Microbubbles combined with ultrasound therapy in ischemic stroke: A systematic review of in-vivo preclinical studies.

BACKGROUND: Microbubbles (MBs) combined with ultrasound sonothrombolysis (STL) appears to be an alternative therapeutic strategy for acute ischemic stroke (IS), but clinical results remain controversial. OBJECTIVE: The aim of this systematic review is to identify the parameters tested; to assess evidence on the safety and efficacy on preclinical data on STL; and to assess the validity and publication bias. METHODS: Pubmed® and Web of ScienceTM databases were systematically searched from January 1995 to April 2017 in French and English. We included studies evaluating STL on animal stroke model. This systematic review was conducted in accordance with the PRISMA guidelines. Data were extracted following a pre-defined schedule by two of the authors. The CAMARADES criteria were used for quality assessment. A narrative synthesis was conducted. RESULTS: Sixteen studies met the inclusion criteria. The result showed that ultrasound parameters and types of MBs were heterogeneous among studies. Numerous positive outcomes on efficacy were found, but only four studies demonstrated superiority of STL versus recombinant tissue-type plasminogen activator on clinical criteria. Data available on safety are limited. LIMITATIONS: Quality assessment of the studies reviewed revealed a number of biases. CONCLUSION: Further in vivo studies are needed to demonstrate a better efficacy and safety of STL compared to currently approved therapeutic options. SYSTEMATIC REVIEW REGISTRATION: http://syrf.org.uk/protocols/.

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

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

Microbubble-mediated ultrasound drug-delivery and therapeutic monitoring.

INTRODUCTION: Recent developments in ultrasound imaging and ultrasound contrast agents (UCAs) improved diagnostic confidence in echography and set into motion their combined use as a tool for drug delivery and therapeutic monitoring. Non-invasive, precise and targeted delivery of drug molecules to pathological tissues by employing different mechanisms of drug release is becoming feasible. Areas covered: We sought to describe: the nature and features of UCAs; outline current contrast-specific imaging modes; before describing a variety of strategies for using ultrasound and microbubbles as a drug delivery system. Our expert opinion focusses on results and prospects of using ultrasound and microbubbles as a dual modality for drug delivery and therapeutic monitoring. Expert opinion: Today, ultrasound and microbubbles present a realistic prospect as drug delivery tools that have been demonstrated in a variety of animal models and clinical indications. Besides delivering drugs, ultrasound and microbubbles have demonstrated added value through therapeutic monitoring and assessment. Successful evaluation of the sonoporation mechanism(s), ultrasound parameters, drug type and dose will need to be addressed before translating this technology for clinic use. Ultimately, the development of a strategy for monitoring targeted delivery and its implementation in clinical practice would advance therapeutic treatment to a new qualitative level.

Surface Charge Measurement of SonoVue, Definity and Optison: A Comparison of Laser Doppler Electrophoresis and Micro-Electrophoresis.

Microbubble (MB) contrast-enhanced ultrasonography is a promising tool for targeted molecular imaging. It is important to determine the MB surface charge accurately as it affects the MB interactions with cell membranes. In this article, we report the surface charge measurement of SonoVue, Definity and Optison. We compare the performance of the widely used laser Doppler electrophoresis with an in-house micro-electrophoresis system. By optically tracking MB electrophoretic velocity in a microchannel, we determined the zeta potentials of MB samples. Using micro-electrophoresis, we obtained zeta potential values for SonoVue, Definity and Optison of -28.3, -4.2 and -9.5 mV, with relative standard deviations of 5%, 48% and 8%, respectively. In comparison, laser Doppler electrophoresis gave -8.7, +0.7 and +15.8 mV with relative standard deviations of 330%, 29,000% and 130%, respectively. We found that the reliability of laser Doppler electrophoresis is compromised by MB buoyancy. Micro-electrophoresis determined zeta potential values with a 10-fold improvement in relative standard deviation.

A Targeting Microbubble for Ultrasound Molecular Imaging.

RATIONALE: Microbubbles conjugated with targeting ligands are used as contrast agents for ultrasound molecular imaging. However, they often contain immunogenic (strept)avidin, which impedes application in humans. Although targeting bubbles not employing the biotin-(strept)avidin conjugation chemistry have been explored, only a few reached the stage of ultrasound imaging in vivo, none were reported/evaluated to show all three of the following properties desired for clinical applications: (i) low degree of non-specific bubble retention in more than one non-reticuloendothelial tissue; (ii) effective for real-time imaging; and (iii) effective for acoustic quantification of molecular targets to a high degree of quantification. Furthermore, disclosures of the compositions and methodologies enabling reproduction of the bubbles are often withheld. OBJECTIVE: To develop and evaluate a targeting microbubble based on maleimide-thiol conjugation chemistry for ultrasound molecular imaging. METHODS AND RESULTS: Microbubbles with a previously unreported generic (non-targeting components) composition were grafted with anti-E-selectin F(ab')2 using maleimide-thiol conjugation, to produce E-selectin targeting microbubbles. The resulting targeting bubbles showed high specificity to E-selectin in vitro and in vivo. Non-specific bubble retention was minimal in at least three non-reticuloendothelial tissues with inflammation (mouse heart, kidneys, cremaster). The bubbles were effective for real-time ultrasound imaging of E-selectin expression in the inflamed mouse heart and kidneys, using a clinical ultrasound scanner. The acoustic signal intensity of the targeted bubbles retained in the heart correlated strongly with the level of E-selectin expression (|r|≥0.8), demonstrating a high degree of non-invasive molecular quantification. CONCLUSIONS: Targeting microbubbles for ultrasound molecular imaging, based on maleimide-thiol conjugation chemistry and the generic composition described, may possess properties (i)-(iii) desired for clinical applications.

Quantitative ultrasound molecular imaging.

Ultrasound molecular imaging using targeting microbubbles is predominantly a semi-quantitative tool, thus limiting its potential diagnostic power and clinical applications. In the work described here, we developed a novel method for acoustic quantification of molecular expression. E-Selectin expression in the mouse heart was induced by lipopolysaccharide. Real-time ultrasound imaging of E-selectin expression in the heart was performed using E-selectin-targeting microbubbles and a clinical ultrasound scanner in contrast pulse sequencing mode at 14 MHz, with a mechanical index of 0.22-0.26. The level of E-selectin expression was quantified using a novel time-signal intensity curve analytical method based on bubble elimination, which consisted of curve-fitting the bi-exponential equation [Formula: see text] to the elimination phase of the myocardial time-signal intensity curve. Ar and Af represent the maximum signal intensities of the retained and freely circulating bubbles in the myocardium, respectively; λr and λf represent the elimination rate constants of the retained and freely circulating bubbles in the myocardium, respectively. Ar correlated strongly with the level of E-selectin expression (|r|>0.8), determined using reverse transcriptase real-time quantitative polymerase chain reaction, and the duration of post-lipopolysaccharide treatment-both linearly related to cell surface E-selectin protein (actual bubble target) concentration in the expression range imaged. Compared with a conventional acoustic quantification method (which used retained bubble signal intensity at 20 min post-bubble injection), this new approach exhibited greater dynamic range and sensitivity and was able to simultaneously quantify other useful characteristics (e.g., the microbubble half-life). In conclusion, quantitative determination of the level of molecular expression is feasible acoustically using a time-signal intensity curve analytical method based on bubble elimination.

Dynamics of targeted microbubble adhesion under pulsatile compared with steady flow.

Hemodynamic flow variations at low fluid shear stress are thought to play a critical role in local atherosclerotic plaque initiation and development and to affect plaque instability. Targeted microbubbles are being developed as intravascular agents for identifying atherosclerotic lesions using ultrasound. How variations in local hydrodynamic flow influence the adhesiveness of targeted microbubbles is not well understood. We postulated that rates of targeted microbubble binding and accumulation differ when subjected to steady flow (SF) as compared with oscillatory or pulsatile flow (PF), because PF imposes non-uniform blood rheology and periodic acceleration and deceleration of blood velocity, when compared with SF. We assessed the binding rates of targeted microbubbles in seven randomly assigned PF and seven matched SF replicate runs at low (<1 Pa) and intermediate (≥1 and <2.5 Pa) wall shear stress (WSS) by drawing 4.8 × 10(6) microbubbles mL(-1) over streptavidin-coated substrates, immobilized within a parallel plate flow chamber at a calculated density of 81 binding sites µm(-2). Selective binding and accumulation of targeted microbubbles was recorded in a single field of view using real-time video microscopy. Microbubble accumulation was modeled to obtain flow-mediated microbubble binding kinetics (amplitude, A, and rate constant, k). PF elicited higher microbubble accumulation rates, in comparison to SF. The rates of microbubble accumulation differed significantly between PF and SF (p < 0.05) at intermediate WSS but not at low WSS (p > 0.05). The rate of microbubble accumulation decreased as WSS increased.

Single bubble acoustic characterization and stability measurement of adherent microbubbles.

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

Effect of ultrasound on adherent microbubble contrast agents.

An investigation into the effect of clinical ultrasound exposure on adherent microbubbles is described. A flow phantom was constructed in which targeted microbubbles were attached using biotin-streptavidin linkages. Microbubbles were insonated by broadband imaging pulses (centred at 2.25 MHz) over a range of pressures (peak negative pressure (PNP) = 60-375 kPa). Individual adherent bubbles were observed optically and classified as either being isolated or with a single neighbouring bubble. It is found that bubble detachment and deflation are two significant effects, even during low amplitude ultrasound exposure. Specifically, while at very low acoustic pressure (PNP < 75 kPa) 95% of the bubbles were not affected, at medium pressure (151 kPa < P < 225 kPa) 53% of the bubbles detached and at higher pressures (301 kPa < P < 375 kPa) 96% of the bubbles detached. In addition, more than 50% of the bubbles underwent deflation at pressures between 301 and 375 kPa. At pressures between 226 and 300 kPa, more adherent bubbles detached when there was a neighbouring bubble, suggesting the role of multiple scattering and secondary Bjerknes force on bubble detachment. The flow shear, primary and secondary Bjerknes forces exerted on each bubble were calculated and compared to the estimated forces acting on the bubble due to oscillations. The oscillation force is shown to be much higher than other forces. The mechanisms of bubble detachment are discussed.

Evaluation of methods for sizing and counting of ultrasound contrast agents.

A precise, accurate and well documented method for the sizing and counting of microbubbles is essential for all aspects of quantitative microbubble-enhanced ultrasound imaging. The efficacy of (a) electro-impedance volumetric zone sensing (ES) also called a Coulter counter/multisizer; (b) optical microscopy (OM); and (c) laser diffraction (LD), for the sizing and counting of microbubbles was assessed. Microspheres with certified mean diameter and number concentration were used to assess sizing and counting reproducibility (precision) and reliability (accuracy) of ES, OM and LD. SonoVue™ was repeatedly (n = 3) sized and counted to validate ES, OM and LD sizing and counting efficacy. Statistical analyses of intra-method variability for the SonoVue™ mean diameter showed that the best microbubble sizing reproducibility was obtained using OM with a mean diameter sizing variability of 1.1%, compared with a variability of 4.3% for ES and 7.1% for LD. The best microbubble counting reproducibility was obtained using ES with a number concentration variability of 8.3%, compared with a variability of 22.4% for OM and 32% for LD. This study showed that no method is fully suited to both sizing and counting of microbubbles.

On sizing and counting of microbubbles using optical microscopy.

Intra- and interobserver (n = 3) variability of sizing and counting microbubbles using optical microscopy (OM) was assessed. The system was calibrated using standardised mono-disperse and poly-disperse microspheres. Results of the calibration show intraobserver variations of number count (C) = 13.0% and arithmetic mean size (MS) = 0.2%, and interobserver variations of C = 18.4% and MS = 0.6%, for the mono-disperse microspheres. For the poly-disperse microspheres, intraobserver variations were: C = 6.9% and MS = 0.8%, and interobserver: C = 10.5% and MS = 0.3%. For SonoVue™ the intraobserver variations were: C = 23.3% and MS = 8.0%, and interobserver C = 6.8% and MS = 3.8%. The results suggest that the higher values of the intraobserver variation for SonoVue™ arise from the natural decay of microbubbles over time. This article presents a detailed protocol and outlines potential pitfalls in our approach. These results are in general agreement with those previously reported and compare well with known size distributions.

Molecular dynamics simulations of liquid condensed to liquid expanded transitions in DPPC monolayers.

We have investigated the phase behavior of DPPC (dipalmitoylphosphatidylcholine) monolayers at the water-air interface using molecular dynamics simulations, where the phospholipids and the water molecules are modeled atomistically. We report pressure-area isotherms in the interval of 273-310 K. Our results show evidence for a liquid condensed (LC) to liquid expanded (LE) phase transition and indicate that ordered condensed phases can nucleate from a starting disordered phase on a time scale of approximately 50 ns. The existence of the phase transition is confirmed with structural analyses of the phospholipid pair correlation functions and of the monolayer thickness. We find that the change in the monolayer thickness associated with the LC-LE transition is largely due to a shortening of the hydrocarbon chains, with little modification in the average tilt angle of the choline head group. This result is compatible with recent sum frequency spectroscopy experiments, which concluded that the transition occurs without major changes in the orientation of the head group with respect to the monolayer plane. The dependence of the simulated pressure-area isotherms on temperature, in particular, the reduction in width of the coexistence plateau with increasing temperature, is consistent with published experimental pressure-area isotherms.

Microbubble stability is a major determinant of the efficiency of ultrasound and microbubble mediated in vivo gene transfer.

In the search for an efficient nonviral gene therapy approach for the treatment of genetic disorders of cardiac and skeletal muscle such as Duchenne muscular dystrophy, ultrasound in combination with contrast enhancing microbubbles has emerged as a promising tool for safe and site-specific enhancement of gene delivery. Indeed, microbubble-enhanced gene transfer (MBGT) has been investigated for a wide variety of target sites using both reporter and therapeutic genes. Although a range of different microbubbles have been used for MBGT studies, comparison of their efficiencies is difficult because microbubble concentration and the ultrasound settings used for the application vary considerably. Only two studies to date have attempted a direct comparison of commercially available microbubbles, and both concluded that not all microbubbles show the same efficiencies with MBGT. Thus far, the reason for this is unclear. Here, the efficiency of three commercially available microbubbles--Optison, SonoVue and Sonazoid--was analyzed to understand the microbubble properties that are important for their function as an effective enhancer for gene transfer in vivo. In this study, plasmid DNA or antisense oligonucleotides were delivered by systemic injection with MBGT, focused on the heart. Gene delivery to the heart with equalized concentrations of the three microbubbles showed that Optison and Sonazoid are more efficient in MBGT compared with SonoVue, which showed the weakest gene transfer to the myocardium. Investigations into the properties of these microbubbles showed that size and shell composition did not directly influence MBGT, whereas the microbubbles with increased stability in an ultrasound field showed better MBGT results than those degrading faster. Moreover, the microbubble concentration used for MBGT was also found to be an important factor influencing the efficiency of MBGT. In conclusion, the stability of a microbubble was shown to be a major influential factor for its performance in MBGT, as is the concentration of the microbubbles used. These findings emphasize the importance of detailed investigations into the properties of microbubbles to allow the production of a microbubble specifically designed for optimum performance with MBGT.

High-speed optical observations and simulation results of SonoVue microbubbles at low-pressure insonation.

Abstract-Modified Rayleigh-Plesset models are commonly used to characterize the acoustic response of microbubbles under ultrasound exposure. In most instances these models have been parameterized through acoustic measurements taken from bulk suspensions of microbubbles. The aim of this study was to parameterize the Hoff model for the commercial contrast agent SonoVue using optically observed oscillations from individual microbubbles recorded with a high-speed camera. The shell elasticity model term was tuned to fit simulation data to the measured oscillations while the shell viscosity parameter was held constant at 1 Pa??s. The results demonstrate a limited ability of the model to predict the microbubble behavior. The shell elasticity parameter was found to vary proportionally between 10 and 80 MPa with the initial microbubble diameter, implying the viscoelastic shell terms are not a constant property of the shell material. Further analysis using a moving window optimization to probe the microbubble responses suggests that the elasticity of the shell can increase by up to 50% over the course of insonation, particularly for microbubbles oscillating nearer to their resonant frequency. Microbubble oscillations were modeled more successfully by incorporating a varying elasticity term into the model.

Membrane-protein crystallization in cubo: temperature-dependent phase behaviour of monoolein-detergent mixtures.

The lipidic cubic phase of monoolein has proved to be a matrix well suited to the production of three-dimensional crystals of membrane proteins. It consists of a single continuous bilayer, which is contorted in three-dimensional space and separates two distinct water channels. It has previously been proposed that on the addition of precipitants, membrane proteins embedded in the cubic phase migrate through the matrix to nucleation sites and that this process is dependent upon the stability of the lipidic cubic phase. Here, the effect of detergent type (C(8)-C(12) glucosides, C(8)-C(12) maltosides and C(7) thioglucoside) and concentration (1-3x the critical micelle concentration; CMC) on cubic phase stability are reported in the form of the temperature-dependent phase behaviour (268-313 K) in 40% aqueous solution. The results are tabulated to show the best monoolein (MO)-detergent mixtures, mixing temperatures and crystallization temperatures identified. Monoolein-detergent mixtures suited for low-temperature in cubo crystallization of temperature-sensitive proteins are also reported for the first time. These mixtures can be prepared at low temperatures (mixed at