Microbubble Populations Research Articles

The effect of surfactant shedding and gas diffusion on pressure wave propagation through an ultrasound contrast agent suspension

Interest in microbubbles as agents for therapeutic and quantitative imaging applications in biomedical ultrasound has increased the need for their accurate modeling. However, effects such as gas diffusion, the properties of the shell, and changes in bubble behavior under repeated exposure to ultrasound pulses are still not well understood. A revised equation for microbubble motion is proposed that includes the effects of gas diffusion as well as a nonlinear surface tension, which depends on a non-constant surfactant surface concentration. This is incorporated into a nonlinear wave propagation model to account for these additional time-dependent effects in the response of microbubble contrast agent populations. The results from the model indicate significant changes in both bubble behavior and the propagated pulse compared with those predicted by existing propagation models; and show better agreement with experimental data. Our analysis indicates that changes in bubble dynamics are dominated both by surfactant shedding on ultrasonic timescales and gas diffusion over longer timescales between pulses. Therefore, incorporating such time-dependent phenomena in ultrasound imaging algorithms should lead to better quantitative agreement with experiments.

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

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

Size distribution of microbubbles as a function of shell composition

The effect of modifying the shell composition of a population of microbubbles on their size demonstrated through experiment. Specifically, these variations include altering both the mole fraction and molecular weight of functionalized polymer, polyethylene glycol (PEG) in the microbubble phospholipid monolayer shell (1–15mol% PEG, and 1000–5000g/mole, respectively). The size distribution is measured with an unbiased image segmentation program written in MATLAB which identifies and sizes bubbles from micrographs. For a population of microbubbles with a shell composition of 5mol% PEG2000, the mean diameter is 1.42μm with a variance of 0.244μm. For the remainder of the shell compositions studied herein, we find that the size distributions do not show a statistically significant correlation to either PEG molecular weight or mole fraction. All the measured distributions are nearly Gaussian in shape and have a monomodal peak.

Quantitation of MRI sensitivity to Quasi-monodisperse microbubble contrast agents for spatially resolved manometry

The direct in-vivo measurement of fluid pressure cannot be achieved with MRI unless it is done with the contribution of a contrast agent. No such contrast agents are currently available commercially, whilst those demonstrated previously only produced qualitative results due to their broad size distribution. Our aim is to quantitate then model the MR sensitivity to the presence of quasi-monodisperse microbubble populations. Lipid stabilised microbubble populations with mean radius 1.2 ± 0.8 μm have been produced by mechanical agitation. Contrast agents with increasing volume fraction of bubbles up to 4% were formed and the contribution the bubbles bring to the relaxation rate was quantitated. A periodic pressure change was also continuously applied to the same contrast agent, until MR signal changes were only due to bubble radius change and not due to a change in bubble density. The MR data compared favourably with the prediction of an improved numerical simulation. An excellent MR sensitivity of 23 % bar(-1) has been demonstrated. This work opens up the possibility of generating microbubble preparations tailored to specific applications with optimised MR sensitivity, in particular MRI based in-vivo manometry.

Scaled-up production of monodisperse, dual layer microbubbles using multi-array microfluidic module for medical imaging and drug delivery

The production of uniform-sized and multilayer microbubbles enables promising medical applications that combine ultrasound contrast and targeted delivery of therapeutics, with improvements in the consistency of acoustic response and drug loading relative to non-uniform populations of microbubbles. Microfluidics has shown utility in the generation of such small multi-phase systems, however low production rates from individual devices limit the potential for clinical translation. We present scaled-up production of monodisperse dual-layered microbubbles in a novel multi-array microfluidic module containing four or eight hydrodynamic flow-focusing orifices. Production reached 1.34 × 10(5) Hz in the 8-channel configuration, and microbubble diameters in the high-speed regime (> 5 × 10(4) Hz) ranged between 18.6-22.3 μm with a mean pooled polydispersity index under 9 percent. Results demonstrate that microfluidic scale-up for high-output production of multilayer bubbles is possible while maintaining consistency in size production, suggesting that this method may be appropriate for future clinical applications.

Simulation of noninvasive blood pressure estimation using ultrasound contrast agent microbubbles

The microbubble ultrasound contrast agent (UCA) has been widely recognized as a potential noninvasive tool for blood pressure measurement. However, UCA indices such as the shift in the resonance frequency and echo amplitude have problems of low resolution, nonlinear relationship with blood pressure, etc. In this paper, a novel UCA index, the shift in the subharmonic optimal driving frequency (SSODF) of microbubbles, is proposed. The effectiveness of the index for estimating blood pressure was evaluated by performing a microbubble acoustic response simulation. The behavior of commercial UCA microbubbles was investigated as a function of the driving acoustic pressure (in kilopascals) and ambient overpressure (in millimeters of mercury). Simulation results showed that for a 1.6-μm-diameter microbubble, SSODF increased linearly with the overpressure in a range of 0 to 200 mmHg and was maximum (2.07 MHz) at 380 kPa. Changes of the overpressure as small as 5 mmHg can be detected using SSODF. For a population of microbubbles with a Gaussian size distribution (mean diameter: 1.6 μm, standard deviation: 0.2 μm), SSODF was 1.7 MHz at 280 kPa. With further experimental validation, the proposed method may be developed as a novel noninvasive technique for accurate blood pressure measurement.

Fluorous materials in microbubble engineering science and technology—Design and development of new bubble preparation and sizing technologies

Microbubbles are being used in ultrasound diagnosis imaging, could serve as contrast agents for multiple imaging modalities and for parenteral oxygen delivery, and have potential in materials science as new multi-scale materials for magnetic, optical and magneto-optical devices. We designed new procedures for the preparation of narrowly size-distributed, monomodal microbubble populations, as well as a new multifrequency acoustical device for microbubble sizing. Methods for microbubble stabilization using fluorinated gases and surfactants are also discussed. Prospects include further use of fluorinated amphiphiles for bubble stabilization and size control, and decoration of bubble surface with functional particles (e.g. magnetic iron oxide nanoparticles).

Premature Cardiac Contractions Produced Efficiently By External High-Intensity Focused Ultrasound

Exposure of myocardium to a mechanical impact may produce premature ventricular contractions (PVCs). High-intensity focused ultrasound was reported to generate PVCs, while microbubbles at the target increased absorption, thus, promoting energy localization and decreased PVC threshold. The objective was to investigate the benefit of a two-stage ultrasonic transmission: (1) asymmetric mostly negative waveform at the focus (microbubbles generation) and (2) asymmetric mostly positive waveform at the microbubbles (impact generation). Optimization of transmission parameters was performed by measuring passive cavitation and attenuation. In vivo intact rat studies were performed while measuring electrocardiograph (ECG) and blood pressure. Most PVCs with blood injection were created while applying 3.06 MPa peak negative pressure during 1 ms, followed by 5.1 MPa peak positive pressure during 50 ms. Increasing the second stage from 5 ms to 50 ms increased the occurrence of PVCs. This study demonstrates that creation of localized microbubble population at the target promotes generation of PVCs without the need to inject contrast agents.

Strasbourg's SOFFT team—Soft functional systems self-assembled from perfluoroalkylated molecular components

The SOFFT (Systèmes Organisés Fluorés Fonctionnels à Finalités Thérapeutiques) team is engaged in the design, building, characterization and assessment of highly fluorinated functional self-assembled molecular systems ( F-SAMS, i.e. fluorinated soft matter). Therefore, it synthesizes dedicated fluorinated building blocks and exploits the specific properties of highly fluorinated chains and components (“hyper”hydrophobicity, lipophobicity, volume, rigidity, surface properties, etc.) to drive self-assembly. The systems investigated include interfacial films and membranes, 2D arrays of surface micelles, composite multilayered films, fluorinated vesicles and tubules, diverse types of emulsions, microbubbles and decorated microbubbles, for which preparation and characterization techniques have been developed. Fluorinated components are used to modify and control phospholipid film and membrane behavior, stabilize and control emulsion and microbubble characteristics, generate micro- and nanocompartments, useful as templates for polymerization or for confinement. Key findings include formation on water and solid supports of surface hemimicelles that provide nanometer-size surface patterns; stacking of self-assembled hemimicelles; reversible vertical phase segregation in surface films; exploration of 2D/3D transitions in surface films; obtaining of facetted fluid vesicles; improved emulsion and bubble stabilization and particle control; determination of the mechanism of emulsion stabilization by ( F-alkyl)alkyl diblocks; characterization of a co-surfactant effect for non-polar molecules; obtaining of narrowly sized populations of stable microbubbles; and correlation of bubble size and stability with wall and internal phase components. Targeted applications include lung surfactant replacement preparations; adhesion control of cells capable of insulin production; parenteral oxygen delivery and pulmonary drug delivery; and tools for biomedical research. Multiscale objects are currently being investigated, such as microbubbles decorated with magnetic particles destined for multimodal diagnostic imaging and, in materials science, for their non-linear magnetic, optical and magneto-optic properties.

Effect of bubble shell nonlinearity on ultrasound nonlinear propagation through microbubble populations

Nonlinear propagation of ultrasound through microbubble populations can generate artifacts and reduce contrast to tissue ratio in ultrasound imaging. The existing propagation model, which underestimates harmonic generation by an order of magnitude, was revised by incorporating a nonlinear constitutive equation for the coating into the description of the microbubble dynamics. Significantly better agreement with experiments was obtained, indicating that coating nonlinearity represents an important contribution to nonlinear propagation of ultrasound in microbubble populations. The results were found to be sensitive to the parameters characterizing the coating nonlinearity and thus accurate measurement of these parameters is required for accurate quantitative predictions.

The Acoustic Scatter from Single biSphere Microbubbles

Single microbubble acoustic acquisitions provide information on the behaviour of microbubble populations by enabling the generation of large amounts of data. Acoustic signals from single polylactide-shelled and albumin coated biSphere™ microbubbles have been acquired. The responses observed from a range of incident frequencies and acoustic pressures varied in duration. Partial echoes shorter than the incident pulse duration have been observed for low frequency pulses of sufficient amplitude, suggesting release of gas from bubbles. The results presented suggest that the mechanism of scatter from hard shelled agents may be shell disruption and gas release, or partly from gas leaking from defected shell sites, which has previously not been observed optically. These results can provide the basis for improved imaging through optimization of incident pulse parameters, with potential benefits to both diagnostic and therapeutic techniques. (E-mail: d.h.thomas@ed.ac.uk)

Determination of postexcitation thresholds for single ultrasound contrast agent microbubbles using double passive cavitation detection

This work presents experimental responses of single ultrasound contrast agents to short, large amplitude pulses, characterized using double passive cavitation detection. In this technique, two matched, focused receive transducers were aligned orthogonally to capture the acoustic response of a microbubble from within the overlapping confocal region. The microbubbles were categorized according to a classification scheme based on the presence or absence of postexcitation signals, which are secondary broadband spikes following the principle oscillatory response of the ultrasound contrast agent and are indicative of the transient collapse of the microbubble. Experiments were conducted varying insonifying frequencies (0.9, 2.8, 4.6, and 7.1 MHz) and peak rarefactional pressures (200 kPa to 6.2 MPa) for two types of contrast agents (Definity and Optison). Results were fit using logistic regression analysis to define pressure thresholds where at least 5% and 50% of the microbubble populations collapsed for each frequency. These thresholds were found to occur at lower pressures for Definity than for Optison over the range of frequencies studied; additionally, the thresholds occurred at lower pressures with lower frequencies for both microbubble types in most cases, though this trend did not follow a mechanical index scaling.

Dual Frequency Method for Simultaneous Translation and Real-Time Imaging of Ultrasound Contrast Agents Within Large Blood Vessels

A dual frequency excitation method for simultaneous translation and selective real-time imaging of microbubbles is presented. The method can distinguish signals originating from free flowing and static microbubbles. This method is implemented on a programmable scanner with a broadband linear array. The programmable interface allows for dynamic variations in the acoustic parameters and aperture attributes, enabling application of this method to large blood vessels located at varying depths. The performance of the method was evaluated in vitro (vessel diameter 2mm) by quantifying the sensitivity of the method to various acoustic, microbubble, and fluid flow parameters. It was observed that the static microbubble response maximized at the approximate resonance frequency of the microbubble population (estimated from a coulter counter measurement), thus, signifying the need for dual frequency excitation. The static microbubble signal declined from 25 to 12 dB with increasing centerline flow velocities (2.65–15.9cm/s); indicating the applicable range of flow velocities. The maximum intensity of the static microbubbles signal scaled with variations in the microbubble concentration. The rate of increment of static microbubble signal was independent of microbubble concentration. It was deduced that the rate of increment of the static microbubble signal is primarily a function of the pulse frequency, whereas the maximum static microbubble signal intensity is dependent on three parameters: (a) the pulse frequency, (b) the flow velocity and (c) the microbubble concentration. The proposed dual frequency sequence may enable the application of radiation force for optimizing the effect of targeted imaging and modulating drug delivery in large blood vessels with high flow velocities. (E-mail: avp2b@virginia.edu)

Microfoam formation in a capillary

The ultrasound-induced formation of bubble clusters may be of interest as a therapeutic means. If the clusters behave as one entity, i.e., one mega-bubble, its ultrasonic manipulation towards a boundary is straightforward and quick. If the clusters can be forced to accumulate to a microfoam, entire vessels might be blocked on purpose using an ultrasound contrast agent and a sound source. In this paper, we analyse how ultrasound contrast agent clusters are formed in a capillary and what happens to the clusters if sonication is continued, using continuous driving frequencies in the range 1–10 MHz. Furthermore, we show high-speed camera footage of microbubble clustering phenomena. We observed the following stages of microfoam formation within a dense population of microbubbles before ultrasound arrival. After the sonication started, contrast microbubbles collided, forming small clusters, owing to secondary radiation forces. These clusters coalesced within the space of a quarter of the ultrasonic wavelength, owing to primary radiation forces. The resulting microfoams translated in the direction of the ultrasound field, hitting the capillary wall, also owing to primary radiation forces. We have demonstrated that as soon as the bubble clusters are formed and as long as they are in the sound field, they behave as one entity. At our acoustic settings, it takes seconds to force the bubble clusters to positions approximately a quarter wavelength apart. It also just takes seconds to drive the clusters towards the capillary wall. Subjecting an ultrasound contrast agent of given concentration to a continuous low-amplitude signal makes it cluster to a microfoam of known position and known size, allowing for sonic manipulation.

Phospholipid-Coated Gas Bubble Engineering: Key Parameters for Size and Stability Control, as Determined by an Acoustical Method

We have recently reported the sampling of differently sized monomodal populations of microbubbles from a polydisperse lipid-coated bubble preparation. The microbubbles were coated with dimyristoylphosphatidylcholine (DMPC) and stabilized by perfluorohexane (PFH). Such microbubbles are useful as contrast agents and, potentially, for oxygen, drug, and gene delivery and as therapeutic devices. Monomodal populations of small bubbles (approximately 1.6 microm in radius) and large bubbles (approximately 5.4 microm) have been obtained, as assessed by acoustical measurement, static light scattering, and optical microscopy. In this paper, we have determined the influence of various preparation parameters on the initial size characteristics (mean radius and radii distribution) of the microbubbles and on their stability upon time. The bubble size was determined acoustically, with a homemade acoustic setup equipped with a low-power emitter, to avoid altering the bubble stability. We have focused on the effects of the bubble flotation time during the fractionation process and on the DMPC concentration. PFH was indispensable for obtaining stable bubbles. The nature of the buffer [Isoton II vs N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)] used as the continuous phase did not significantly impact the bubble characteristics and stability. In both buffers, the half-lives of small bubbles (approximately 1.6 microm in radius in Isoton II and approximately 2.1 microm in HEPES) were found to be longer than those of larger ones (approximately 5.4 and approximately 5.9 microm in Isoton II and HEPES, respectively). The bubble stability study revealed that in both buffers, the average radius of the population of large bubbles progressively increased with time. On the other hand, the average radius of the population of small bubbles decreased slightly in Isoton II and remained constant in HEPES. This suggests that the dissolution behavior of small and large bubbles is governed by different mechanisms.

Changes in Lipid-Encapsulated Microbubble Population During Continuous Infusion and Methods to Maintain Consistency

Stabilized microbubbles are used as ultrasound contrast agents. These micron-sized gas capsules are injected into the bloodstream to provide contrast enhancement during ultrasound imaging. Some contrast imaging strategies, such as destruction-reperfusion, require a continuous injection of microbubbles over several minutes. Most quantitative imaging strategies rely on the ability to administer a consistent dose of contrast agent. Because of the buoyancy of these gas-filled agents, their spatial distribution within a syringe changes over time. The population of microbubbles that is pumped from a horizontal syringe outlet differs from initial population as the microbubbles float to the syringe top. In this manuscript, we study the changes in the population of a contrast agent that is pumped from a syringe caused by microbubble floatation. Results are presented in terms of change in concentration and change in mean diameter, as a function of time, suspension medium and syringe diameter. Data illustrate that the distribution of contrast agents injected from a syringe changes in both concentration and mean diameter over several minutes without mixing. We discuss the application of a mixing system and viscosity agents to keep the contrast solution more evenly distributed in a syringe. These results are significant for researchers using microbubble contrast agents in continuous-infusion applications where it is important to maintain consistent contrast agent delivery rate, or in situations where the injection syringe cannot be mixed immediately before administration. (E-mail: padayton@bme.unc.edu)

Changes in Lipid-Encapsulated Microbubble Population During Continuous Infusion and Methods to Maintain Consistency

Changes in Lipid-Encapsulated Microbubble Population During Continuous Infusion and Methods to Maintain Consistency

Sonoporation by Ultrasound-Activated Microbubble Contrast Agents: Effect of Acoustic Exposure Parameters on Cell Membrane Permeability and Cell Viability

This work investigates the effect of ultrasound exposure parameters on the sonoporation of KHT-C cells in suspension by perflutren microbubbles. Variations in insonating acoustic pressure (0.05 to 3.5 MPa), pulse frequency (0.5 to 5.0 MHz), pulse repetition frequency (10 to 3000 Hz), pulse duration (4 to 32 micros) and insonation time (0.1 to 900 s) were studied. The number of cells permeabilised to a fluorescent tracer molecule (70 kDa FITC-dextran) and the number of viable cells were measured using flow cytometry. The effect of exposure on the microbubble population was measured using a Coulter counter. Cell viability and membrane permeability were found to depend strongly on the acoustic exposure conditions. Cell permeability increased and viability decreased with increasing peak negative pressure, pulse repetition frequency, pulse duration and insonation time and with decreasing pulse centre frequency. The highest therapeutic ratio (defined as the ratio of permeabilised to nonviable cells) achieved was 8.8 with 32 +/- 4% permeabilization and 96 +/- 1% viability at 570 kPa peak negative pressure, 8 micros pulse duration, 3 kHz pulse repetition frequency, 500 kHz centre frequency and 12 s insonation time with microbubbles at 3.3% volume concentration. These settings correspond to an acoustic energy density (E(SPPA)) of 3.12 J/cm(2). Cell permeability and viability did not correlate with bubble disruption. The results indicate that ultrasound exposure parameters can be optimized for therapeutic sonoporation and that bubble disruption is a necessary but insufficient indicator of ultrasound-induced permeabilization.

Microbubble Destruction During Intravenous Administration: A Preliminary Study

The concentration and size distribution of microbubble suspensions are important parameters for both diagnostic and therapeutic applications. The aim of this preliminary study was to investigate the relationship between changes in the microbubble population and the administration process variables, specifically syringe inner diameter, needle inner diameter, volume flow rate and the liquid in which the microbubbles are suspended. It was found that reducing either the syringe or needle inner diameter produced large reductions in microbubble concentration during administration, as much as 99.9% for needle inner diameters <0.24 mm. Increasing the volume flow rate up to 3 mL/min and changing the suspending fluid from distilled water to glycerol, however, were both found to reduce the degree of microbubble destruction. Further work is needed to fully explain these observations. However, investigation of the response of microbubbles to changes in hydrostatic pressure only, indicated that this was unlikely to be the main mechanism of destruction and hence that shear stress was a more important factor. Comparison with findings from another recent study of microbubble stability indicated that microbubble size, concentration and composition were also important parameters and should be taken into account in designing administration procedures for microbubble agents. It was concluded that current procedures should be reviewed, particularly for therapeutic applications, and that the results should also be taken into account when assessing the accuracy of microbubble size distribution measurements obtained using automatic particle sizing equipment in which microbubbles are made to flow under pressure. (E-mail: e_stride@meng.ucl.ac.uk)

Stability and rheological behavior of concentrated monodisperse food emulsifier coated microbubble suspensions

Nearly monodispersed populations of microbubbles were produced using flow focusing with a food grade emulsifier. The microbubbles produced by this technique have diameters in the range of 120–200 μm. The flow focusing device uses metered streams of air and liquid to produce a jet that periodically pinches to make individual microbubbles. The size of the microbubbles can be controlled by changing the relative flow rates of the gas and the liquid. The emulsifier consists of a mixture of monoglycerides, diglycerides and sodium stearoyl lactylate in combination with polyethylene glycol (PEG)-40 stearate. The emulsifier forms a thin shell that stabilizes the microbubbles. The microbubbles are stable over time with their sizes remaining roughly constant over 2 h. Such stability allows suspensions of microbubbles to be formed and their rheological properties tested. The sizes of the microbubbles are also monitored off-line while testing, examining the effect of shearing on the bubble sizes, as well as their stability over time. These results show that the microbubble suspensions are viscoelastic and exhibit power law behavior. The relationship between the air fraction of the suspension and fluid viscosity is determined.