A comparative study on detection of polymer-shelled microbubbles by different excitation pulses.

Contrast agents are widely used in ultrasound imaging. Many imaging techniques have been developed to improve the contrast between tissue and the agents, based on the nonlinear response of microbubbles. In this study, heterodyne excitation was introduced and was compared with traditional sinusoidal signal and chirp excitation for visualizing polymer-shelled microbubbles and degassed water in a tissue-mimicking phantom. Pulse inversion technique was implemented under plane wave (PW) and focused imaging mode. Image enhancement was evaluated by contrast-to-tissue ratio (CTR) at different transmitting peak negative pressures (PNPs). Experimental results showed that heterodyne excitation had a better suppression effect on tissue signals in PW imaging. The CTR reached an approximation of 17 dB at a low peak negative pressure, which was much higher compared to other excitations. In focused wave imaging, a saturation threshold of CTR was observed for the sinusoidal wave burst and chirp excitation at high PNPs. Heterodyne excitation showed considerable contrast-to-noise ratio under both imaging modes. The response of a polymer-shelled microbubble under heterodyne excitation was simulated. Simulations suggest that in future work, specific filters are required to extract the nonlinear components, such as at the two-peak frequencies around fundamental frequency, to achieve a better image enhancement effect.

Cellulose Nanofiber-Coated Perfluoropentane Droplets: Fabrication and Biocompatibility Study.

Purpose: To study the effect of cellulose nanofiber (CNF)-shelled perfluoropentane (PFP) droplets on the cell viability of 4T1 breast cancer cells with or without the addition of non-encapsulated paclitaxel. Methods: The CNF-shelled PFP droplets were produced by mixing a CNF suspension and PFP using a homogenizer. The volume size distribution and concentration of CNF-shelled PFP droplets were estimated from images taken with an optical microscope and analyzed using Fiji software and an in-house Matlab script. The thermal stability was qualitatively assessed by comparing the size distribution and concentration of CNF-shelled PFP droplets at room temperature (~22°) and 37°C. The cell viability of 4T1 cells was measured using a 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, a hemolysis assay was performed to assess blood compatibility of CNF-shelled PFP droplets. Results: The droplet diameter and concentration of CNF-shelled PFP droplets decreased after 48 hours at both room temperature and 37°C. In addition, the decrease in concentration was more significant at 37°C, from 3.50 ± 0.64×106 droplets/mL to 1.94 ± 0.10×106 droplets/mL, than at room temperature, from 3.65 ± 0.29×106 droplets/mL to 2.56 ± 0.22×106 droplets/mL. The 4T1 cell viability decreased with increased exposure time and concentration of paclitaxel, but it was not affected by the presence of CNF-shelled PFP droplets. No hemolysis was observed at any concentration of CNF-shelled PFP droplets. Conclusion: CNF-shelled PFP droplets have the potential to be applied as drug carriers in ultrasound-mediated therapy.

Safety of arterial shear wave elastography-ex-vivoassessment of induced strain and strain rates.

Shear wave elastography (SWE) is a promising technique for characterizing carotid plaques and assessing local arterial stiffness. The mechanical stress to which the tissue is subjected during SWE using acoustic radiation force (ARF), leading to strain at a certain strain rate, is still relatively unknown. Because SWE is increasingly used for arterial applications where the mechanical stress could potentially lead to significant consequences, it is important to understand the risks of SWE-induced strain and strain rate. The aim of this study was to investigate the safety of SWE in terms of induced arterial strain and strain rateex-vivoand in a human carotid arteryin-vivo. SWE was performed on six porcine aortae as a model of the human carotid artery using different combinations of ARF push parameters (push voltage: 60/90 V, aperture width: f/1.0/1.5, push length: 100/150/200µs) and distance to push position. The largest induced strain and strain rate were 1.46% and 54 s-1(90 V, f/1.0, 200µs), respectively. Moreover, the SWE-induced strains and strain rates increased with increasing push voltage, aperture, push length, and decreasing distance between the region of interest and the push. In the human carotid artery, the SWE-induced maximum strain was 0.06% and the maximum strain rate was 1.58 s-1, compared with the maximum absolute strain and strain rate of 12.61% and 5.12 s-1, respectively, induced by blood pressure variations in the cardiac cycle. Our results indicate thatex-vivoarterial SWE does not expose the artery to higher strain rate than normal blood pressure variations, and to strain one order of magnitude higher than normal blood pressure variations, at the push settings and distances from the region of interest used in this study.

Fundamentals, biomedical applications and future potential of micro-scale cavitation-a review.

Thanks to the developments in the area of microfluidics, the cavitation-on-a-chip concept enabled researchers to control and closely monitor the cavitation phenomenon in micro-scale. In contrast to conventional scale, where cavitation bubbles are hard to be steered and manipulated, lab-on-a-chip devices provide suitable platforms to conduct smart experiments and design reliable devices to carefully harness the collapse energy of cavitation bubbles in different bio-related and industrial applications. However, bubble behavior deviates to some extent when confined to micro-scale geometries in comparison to macro-scale. Therefore, fundamentals of micro-scale cavitation deserve in-depth investigations. In this review, first we discussed the physics and fundamentals of cavitation induced by tension-based as well as energy deposition-based methods within microfluidic devices and discussed the similarities and differences in micro and macro-scale cavitation. We then covered and discussed recent developments in bio-related applications of micro-scale cavitation chips. Lastly, current challenges and future research directions towards the implementation of micro-scale cavitation phenomenon to emerging applications are presented.

Measuring the Compressibility of Cellulose Nanofiber-Stabilized Microdroplets Using Acoustophoresis.

Droplets with a liquid perfluoropentane core and a cellulose nanofiber shell have the potential to be used as drug carriers in ultrasound-mediated drug delivery. However, it is necessary to understand their mechanical properties to develop ultrasound imaging sequences that enable in vivo imaging of the vaporization process to ensure optimized drug delivery. In this work, the compressibility of droplets stabilized with cellulose nanofibers was estimated using acoustophoresis at three different acoustic pressures. Polyamide particles of known size and material properties were used for calibration. The droplet compressibility was then used to estimate the cellulose nanofiber bulk modulus and compare it to experimentally determined values. The results showed that the acoustic contrast factor for these droplets was negative, as the droplets relocated to pressure antinodes during ultrasonic actuation. The droplet compressibility was 6.6-6.8 ×10-10 Pa-1, which is higher than for water (4.4×10-10 Pa-1) but lower than for pure perfluoropentane (2.7×10-9 Pa-1). The compressibility was constant across different droplet diameters, which was consistent with the idea that the shell thickness depends on the droplet size, rather than being constant.

Deriving acoustic properties for perfluoropentane droplets with viscoelastic cellulose nanofiber shell via numerical simulations.

Perfluoropentane droplets with cellulose nanofibers (CNF) shells have demonstrated better stability and easier surface modification as ultrasound contrast agents and drug delivery vehicles. This paper presents a theoretical model assuming a four-phase state "inverse antibubble," with the core filled with gas perfluoropentane surrounded by liquid perfluoropentane. A continuous, incompressible, and viscoelastic stabilizing layer separates the core from the surrounding water. A parametric study is performed to predict the frequency-dependent attenuation coefficient, the speed of sound, and the resonance frequency of the droplets which have a mean diameter of 2.47 ± 0.95 µm. Results reveal that the CNF-stabilized perfluoropentane droplets can be modeled in a Rayleigh-Plesset like equation. We conclude that the shell strongly influences the acoustic behavior of the droplets and the resonance frequency largely depends on the initial gas cavity radius. More specifically, the peak attenuation coefficient and peak-to-peak speed of sound decrease with increasing shear modulus, shear viscosity, and shell thickness, while they increase with increasing gas cavity radius and concentration. The resonance frequency increases as shear modulus and shell thickness increase, while it decreases as shear viscosity and gas cavity radius increase. It is worth mentioning that droplet concentration has no effect on the resonance frequency.

Design and fabrication of a vigorous "cavitation-on-a-chip" device with a multiple microchannel configuration.

Hydrodynamic cavitation is one of the major phase change phenomena and occurs with a sudden decrease in the local static pressure within a fluid. With the emergence of microelectromechanical systems (MEMS), high-speed microfluidic devices have attracted considerable attention and been implemented in many fields, including cavitation applications. In this study, a new generation of 'cavitation-on-a-chip' devices with eight parallel structured microchannels is proposed. This new device is designed with the motivation of decreasing the upstream pressure (input energy) required for facile hydrodynamic cavitation inception. Water and a poly(vinyl alcohol) (PVA) microbubble (MB) suspension are used as the working fluids. The results show that the cavitation inception upstream pressure can be reduced with the proposed device in comparison with previous studies with a single flow restrictive element. Furthermore, using PVA MBs further results in a reduction in the upstream pressure required for cavitation inception. In this new device, different cavitating flow patterns with various intensities can be observed at a constant cavitation number and fixed upstream pressure within the same device. Moreover, cavitating flows intensify faster in the proposed device for both water and the water-PVA MB suspension in comparison to previous studies. Due to these features, this next-generation 'cavitation-on-a-chip' device has a high potential for implementation in applications involving microfluidic/organ-on-a-chip devices, such as integrated drug release and tissue engineering.

A Study on the Acoustic Response of Pickering Perfluoropentane Droplets in Different Media.

Acoustic droplet vaporization (ADV) is the physical process of liquid-to-gas phase transition mediated by pressure variations in an ultrasound field. In this study, the acoustic response of novel particle-stabilized perfluoropentane droplets was studied in bulk and confined media. The oil/water interface was stabilized by cellulose nanofibers. First, their acoustic responses under idealized conditions were examined to assess their susceptibility to undergo ADV. Second, the droplets were studied in a more realistic setting and placed in a confined medium. Lastly, an imaging setup was developed and tested on the droplets. The acoustic response could be seen when the amplitude of the peak negative pressure (PNP) was above 200 kPa, suggesting that this is the vaporization pressure threshold for these droplets. Increasing the PNP resulted in a decrease in signal intensity over time, suggesting a more destructive behavior. The imaging setup was able to differentiate between the droplets and the surrounding tissue. Results obtained within this study suggest that these droplets have potential in terms of ultrasound-mediated diagnostics and therapy.

On the Development of a Novel Contrast Pulse Sequence for Polymer-Shelled Microbubbles.

Contrast agents are routinely used in ultrasound examinations. Nonlinear ultrasound imaging techniques have been developed over decades to enhance the contrast between the tissue and the blood pool after the injection of ultrasound contrast agents (UCAs). In this study, we introduce a new contrast pulse sequence, CPS4. The CPS4 combines pulse inversion (PI), subharmonic (SH), and ultraharmonic (UH) techniques to remove propagation distortion while capturing the unique SH and UH responses from UCAs. The novel CPS4 and conventional PI, SH, and UH techniques were used to detect the presence of a research-grade, thick-shell, polymer microbubble in a tissue-mimicking flow phantom. The contrast-to-tissue ratios (CTRs) obtained from the applications of all techniques were compared. The results show that the highest CTR of approximately 16 dB was obtained using CPS4, which was superior to the individual reference techniques: PI, SH, and UH techniques, in all scenarios considered in this study.

Unravelling the Acoustic and Thermal Responses of Perfluorocarbon Liquid Droplets Stabilized with Cellulose Nanofibers.

The attractive colloidal and physicochemical properties of cellulose nanofibers (CNFs) at interfaces have recently been exploited in the facile production of a number of environmentally benign materials, e.g. foams, emulsions, and capsules. Herein, these unique properties are exploited in a new type of CNF-stabilized perfluoropentane droplets produced via a straightforward and simple mixing protocol. Droplets with a comparatively narrow size distribution (ca. 1-5 µm in diameter) were fabricated, and their potential in the acoustic droplet vaporization process was evaluated. For this, the particle-stabilized droplets were assessed in three independent experimental examinations, namely temperature, acoustic, and ultrasonic standing wave tests. During the acoustic droplet vaporization (ADV) process, droplets were converted to gas-filled microbubbles, offering enhanced visualization by ultrasound. The acoustic pressure threshold of about 0.62 MPa was identified for the cellulose-stabilized droplets. A phase transition temperature of about 22 °C was observed, at which a significant fraction of larger droplets (above ca. 3 µm in diameter) were converted into bubbles, whereas a large part of the population of smaller droplets were stable up to higher temperatures (temperatures up to 45 °C tested). Moreover, under ultrasound standing wave conditions, droplets were relocated to antinodes demonstrating the behavior associated with the negative contrast particles. The combined results make the CNF-stabilized droplets interesting in cell-droplet interaction experiments and ultrasound imaging.

Review on Acoustic Droplet Vaporization in Ultrasound Diagnostics and Therapeutics.

Acoustic droplet vaporization (ADV) is the physical process in which liquid undergoes phase transition to gas after exposure to a pressure amplitude above a certain threshold. In recent years, new techniques in ultrasound diagnostics and therapeutics have been developed which utilize microformulations with various physical and chemical properties. The purpose of this review is to give the reader a general idea on how ADV can be implemented for the existing biomedical applications of droplet vaporization. In this regard, the recent developments in ultrasound therapy which shed light on the ADV are considered. Modern designs of capsules and nanodroplets (NDs) are shown, and the material choices and their implications for function are discussed. The influence of the physical properties of the induced acoustic field, the surrounding medium, and thermophysical effects on the vaporization are presented. Lastly, current challenges and potential future applications towards the implementation of the therapeutic droplets are discussed.

Modeling and parametric investigation of thick encapsulated microbubble's nonspherical oscillations.

Numerous studies have been carried out in the past few decades to investigate the radial oscillations of encapsulated microbubbles (MBs). Nonspherical oscillations also have gained attention, being unavoidable in actual applications of these bubbles. The present paper is intended to describe the nature of resonance trends of such spherical and nonspherical modes of a thick encapsulated MB filled with air and suspended in water. The shell material is assumed to be linear viscoelastic and quasi-incompressible. The considered isotropic and spherically isotropic material parametric range is limited to thick polymer shelled MBs. For the case of an isotropic material, shell viscosity has a major influence on the fundamental modes with meridional wave number n = 0, 4, especially for thicker bubbles, unlike for the case of the spherically isotropic material case considered, where the viscosity has very little influence. For most of the parametric range, n = 2, 3 modes are underdamped and their frequency is found to be lower than the n = 0, 4 modes, for both material cases. An interesting case is found for a spherically isotropic quasi-incompressible material case, where the first few nonspherical mode resonances are very close to radial mode resonance frequency.

Graphene Meets Microbubbles: A Superior Contrast Agent for Photoacoustic Imaging.

Coupling graphene with a soft polymer surface offers the possibility to build hybrid constructs with new electrical, optical, and mechanical properties. However, the low reactivity of graphene is a hurdle in the synthesis of such systems which is often bypassed by oxidizing its carbon planar structure. However, the defects introduced with this process jeopardize the properties of graphene. In this paper we present a different approach, applicable to many different polymer surfaces, which uses surfactant assisted ultrasonication to exfoliate, and simultaneously suspend, graphene in water in its intact form. Tethering pristine graphene sheets to the surfaces is accomplished by using suitable reactive functional groups of the surfactant scaffold. We focused on applying this approach to the fabrication of a hybrid system, made of pristine graphene tethered to poly(vinyl alcohol) based microbubbles (PVA MBs), designed for enhancing photoacoustic signals. Photoacoustic imaging (PAI) is a powerful preclinical diagnostic tool which provides real time images at a resolution of 40 µm. The leap toward clinical imaging has so far been hindered by the limited tissues penetration of near-infrared (NIR) pulsed laser radiation. Many academic and industrial research laboratories have met this challenge by designing devices, each with pros and cons, to enhance the photoacoustic (PA) signal. The major advantages of the hybrid graphene/PVA MBs construct, however, are (i) the preservation of graphene properties, (ii) biocompatibility, a consequence of the robust anchoring of pristine graphene to the bioinert surface of the PVA bubble, and (iii) a very good enhancement in a NIR spectral region of the PA signal, which does not overlap with the signals of PA active endogenous molecules such as hemoglobin.

Investigation of polymer-shelled microbubble motions in acoustophoresis.

The objective of this paper is to explore the trajectory motion of microsize (typically smaller than a red blood cell) encapsulated polymer-shelled gas bubbles propelled by radiation force in an acoustic standing-wave field and to compare the corresponding movements of solid polymer microbeads. The experimental setup consists of a microfluidic chip coupled to a piezoelectric crystal (PZT) with a resonance frequency of about 2.8MHz. The microfluidic channel consists of a rectangular chamber with a width, w, corresponding to one wavelength of the ultrasound standing wave. It creates one full wave ultrasound of a standing-wave pattern with two pressure nodes at w/4 and 3w/4 and three antinodes at 0, w/2, and w. The peak-to-peak amplitude of the electrical potential over the PZT was varied between 1 and 10V. The study is limited to no-flow condition. From Gor'kov's potential equation, the acoustic contrast factor, Φ, for the polymer-shelled microbubbles was calculated to about -60.7. Experimental results demonstrate that the polymer-shelled microbubbles are translated and accumulated at the pressure antinode planes. This trajectory motion of polymer-shelled microbubbles toward the pressure antinode plane is similar to what has been described for other acoustic contrast particles with a negative Φ. First, primary radiation forces dragged the polymer-shelled microbubbles into proximity with each other at the pressure antinode planes. Then, primary and secondary radiation forces caused them to quickly aggregate at different spots along the channel. The relocation time for polymer-shelled microbubbles was 40 times shorter than that for polymer microbeads, and in contrast to polymer microbeads, the polymer-shelled microbubbles were actuated even at driving voltages (proportional to radiation forces) as low as 1V. In short, the polymer-shelled microbubbles demonstrate the behavior attributed to the negative acoustic contrast factor particles and thus can be trapped at the antinode plane and thereby separated from particles having a positive acoustic contrast factor, such as for example solid particles and cells. This phenomenon could be utilized in exploring future applications, such as bioassay, bioaffinity, and cell interaction studies in vitro in a well-controlled environment.

In Search of the Optimal Heart Perfusion Ultrasound Imaging Platform.

OBJECTIVES: Quantification of myocardial perfusion by contrast echocardiography remains a challenge. Existing imaging phantoms used to evaluate the performance of ultrasound scanners do not comply with perfusion basics in the myocardium, where perfusion and motion are inherently coupled. To contribute toward an improvement, we developed a contrast echocardiographic perfusion imaging platform based on an isolated rat heart coupled to an ultrasound scanner. METHODS: Perfusion was assessed by using 3 different types of contrast agents: dextran-based Promiten (Meda AB, Solna, Sweden), phospholipid-shelled SonoVue (Bracco Diagnostics, Inc, Princeton, NJ), and polymer-shelled MB-pH5-RT, developed in-house. The myocardial video intensity was monitored over time from contrast agent administration to peak, and 2 characteristic constants were calculated by using an exponential fit: A, representing capillary volume; and ß, representing inflow velocity. RESULTS: Acquired experimental evidence demonstrates that the application of all 3 contrast agents allows sonographic estimation of myocardial perfusion in the isolated rat heart. Video intensity maps show that an increase in contrast concentration increases the late-plateau values, A, mimicking increased capillary volume. Estimated values of the flow, proportional to A × ß, increase when the pressure of the perfusate column increases from 80 to 110 cm of water. This finding is in agreement with the true values of the coronary flow increase measured by a flowmeter attached to the aortic cannula. CONCLUSIONS: The contrast echocardiographic perfusion imaging platform described holds promise for standardized evaluation and optimization of contrast perfusion ultrasound imaging in which real-time inflow curves at low acoustic power semiquantitatively reflect coronary flow.

Ultrasound contrast agent loaded with nitric oxide as a theranostic microdevice.

The current study describes novel multifunctional polymer-shelled microbubbles (MBs) loaded with nitric oxide (NO) for integrated therapeutic and diagnostic applications (ie, theranostics) of myocardial ischemia. We used gas-filled MBs with an average diameter of 4 µm stabilized by a biocompatible shell of polyvinyl alcohol. In vitro acoustic tests showed sufficient enhancement of the backscattered power (20 dB) acquired from the MBs' suspension. The values of attenuation coefficient (0.8 dB/cm MHz) and phase velocities (1,517 m/s) were comparable with those reported for the soft tissue. Moreover, polymer MBs demonstrate increased stability compared with clinically approved contrast agents with a fracture threshold of about 900 kPa. In vitro chemiluminescence measurements demonstrated that dry powder of NO-loaded MBs releases its gas content in about 2 hours following an exponential decay profile with an exponential time constant equal to 36 minutes. The application of high-power ultrasound pulse (mechanical index =1.2) on the MBs resuspended in saline decreases the exponential time constant from 55 to 4 minutes in air-saturated solution and from 17 to 10 minutes in degassed solution. Thus, ultrasound-triggered release of NO is achieved. Cytotoxicity tests indicate that phagocytosis of the MBs by macrophages starts within 6-8 hours. This is a suitable time for initial diagnostics, treatment, and monitoring of the therapeutic effect using a single injection of the proposed multifunctional MBs.

Unique pumping-out fracturing mechanism of a polymer-shelled contrast agent: an acoustic characterization and optical visualization.

This work describes the fracturing mechanism of air-filled microbubbles (MBs) encapsulated by a cross-linked poly(vinyl alcohol) (PVA) shell. The radial oscillation and fracturing events following the ultrasound exposure were visualized with an ultrahigh-speed camera, and backscattered timedomain signals were acquired with the acoustic setup specific for harmonic detection. No evidence of gas emerging from defects in the shell with the arrival of the first insonation burst was found. In optical recordings, more than one shell defect was noted, and the gas core was drained without any sign of air extrusion when several consecutive bursts of 1 MPa amplitude were applied. In acoustic tests, the backscattered peak-to-peak voltage gradually reached its maximum and exponentially decreased when the PVA-based MB suspension was exposed to approximately 20 consecutive bursts arriving at pulse repetition frequencies of 100 and 500 Hz. Taking into account that the PVA shell is porous and possibly contains large air pockets between the cross-linked PVA chains, the aforementioned acoustic behavior might be attributed to pumping gas from these pockets in combination with gas release from the core through shell defects. We refer to this fracturing mechanism as pumping-out behavior, and this behavior could have potential use for the local delivery of therapeutic gases, such as nitric oxide.

Assessment of the viscoelastic and oscillation properties of a nano-engineered multimodality contrast agent.

Combinations of microbubbles (MBs) and superparamagnetic iron oxide nanoparticles (SPIONs) are used to fabricate dual contrast agents for ultrasound and MRI. This study examines the viscoelastic and oscillation characteristics of two MB types that are manufactured with SPIONs and either anchored chemically on the surface (MBs-chem) or physically embedded (MBs-phys) into a polymer shell. A linearized Church model was employed to simultaneously fit attenuation coefficients and phase velocity spectra that were acquired experimentally. The model predicted lower viscoelastic modulus values, undamped resonance frequencies and total damping ratios for MBs-chem. MBs-chem had a resonance frequency of approximately 13 MHz and a damping ratio of approximately 0.9; thus, MBs-chem can potentially be used as a conventional ultrasound contrast agent with the combined functionality of MRI detection. In contrast, MBs-phys had a resonance frequency and damping of 28 MHz and 1.2, respectively, and requires further modification of clinically available contrast pulse sequences to be visualized.

On the interplay of shell structure with low- and high-frequency mechanics of multifunctional magnetic microbubbles.

Polymer-shelled magnetic microbubbles have great potential as hybrid contrast agents for ultrasound and magnetic resonance imaging. In this work, we studied US/MRI contrast agents based on air-filled poly(vinyl alcohol)-shelled microbubbles combined with superparamagnetic iron oxide nanoparticles (SPIONs). The SPIONs are integrated either physically or chemically into the polymeric shell of the microbubbles (MBs). As a result, two different designs of a hybrid contrast agent are obtained. With the physical approach, SPIONs are embedded inside the polymeric shell and with the chemical approach SPIONs are covalently linked to the shell surface. The structural design of hybrid probes is important, because it strongly determines the contrast agent's response in the considered imaging methods. In particular, we were interested how structural differences affect the shell's mechanical properties, which play a key role for the MBs' US imaging performance. Therefore, we thoroughly characterized the MBs' geometric features and investigated low-frequency mechanics by using atomic force microscopy (AFM) and high-frequency mechanics by using acoustic tests. Thus, we were able to quantify the impact of the used SPIONs integration method on the shell's elastic modulus, shear modulus and shear viscosity. In summary, the suggested approach contributes to an improved understanding of structure-property relations in US-active hybrid contrast agents and thus provides the basis for their sustainable development and optimization.