SonoVue Microbubbles Research Articles

Abstract 2899: Ultrasound Exposed Microbubbles Cause Local Hyperpolarization of the Cell Membrane.

Background Ultrasound (US) and microbubbles have gained wide interest as a tool for local delivery of drugs and genes. However, the exact mechanisms underlying increased cellular uptake of drugs and genes are still not clear. Recently, we showed an increase in cell membrane permeability for Ca 2+ in rat cardiomyoblast (H9c2) cells exposed to US and microbubbles. In this study, we hypothesized that the Ca 2+ influx will have an effect on the membrane potential, and investigated whether Ca 2+ dependent potassium channels (BK Ca ) are involved. We particularly focused on local events where the microbubble was in contact with the cell membrane. Material and Methods H9c2 cells were cultured on US transparent membranes. US exposure consisted of bursts of 1 MHz with a peak negative pressure of 50 or 250 kPa. Pulse repetition frequency was 20 Hz, duty cycle 0.2%. Cells were exposed during 30s in the presence of Sonovue microbubbles. The membrane potential was monitored during US exposure using the fluorescent dye di-4-ANEPPS. The experiments were repeated in the presence of iberiotoxin (100 nM), a specific inhibitor of BK Ca channels. Results Surprisingly, despite the previously reported Ca 2+ influx, we found patches of hyperpolarization of the cell membrane, as reflected by local increases in di-4-ANEPPS mean fluorescent intensity (MIF), shown in table I as percentage of control (%MIF/control). This hyperpolarization was caused by the activation of BK Ca channels, as iberiotoxin completely prevented hyperpolarization. Conclusion US and microbubbles elicit a Ca 2+ influx, which leads to activation of BK Ca channels and a subsequent, local hyperpolarization of the cell membrane. This local hyperpolarization of the cell membrane may facilitate uptake of macromolecules through endocytosis and macropinocytosis. Table I

Grafting of abciximab to a microbubble-based ultrasound contrast agent for targeting to platelets expressing GP IIb/IIIa – Characterization and in vitro testing

Abciximab-grafted ultrasound sensitive microbubbles were developed for the diagnosis of stroke. The antibody fragment abciximab, which binds to the GP IIb/IIIa and α vβ 3 receptors expressed by activated platelets, was chosen because most ischemic strokes are due to arterial thrombi that are mainly composed of platelets. The abciximab antibody fragment was activated by reduction of the disulfide bond for grafting on the microbubbles. The suspension was freeze-dried after the grafting was performed directly on the formed microbubbles. Quantification of the amounts of abciximab present on the surface of the microbubbles was assessed semi-quantitatively by flow cytometry, and quantitatively using radio-labeled abciximab. A protocol for human and rat platelets deposition and fixation was implemented and the expression of the GP IIb/IIIa receptor was validated by immunostaining. The abciximab-grafted microbubbles showed high static and dynamic binding to fixed platelets. Detection by ultrasonography of microbubbles bound on white and red clots gave higher signals compared to SonoVue microbubbles.

Accuracy of contrast-enhanced breast ultrasound for pre-operative tumor size assessment in patients diagnosed with invasive ductal carcinoma of the breast

Our aim was to assess the feasibility and accuracy of contrast-enhanced ultrasound (CEUS) of the breast with SonoVue microbubbles for pre-operative size measurement of invasive breast carcinomas. Seven patients diagnosed with nine invasive breast carcinomas prospectively underwent gray-scale ultrasound and CEUS of the breast according to a standardized protocol. CEUS of the breast was performed by a Philips iU22 scanner equipped with a 4–8 MHz linear array transducer. We used a single dose of 2.4 ml SonoVue as contrast agent. Breast lesion morphology was scored according to the sonographic BI-RADS lexicon criteria and classified accordingly. The greatest tumor dimensions on gray-scale ultrasound and CEUS of the breast were finally compared with the greatest histopathologic tumor sizes. Gray-scale ultrasound underestimated the histopathologic tumor size in 6/9 cases (67%), whereas CEUS of the breast underestimated tumor size in only 3/9 (33%) cases. CEUS of the breast was significantly more accurate for tumor size assessment. Greatest tumor dimension as measured with gray-scale ultrasound of the breast was within 2 mm of the pathologic tumor size in only 2/9 cases (22%), whereas CEUS of the breast accurately assessed tumor size within 2 mm of pathologic tumor size in 6/9 (67%) of the cases (P<0.05). CEUS of the breast proved to be a feasible and safe procedure. It is more accurate than gray-scale ultrasound of the breast for pre-operative size assessment of invasive ductal breast carcinomas.

Effect of ultrasound-activated microbubbles on the cell electrophysiological properties

New clinical applications of ultrasound contrast microbubbles extend beyond imaging and diagnosis toward therapeutic applications. Cell membrane permeability and the uptake of substances have been shown to be enhanced by microbubbles under ultrasound stimulation. However, the mechanisms of action of ultrasound-activated microbubbles are still unknown. The aim of our study was to examine how microbubbles and ultrasound interact with cells in an attempt to understand the sonoporation mechanism. The ruptured-patch-clamp whole-cell technique was used to measure membrane potential variations of a single cell. SonoVue microbubbles and mammary breast cancer cell line MDA-MB-231 were used. Ultrasound was applied using single-element transducers of 1 MHz. Microbubbles and cells were simultaneously video monitored during ultrasound exposure. Our results showed that, during sonoporation, a marked cell membrane hyperpolarization occurs ( n = 6 cells) at negative pressures above 150 kPa, indicating the activation of specific ion channels while the cell and the microbubbles remain viable. The hyperpolarization was sustained for as long as the microbubbles are in a direct contact with the cell and the ultrasound waves are transmitted. Smaller acoustic amplitudes induced only mild hyperpolarization, whereas shutting off the ultrasound brings the cell membrane potential to its resting value. However, ultrasound alone did not affect the cell membrane potential. A similar hyperpolarization of the cell membrane was observed when a mechanical pressure was applied on the cell through a glass probe. In conclusion, the results demonstrate that microbubbles’ oscillations under ultrasound activation entail modifications of the electrophysiologic cell activities by triggering the modulation of ionic transports through the plasmic cell membrane. However, only cells in direct contact with the microbubbles are impacted. The mechanisms involved are likely related to activation of specific channels sensitive to mechanical stresses (stretch-activated channels) and possibly nonspecific ion channels. (E-mail: bouakaz@med.univ-tours.fr)

Contrast-Enhanced Breast Ultrasonography Reveals an Unusual Breast Tumor in a Male Patient With Gynecomastia

Pseudoangiomatous stromal hyperplasia (PASH), first described in 1986, is a benign breast lesion consisting of complex, anastomosing, slitlike pseudovascular spaces, which are either acellular or lined by slender, spindle-shaped cells. 1,2 The exact etiology of PASH is unknown. It usually presents as a breast mass, affecting women in their reproductive years. Pseudoangiomatous stromal hyperplasia has been reported in at least 25% of cases of gynecomastia. 2 Although PASH is a benign lesion, it has a propensity for growth over time and local recurrence after excision.3 Several small-scale studies already described the mammographic and gray scale ultrasonographic findings of PASH. 3,4,5 To our knowledge, the appearance of PASH on contrast-enhanced ultrasonography (CEUS) of the breast has never been described before. In past years, CEUS has been used increasingly in clinical practice to characterize indeterminate breast lesions. 6-14 In CEUS, a contrast agent consisting of a suspension of microbubbles is used to increase the blood echogenicity and to improve detection of slow and low-volume blood flow in microvessels (<5 μm). Because malignant breast tumors display neoangiogenesis that can be depicted by the contrast agent, it is believed that CEUS can be used to characterize breast masses and distinguish benign from malignant lesions. In this report, we describe the case of an elderly male patient with gynecomastia in whom conventional mammography and gray scale ultrasonography revealed a solid tumor in the left breast that was suggestive of malignancy. Additional workup of the lesion included CEUS of the breast with SonoVue (Bracco SpA, Milan, Italy) microbubbles to analyze tumor vascularization. On the basis of the CEUS findings, the lesion was down-staged to a benign breast lesion, probably a PASH. 6-13 After ultrasonographically guided biopsy of the lesion, histologic examination of the tissue confirmed the diagnosis. We describe the imaging characteristics of PASH on CEUS and provide an in-depth review of the literature.

Spatial and acoustic pressure dependence of microbubble‐mediated gene delivery targeted using focused ultrasound

Ultrasound/microbubble-mediated gene delivery has the potential to be targeted to tissue deep in the body by directing the ultrasound beam following vector administration. Application of this technology would be minimally invasive and benefit from the widespread clinical experience of using ultrasound and microbubble contrast agents. In this study we evaluate the targeting ability and spatial distribution of gene delivery using focused ultrasound. Using a custom-built exposure tank, Chinese hamster ovary cells in the presence of SonoVue microbubbles and plasmid encoding beta-galactosidase were exposed to ultrasound in the focal plane of a 1 MHz transducer. Gene delivery and cell viability were subsequently assessed. Characterisation of the acoustic field and high-resolution spatial analysis of transfection were used to examine the relationship between gene delivery efficiency and acoustic pressure. In contrast to that seen in the homogeneous field close to the transducer face, gene delivery in the focal plane was concentrated on the ultrasound beam axis. Above a minimum peak-to-peak value of 0.1 MPa, transfection efficiency increased as acoustic pressure increased towards the focus, reaching a maximum above 1 MPa. Delivery was microbubble-dependent and cell viability was maintained. Gene delivery can be targeted using focused ultrasound and microbubbles. Since delivery is dependent on acoustic pressure, the degree of targeting can be determined by appropriate transducer design to modify the ultrasound field. In contrast to other physical gene delivery approaches, the non-invasive targeting ability of ultrasound makes this technology an attractive option for clinical gene therapy.

Transient permeabilization of cell membranes by ultrasound-exposed microbubbles is related to formation of hydrogen peroxide

In the present study, we addressed the interactions among ultrasound, microbubbles, and living cells as well as consequent arising bioeffects. We specifically investigated whether hydrogen peroxide (H(2)O(2)) is involved in transient permeabilization of cell membranes in vitro after ultrasound exposure at low diagnostic power, in the presence of stable oscillating microbubbles, by measuring the generation of H(2)O(2) and Ca(2+) influx. Ultrasound, in the absence or presence of SonoVue microbubbles, was applied to H9c2 cells at 1.8 MHz with a mechanical index (MI) of 0.1 or 0.5 during 10 s. This was repeated every minute, for a total of five times. The production of H(2)O(2) was measured intracellularly with CM-H(2)DCFDA. Cell membrane permeability was assessed by measuring real-time changes in intracellular Ca(2+) concentration with fluo-4 using live-cell fluorescence microscopy. Ultrasound, in the presence of microbubbles, caused a significant increase in intracellular H(2)O(2) at MI 0.1 of 50% and MI 0.5 of 110% compared with control (P < 0.001). Furthermore, we found increases in intracellular Ca(2+) levels at both MI 0.1 and MI 0.5 in the presence of microbubbles, which was not detected in the absence of extracellular Ca(2+). In addition, in the presence of catalase, Ca(2+) influx immediately following ultrasound exposure was completely blocked at MI 0.1 (P < 0.01) and reduced by 50% at MI 0.5 (P < 0.001). Finally, cell viability was not significantly affected, not even 24 h later. These results implicate a role for H(2)O(2) in transient permeabilization of cell membranes induced by ultrasound-exposed microbubbles.

Bubbles in echocardiography: climbing the learning curve

The past few years have seen the rapid development of novel imaging modes tuned to the specific detection of microbubbles; these include harmonic 13-mode, phase or pulse inversion, power modulation, coherent contrast imaging, and recently power pulse inversion. These contrast-specific imaging modes, combined with insonation at low incident pressure, result in greatly enhanced contrast between the vessels that contain the ultrasound contrast agent and the surrounding tissues, rendering perfusion defects highly visible. SonoVue (Bracco, Milan, Italy) is a new echocontrast agent with outstanding stability. Thanks to their very flexible shell, the SonoVue microbubbles are able to generate strong harmonics even when insonated at extremely low mechanical index (MI). The present review illustrates the different modalities of contrast bubble insonation (at high and low MI), which allow visualization of fixed defects and ischaemic or infarcted areas of the myocardium in real-time.

Influence of bubble size distribution on the echogenicity of ultrasound contrast agents: a study of SonoVue.

To study the relative contributions of different bubble size classes to SonoVue's echogenicity in fundamental acoustic imaging modes. SonoVue is a contrast agent, previously known as BR1, with a bubble size distribution extending from approximately 0.7 to 10 microm. A model for the acoustic response of SonoVue was determined and validated for a set of experimental data. This model was used to simulate the acoustic response of a standard batch of SonoVue as the sum of responses of non-overlapping bubble size classes. The simulation was first validated for a standard SonoVue bubble size distribution. When this distribution was considered as five size classes with equal numbers of bubbles, it was found that bubbles smaller than 2 microm accounted for 60% of the total number but contained only 5% of the total gas volume. The simulation results indicated marked differences in the acoustic contributions from these classes, with 80% of the acoustic efficacy provided by bubbles 3 to 9 microm in diameter. The study also compared bubble distributions in number, surface, and volume, with the distribution computed in terms of acoustic efficacy. This study shows why bubble volume is a much better indicator of SonoVue's efficacy than is bubble count. A low threshold in diameter was found for SonoVue microbubbles at approximately 2 microm, under which size bubbles do not contribute appreciably to the echogenicity at medical ultrasound frequencies.