Role of endocytosis in sonoporation-mediated membrane permeabilization and uptake of small molecules: a electron microscopy study.

Sonoporation is a physical method that has been successfully used to deliver drugs into living cells both in vitro and in vivo for experimental and therapeutic purposes. Despite numerous studies on this topic, often reporting successful outcomes, very little is known about the mechanisms involved in the hypothesized membrane permeabilization processes. In this study, electron microscopy was used to investigate the ultra-structural modifications of cell membranes, induced by sonoporation. Here, we demonstrate that sonoporation in the presence of microbubbles induces the formation of a significant number of transient and permeant structures at the membrane level. These structures were transient with a half-life of 10 min and had a heterogeneous size distribution ranging from a few nanometers to 150 nm. We demonstrated that the number and the size of these structures were positively correlated with the enhanced intracellular uptake of small molecules. In addition, we showed that these structures were associated with caveolae-dependent endocytosis for two thirds of the recorded events, with the remaining one third related to non-specific routes such as membrane disruptions as well as caveolae-independent endocytosis. In conclusion, our observations provide direct evidences of the involvement of caveolae-endocytosis in cell membrane permeabilization to small molecules after sonoporation.

Irinotecan delivery by microbubble-assisted ultrasound: in vitro validation and a pilot preclinical study.

Irinotecan is a powerful anticancer drug with severe systemic side effects that limit its clinical application. Drug-targeted delivery with noninvasive methods is required to enhance the drug concentration locally and to reduce these undesirable events. Microbubble-assisted ultrasound has become a promising method for noninvasive targeted drug delivery. The aim of this study is to evaluate the therapeutic effectiveness of in vitro and in vivo irinotecan delivery based on the combination of ultrasound and microbubbles. In the present study, in vitro results showed that the irinotecan treatment with microbubble-assisted ultrasound induced a significant decrease in cell viability of human glioblastoma cells. Moreover, using subcutaneous glioblastoma xenografts, the in vivo preclinical study in nude mice demonstrated that this therapeutic protocol led to a decrease in tumor growth and perfusion and an increase of tumor necrosis. The conclusions drawn from this study demonstrate the promising potential of this therapeutic approach for the anticancer targeted therapy.

Doxorubicin delivery into tumor cells with ultrasound and microbubbles.

Doxorubicin is a potent chemotherapeutic whose severe side effects limit its application. Drug-targeted delivery with noninvasive techniques is required to increase the drug concentration locally and to reduce systemic side effects. Microbubble-assisted ultrasound has become a promising strategy for noninvasive local drug delivery. The aim of this study is to evaluate the applicability and the effectiveness of administration of doxorubicin combined with microbubble-assisted ultrasound in human U-87MG glioblastoma and MDA-MB-231 breast cancer cells. In the present study, the doxorubicin delivery aided by microbubble-assisted ultrasound enhanced the death of breast cancer and glioblastoma cells, including the induction of apoptosis. Various microbubbles were evaluated including Vevo Micromarker, BR14, SonoVue and experimental polymer shelled microbubbles. The results showed that Vevo Micromarker microbubble-assisted ultrasound could induce an enhancement of doxorubicin in glioblastoma and breast cancer cell death. Polylactide-Shelled PEG and Vevo Micromarker microbubbles were the best microbubbles for efficient doxorubicin delivery in the U-87 MG and MDA-MB-231 cells, respectively. Moreover, the induction of apoptosis by doxorubicin and Vevo Micromarker microbubble-assisted ultrasound was examined and results showed a positive increment for acoustic pressures above 600 kPa. The conclusions drawn from in vitro study show the potential of this strategy for an in vivo application.

The Role of Hemodynamics in Intracranial Bifurcation Arteries after Aneurysm Treatment with Flow-Diverter Stents.

BACKGROUND AND PURPOSE: Treatment of intracranial bifurcation aneurysms with flow-diverter stents can lead to caliber changes of the distal vessels in a subacute phase. This study aims to evaluate whether local anatomy and flow disruption induced by flow-diverter stents are associated with vessel caliber changes in intracranial bifurcations. MATERIALS AND METHODS: Radiologic images and demographic data were acquired for 25 patients with bifurcation aneurysms treated with flow-diverter stents. Whisker plots and Mann-Whitney rank sum tests were used to evaluate if anatomic data and caliber changes could be linked. Symmetry/asymmetry were defined as diameter ratio 1 = symmetric and diameter ratio <1 = asymmetric. Computational fluid dynamics was performed on idealized and patient-specific anatomies to evaluate flow changes induced by flow-diverter stents in the jailed vessel. RESULTS: Statistical analysis identified a marked correspondence between asymmetric bifurcation and caliber change. Symmetry ratios were lower for cases showing narrowing or subacute occlusion (medium daughter vessel diameter ratio = 0.59) compared with cases with posttreatment caliber conservation (medium daughter vessel diameter ratio = 0.95). Computational fluid dynamics analysis in idealized and patient-specific anatomies showed that wall shear stress in the jailed vessel was more affected when flow-diverter stents were deployed in asymmetric bifurcations (diameter ratio <0.65) and less affected when deployed in symmetric anatomies (diameter ratio ∼1.00). CONCLUSIONS: Anatomic data analysis showed statistically significant correspondence between caliber changes and bifurcation asymmetry characterized by diameter ratio <0.7 (P < .001). Similarly, computational fluid dynamics results showed the highest impact on hemodynamics when flow-diverter stents are deployed in asymmetric bifurcations (diameter ratio <0.65) with noticeable changes on wall sheer stress fields. Further research and clinical validation are necessary to identify all elements involved in vessel caliber changes after flow-diverter stent procedures.

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.

[Sonopermeabilization: therapeutic alternative with ultrasound and microbubbles]. / Sonoperméabilisation: alternative thérapeutique par ultrasons et microbulles.

Future applications of ultrasound and microbubbles extend to more than imaging applications. Over the last few years, it was reported that sonographic contrast agent effects under ultrasound, modulate transiently cell membrane permeability. This process, named sonoporation and classified as a new physical method to transfer genes or drugs, consists of using a physical energy source to modulate membrane integrity. The possibility to transfer therapeutic genes would be a new tool for gene therapy and could constitute an alternative method. After in vitro and in vivo studies presentation, the therapeutic potential of sonoporation will be investigated in this paper.

Linear and nonlinear characterization of microbubbles and tissue using the Nakagami statistical model.

The goal of this work is to exploit the statistical signatures for discrimination between biological tissues and contrast microbubbles in order to develop new strategies for contrast imaging and tissue characterization. For this purpose, the efficiency of the Nakagami statistical model, for describing the ultrasonic echoes of both contrast microbubbles and tissues, was investigated. Experimental measurements have been performed using a linear array probe connected to an open research platform. A commercially available in vitro phantom was used to mimic biological tissue in which SonoVue contrast microbubbles were flowing. Experimental ultrasound echoes have been filtered around the transmitted frequency (fundamental at 2.5MHz) and around twice the transmitted frequency (at 5MHz) for 2nd harmonic analysis, and a logarithmic compression was applied. The signals have been analyzed in order to evaluate the Nakagami parameter m, the scaling parameter Ω and the probability density function at both frequencies. Parametric images based on the Nakagami parameters map (Nakagami-mode images) were reconstructed and compared to B-mode images. Contrary to the B-mode image which is influenced by the system settings and user operations, the Nakagami parametric image is only based on the backscattered statistics of the ultrasonic signals in a local phantom. Such an imaging principle allows the Nakagami image to quantify the local scatterer concentrations in the phantom and to extract the backscattering information from the regions of the weakest echoes that may be lost in the conventional B-mode image. Results show that the tissue and microbubbles characterization is more sensitive in the 2nd harmonic mode when a logarithmic transform is used. These results would be useful for improving the ultrasound image quality and contrast detection in nonlinear mode.

Detecting broken struts of a Björk-Shiley heart valve using ultrasound: a feasibility study.

The Björk-Shiley (BScc) mechanical heart valve has extensively been used in surgery from 1979 to 1986. There is, compared with equivalent valve types, increased occurrence of unexpected mechanical failure of the outlet strut of the valve, with a high incidence of mortality, when it occurs. Many approaches have been attempted to noninvasively determine BScc valve integrity. None of the approaches resulted in adequate assessment, mostly due to a lack of either sensitivity or specificity demonstrated in in vitro and/or in vivo studies. In our study we analyze leg movement of the BScc valves outlet strut during the cardiac cycle with ultrasound. For a broken strut, the movement of both legs will be significantly different, whereas the difference will be negligible for an intact strut. BScc valves were mounted in the mitral position in an in vitro pulse duplicator system. A focused single-element transducer was used to direct ultrasound on a leg of the outlet strut. Correlation-based time delay estimation was used to estimate differences in time of flight of the outlet strut echoes to determine outlet strut leg movement. The movement of an intact valve and a valve with a single-leg fracture with both ends grating against each other (SLF), the most difficult fracture to diagnose, has been studied. The results showed no significant difference in movement between both legs of the outlet strut of the intact BScc valve (amplitude of movement 9.2 microm +/- 0.1 microm). Whereas for the defective valve, the amplitude of movement of the broken leg of the SLF valve was 12 microm +/- 1.6 microm vs. 8.6 microm +/- 0.1 microm for the intact leg. In conclusion, the proposed method has shown to be feasible in vitro and has potentials for in vivo detection of BScc valve outlet strut fracture.

Low back pain, the stiffness of the sacroiliac joint: a new method using ultrasound.

Abnormal biomechanical properties of the sacroiliac joints are believed to be related to low back and pelvic pain. Presently, physiotherapists judge the condition of the sacroiliac joints by function and provocation tests, and palpation. No objective measuring device is available. Research is ongoing to identify the biomechanical properties of the sacroiliac joints from the dynamic behaviour of the pelvic bones. A new concept based on ultrasound (US) for the measurement of bone vibration is under investigation. The objective of this study was to validate this concept on a physical model and to assess the applicability in vivo. A model consisting of a piezo shaker covered by a layer of US transmission gel (representing bone and soft tissue) has been used. A packet of US detection signals is directed onto the shaker and correlation-based processing is used to estimate the difference in time-of-flight of their echoes. These variations of time are used to compute the displacement of the shaker at each pulse reflection. To assess the validity of our US technique, we compared the obtained measurements with the readings of the built-in strain gauge sensor. The experimental procedure has been tested on a volunteer where low-frequency excitation was provided through the ilium and vibration detected on the sacrum and ilia. The results demonstrated that the correlation-based approach is capable of reproducing the piezo shaker displacements with high accuracy (+/- 7%). Vibration amplitudes from 0.25 microm to 3 microm could be measured. The US technique was able to detect bone vibration in vivo. In conclusion, the principle based on US waves can be used to develop a new measurement tool, instrumental in studying the relation between the biomechanical properties of the sacroiliac joints and low back pain.

Comparison of native and contrast-enhanced harmonic echocardiography for visualization of left ventricular endocardial border.

Our study was designed to compare the utility of fundamental and second harmonic imaging (SH) for visualization of the left ventricular (LV) endocardial border. SH is a new imaging modality using nonlinear acoustic response, which may provide better endocardial border delineation. Standard apical views were studied in 42 patients using fundamental frequency (FF), SH without contrast (1.6- to 1.8-MHz and 2.1- to 2.5-MHz transmission frequencies), and SH after an intravenous injection of 2.5 g of Levovist. The quality of endocardial delineation in 16 standard segments was scored from 0 to 2. The endocardial visualization index was calculated as a mean of the scores. SH with and without contrast significantly improved LV endocardial border detection (endocardial visualization index 1.25+/-0.53, 1.64+/-0.67, 1.55+/-0.69, and 1.73+/-0.28 for fundamental, lower, and higher frequency harmonic and contrast-harmonic mode, respectively, p <0.005). Improvement was found in all LV segments. The number of invisible segments decreased from 142 (FF) to 54, 112, and 61 (in lower, higher, and contrast SH mode, respectively, p <0.001). Endocardial delineation in the apical segments using SH was optimal after contrast injection. In the basal LV area, contrast-enhanced images were less informative because of signal attenuation. Thus, SH significantly improves visualization of the LV endocardial border. Contrast enhancement with Levovist improves imaging of the apical segments but has no additional advantage in the basal segments. SH emerges as first-line modality for studies of LV function.

Standard properties of ultrasound contrast agents.

A standardization procedure for in vitro acoustic characterization of ultrasound contrast agents is presented. One new acoustic parameter for particular importance is retained: This is STAR, scattering-to-attenuation ratio, for quantification of the effectiveness of the contrast agent. The STAR expresses the ability of the contrast agent to enhance the visualization of the tissue containing the contrast agent and, at the same time, represents the degree of its absorption. So, it is desirable to produce a contrast agent with high STAR, having good scattering properties to improve the image visualization, and low attenuation to image the underlying biological structures and to avoid shadowing. In this study, we present methods for calculations and measurements of the STAR and comparison between different contrast agents.

Emboli detection using a new transducer design.

We have presented, in a previous study, a new approach to detect, characterize and estimate the size of gaseous emboli, based on the nonlinear behavior of gaseous bubbles. In this study, a specific transducer design has been developed to be used for such a purpose. It is composed of two separate transmitting and receiving capabilities. The transmit part, consisting of a lead zirconate-titanate (PZT) material, emits at a frequency of 500 kHz and could generate pressures up to 410 kPa. On the top of the transmit surface, a thin polyvinylidene difluoride (PVDF) layer is glued and used for receiving frequencies from 250 kHz (f0/2) up to 2.5 MHz (5 f0). To evaluate this new design, ultrasonic measurements were carried out with gas bubbles with diameters ranging from 10 microm up to 90 microm and solid particles between 350 microm and 550 microm. The experimental results confirmed our previous findings: gaseous emboli with a diameter close to the resonance size scatter significantly at higher harmonic components (from the second harmonic up to the fifth), and bubbles with a diameter around twice the resonance size produce a subharmonic and/or an ultraharmonic component. Meanwhile, solid particles and other bubble sizes behave only linearly and their scattered spectrum appeared without any harmonics. The study demonstrates the utility of this approach in using a single transducer to detect and characterize selective gaseous emboli from other particles using their nonlinear behavior.

Subharmonic and ultraharmonic emissions for emboli detection and characterization.

Emboli detection and characterization is of importance for different patients, such as those undergoing carotid or cardiac surgery. The emboli occur as particulate or gaseous matters. To select the appropriate treatment and reduce the risk of embolism, it is essential to first detect and then classify and, ultimately, size the emboli. We propose, in this study, an approach to characterize and size the emboli based on the nonlinear properties of the emboli. Gaseous emboli were produced by generating single and uniform air bubbles. These bubbles had diameters ranging from 40 microm to 120 microm. Acoustic measurements were carried out and special attention was devoted to the generation of subharmonic and first ultraharmonic components for gas bubbles of different sizes and at different acoustic pressures. For the scanning frequency and the applied acoustic pressures used in this study, only bubbles ranging from 58 microm up to 110 microm are capable of generating a subharmonic and an ultraharmonic frequency component. However, gas emboli outside this range behave differently. In conclusion, such an approach can be used to provide information needed to classify and size emboli.

Noninvasive measurement of the hydrostatic pressure in a fluid-filled cavity based on the disappearance time of micrometer-sized free gas bubbles.

A new method for noninvasive pressure measurement, based on the disappearance time of micrometer-sized free gas bubbles, is described in this article. An ultrasound (US) contrast agent, consisting of encapsulated gas bubbles, is used as a vehicle to transport the free gas bubbles to the desired region where the pressure is to be measured. The small free gas bubbles are generated at the region of interest (e.g., heart chambers), from the encapsulated gas bubbles, which rupture when they are exposed to a single low-frequency (e.g., 0.5 MHz), high acoustic amplitude US burst. The released gas bubbles persist for only a few ms and dissolve in the liquid, depending on their size, the gas, the liquid characteristics and ambient parameters such as temperature, gas concentration and pressure. A pressure-disappearance time relationship is determined using a sequence of high-frequency (e.g., 10 MHz), low acoustic amplitude US pulses. From in vitro experiments, reproducible results show a significant difference between the disappearance time of the bubbles as function of the local pressure, resulting in a quicker disappearance of the bubble for higher values of the pressure. The sensitivity of the method to small pressure changes (50 mmHg) is demonstrated.

Ultrasound contrast agent in intravascular echography: an in vitro study.

The intravascular ultrasound image of the intraluminal contour depends on the difference between acoustic impedances of the media which create the endoluminal interface. There are several limitations to the visualization and detection of this interface. These limitations are due to artifacts encountered during image formation and to anatomical complexity. The purpose of this study is to obtain intraluminal contour enhancement using ultrasound contrast agent (UCA). Therefore, our objective was to address the feasibility of this technique by documenting the following: (i) the acoustic properties of UCA at 30 MHz; (ii) in vitro experimentation with tube or postnecrotic artery; and (iii) suitable digital processing. The images obtained with UCA (enhanced image quality) and subtracted from those without UCA provided, after simple digital processing, accurate visualization of the arterial lumen. The image obtained exhibits an even, high-contrast intraluminal edge. Such characteristics facilitate contour extraction by the automated contour detection procedures.

New technique for emboli detection and discrimination based on nonlinear characteristics of gas bubbles.

Detection and characterization of emboli in the blood stream is of high clinical importance for making decisions after surgery. In this study, a new technique based on the nonlinear oscillations of gas bubbles was applied to gaseous emboli detection, characterization and sizing. To simulate gaseous emboli, an experimental system was developed to produce air bubbles of uniform diameters ranging from 19 microm up to 200 microm. The ultrasonic setup consisted of low-frequency transducers operating at 130 kHz and 250 kHz and using low acoustic pressures (30 kPa and 55 kPa). The experimental and theoretical results show that, depending on the transmitted frequency and the bubble sizes, higher harmonic components were produced in the frequency spectrum of the backscattered echo. Nonresonating bubbles scatter either linearly when their sizes are far away from the resonance size or nonlinearly at the second or third harmonic frequency when their sizes are getting close to the resonance size. Only resonant bubbles or bubbles very close to the resonance size are able to scatter at higher harmonic frequencies (fourth and fifth). This property is used to discriminate resonating bubbles from other bubble sizes. The appearance of harmonic component in the frequency spectrum seems to be an unambiguous tool to differentiate gaseous emboli from solid emboli that scatter linearly.

Optical imaging of contrast agent microbubbles in an ultrasound field with a 100-MHz camera.

Ultrasound (US) contrast agents, used in the field of medical diagnosis, contain small microbubbles of a mean diameter of about 3 microm. The acoustic behavior of these bubbles in US field has been subject to many investigations. In this study, we propose a method to visualize the behavior of the bubbles in a 0.5-MHz US field under a microscope with a frame rate of 4 MHz. For low acoustic pressures (peak negative pressure of 0.12 MPa), the radius-time curve as measured from the optical images is in agreement with the theory. For higher acoustic pressures (peak negative pressure of 0.6 MPa), the recorded radius is significantly larger than predicted by theory and sudden change in the bubbles shapes has been noticed. The proposed method enables the study and characterization of individual bubbles and their encapsulation. It is expected that this will open new areas for quality control, US contrast imaging and US-guided drug delivery.

Ultrasound contrast imaging: current and new potential methods.

For 10 years, it was thought that ultrasound (US) contrast agents could be sufficiently detected and imaged with the conventional imaging techniques, now referred to as fundamental imaging. However, it turned out that fundamental imaging was not sensitive enough to detect the contrast agents in the presence of tissue. New imaging techniques that are based on specific properties of the contrast agents, such as nonlinear and transient scattering, proved to be more sensitive. US contrast imaging modalities used today are fundamental, second harmonic, harmonic power Doppler, and pulse inversion; new modalities, such as release burst and subharmonic imaging are emerging. Second harmonic imaging is still not optimal for perfusion imaging applications. However, in combination with Doppler techniques such as power Doppler, it is one of the most sensitive techniques currently available. A complete understanding of the US-contrast agent interaction is essential for further improvements of current detection methods, and the development of new imaging techniques.

On the effect of lung filtering and cardiac pressure on the standard properties of ultrasound contrast agent.

The goal standard of contrast echocardiography is the absolute measure of myocardial perfusion using a contrast agent. Actually, several contrast agents are developed. All these agents show left ventricular opacification after intravenous injection. However, none of these agents shows an acceptable enhancement of the myocardium yet using conventional imaging techniques. The explanation of this phenomenon should be easy by measuring the acoustic characteristics of the contrast agent and then making a comparison of these characteristics with those of the myocardium. In this study we present definitions of standard acoustic parameters of ultrasound contrast agent, the backscatter coefficient Bs and the scattering-to-attenuation ratio STAR. Afterwards, considering an intravenous injection of the contrast agent, and taking into account the effects of lung filtering and cardiac pressure, the standard properties of contrast agents are determined in different sites: right ventricle (before lung passage), left ventricle (after lung passage and taking into account the pressure effect) and in the coronary system. Calculations showed that the acoustic properties are considerably influenced by these two effects: lung filtering and cardiac pressure. Comparison of these properties with the tissue properties (myocardium) is then performed. This determines the contribution of the contrast agent to the enhancement of the tissue visualization. The simulations are performed on Albunex microspheres. The results reveal that the difference between scattering of the myocardium and scattering of intravenously injected Albunex is too slight to be visible on an echographic image.

Detection procedures of ultrasound contrast agents.

In the early days, it was believed that ultrasound contrast agents (UCA) could be sufficiently detected and imaged with the conventional imaging methods nowadays referred to as fundamental imaging. Newer imaging techniques proved to be more sensitive and are based on specific properties of the UCA. In general, these new characteristics involve non-linear and transient characteristics of contrast agents that appear at the high end of the diagnostic acoustic intensity. Imaging modalities used today for UCA are, besides fundamental imaging, second harmonic imaging, power Doppler, harmonic power Doppler, pulse inversion and pulse inversion Doppler, multi-pulse imaging and subharmonic imaging. Although the results of conventional second harmonic imaging are still not optimal for perfusion imaging applications, in combination with Doppler techniques (colour Doppler, power Doppler) it is one of the most sensitive techniques currently available in terms of agent-to-tissue ratio. Further improvements in current and future detection methods demand a complete understanding of the ultrasound-UCA interaction.