FDLN has any affect on the ability to detect contrast-enhanced FDLNs. Thirteen swine (25-32 kg) were anesthetized during examinations and euthanized afterward. In every swine, 1 mL of a sulfur hexafluoride microbubble ultrasound contrast agent was bilaterally injected subcutaneously below a mammilla. The examined distances varied from 6 to 36 cm. The contrast-enhanced lymphatic channels were visualized with low-mechanical index CEUS and mapped from the injection site to the FDLN. After CEUS was performed, blue dye was injected in the same locations as the contrast agent, and dye-guided surgery was used to localize the FDLNs. To ensure the lymph node detected with the blue dye technique was the same found with CEUS, it was scanned again to confirm contrast enhancement. After 26 injections, 22 inguinal FDLNs were detected with CEUS and the blue dye technique. After 4 injections in 2 swine, contrast-filled lymphatic channels were identified with a course running toward the neck. In all cases but 1, the FDLNs received the contrast agent within 5 minutes. In this swine model, it was possible to use CEUS to locate the FDLNs. In these preliminary experiments, the distances from the contrast agent injection site to the FDLN did not affect the ability to detect the contrast-enhanced FDLNs.

Ultrasound microbubble contrast agent enhances TRAIL gene transfection into hepatocellular carcino-ma cells

Objective To evaluate the impact of the recombined plasmid vector with enhanced green fluorescent protein (EGFP) encoding soluble tumor necrosis factor related apoptesis inducing ligand (pIRES-EGFP-sTRAIL) on proliferation and apoptosis of human hepatocellular carcinoma cell line HepG2, and investi-gate the feasibility and efficiency of the transfection of pIRES- EGFP- sTRAIL into HepG2 by ultrasound micro-bubble contrast agent. Methods pIRES-EGFP-sTRAIL was constructed and transfected into HepG2 cells by using different types of mediated methods: microbubble echocontrast agent combining appropriate dose of ultra-sound irradiation, liposome method, microbubble echocontrast agent only or blank medium treatment. Transfec-tion efficiency was evaluated by EGFP-expressed cell count; proliferation-lnhibiting rate and the apoptosis rate of HepG2 cells were determined by MTT method and flow cytometry analysis; changes of cell morphology were examined by microscopy with Hoechst33258 dyeing; expression of caspase-8 and caspase-3 was detected by Western blot. Results Ultrasound microbubbh enhanced pIKES-EGFP-sTRAIL uptake by HepG2 cells, and the transfection efficiency was significantly higher in ultrasound microlmbble group than that in other groups( P<0.05 ) ; pIRES- EGFP- sTBAIL effectively inhibited HepG2 cell proliferation and induced cell apoptosis by triggering caspase cascade. Both the inhibiting rate and apoptosis rate were significantly higher in ultrasound microbubble group than those in other groups(P<0.05). Conclusion pIRES-EGFP-sTRAIL expresses ef-fectively in HepG2 cells, sTRAIL has a potential role on the inhibiting proliferation and inducing apoptosis of HepG2 cells by triggering caspase cascade, and this role can be enhanced by the administration of low-intensity ultrasound and microbubble echecontrast agent. Key words: Liver neoplasmas; Sonication; Microbubbles; Contrast media; Tumor necrosis factors

Secondary Bjerknes forces between ultrasound contrast agent microbubbles

Acoustic radiation forces arise on UCA microbubbles from pressure gradients in the ultrasound pressure field. The ultrasound wave emitted by an oscillating UCA microbubble and experienced by a neighboring bubble results in the so-called secondary Bjerknes force. Typically, only the (time) averaged value of the secondary Bjerknes force is estimated from experiments. Here, the ability to resolve in time the radial dynamics of UCAs and the evolution of their relative distances, allows us to obtain a quantification of the instantaneous secondary Bjerknes force. Two bubbles were selected and confined within two separate optical traps and positioned away from the sample chamber wall prior to insonation. The radius-time curves and distance-time curves were then extracted from high-speed optical recordings. The camera fully resolved the alternating attractive-repulsive features of the secondary Bjerknes force in time. The effect of the time averaged secondary Bjerknes force results in a net attraction of the two bubbles, with a typical observed displacement of 1-2 μm, with an instantaneous peak value of the secondary Bjerknes force of up to 10-6 N. We predict the mutual interaction of the two coated bubbles in their translation with an accuracy better than 10%.