Phospholipid-coated Microbubbles Research Articles

Are monodisperse phospholipid-coated microbubbles “mono-acoustic?”

Phospholipid-coated microbubbles with a uniform acoustic response are a promising avenue for functional ultrasound sensing. A uniform acoustic response requires both a monodisperse size distribution and uniform viscoelastic shell properties. Monodisperse microbubbles can be produced in a microfluidic flow focusing device. Here, we investigate whether such monodisperse microbubbles have uniform viscoelastic shell properties and thereby a uniform “mono-acoustic” response. To this end, we visualized phase separation of the DSPC and DPPE-PEG5000 lipid shell components and measured the resonance curves of nearly 2000 single and freely floating microbubbles using a high-frequency acoustic scattering technique. The results demonstrate inhomogeneous phase-separated shell microdomains across the monodisperse bubble population, which may explain the measured inhomogeneous viscoelastic shell properties. The shell viscosity varied over an order of magnitude and the resonance frequency by a factor of two indicating both a variation in shell elasticity and in initial surface tension despite the relatively narrow size distribution.

Modulating Nonlinear Acoustic Response of Phospholipid-Coated Microbubbles with pH for Ultrasound Imaging.

Activatable microbubble contrast agents for contrast-enhanced ultrasound have a potential role for measuring physiologic and pathologic states in deep tissues, including tumor acidosis. In this study, we describe a novel observation of increased harmonic oscillation of phosphatidylcholine microbubbles (PC-MBs) in response to lower ambient pH using a clinical ultrasound scanner. MB echogenicity and nonlinear echoes were monitored at neutral and acidic pH using B-mode and Cadence contrast pulse sequencing (CPS), a harmonic imaging technique at 7.0 and 1.5 MHz. A 3-fold increase in harmonic signal intensity was observed when the pH of PC-MB suspensions was decreased from 7.4 to 5.5 to mimic normal and pathophysiological levels that can be encountered in vivo. This pH-mediated activation is tunable based on the chemical structure and length of phospholipids composing the MB shell. It is also reliant on the presence of phosphate groups, as the use of lipids without phosphate instead of phospholipids completely abrogated this phenomenon. The increased harmonic signal likely is the result of increased MB oscillation caused by a decrease of the interfacial tension induced at a lower pH, altering the lipid conformation. While relative signal changes are interpreted clinically as mostly related to blood flow, pH effects could be significant contributors, particularly when imaging tumors. While our observation can be used clinically, it requires further research to isolate the effect of pH from other variables. These findings could pave the way toward for the development of new smart ultrasound contrast agents that expand the clinical utility of contrast-enhanced ultrasound.

Effect of Poly(ethylene glycol) Configuration on Microbubble Pharmacokinetics.

Microbubbles (MBs) hold substantial promise for medical imaging and therapy; nonetheless, knowledge gaps persist between composition, structure, and in vivo performance, especially with respect to pharmacokinetics. Of particular interest is the role of the poly(ethylene glycol) (PEG) layer, which is thought to shield the MB against opsonization and rapid clearance but is also known to cause an antibody response upon multiple injections. The goal of this study was, therefore, to elucidate the role of the PEG layer in circulation persistence of MBs in the naïve animal (prior to an adaptive immune response). Here, we directly observe the number and size of individual MBs obtained from blood samples, unifying size and concentration into the microbubble volume dose (MVD) parameter. This approach enables direct evaluation of the pharmacokinetics of intact MBs, comprising both the lipid shell and gaseous core, rather than separately assessing the lipid or gas components. We examined the in vivo circulation persistence of 3 μm diameter phospholipid-coated MBs with three different mPEG2000 content: 2 mol % (mushroom), 5 mol % (intermediate), and 10 mol % (brush). MB size and concentration in the blood were evaluated by a hemocytometer analysis over 30 min following intravenous injections of 20 and 40 μL/kg MVD in Sprague-Dawley rats. Interestingly, pharmacokinetic analysis demonstrated that increasing PEG concentration on the MB surface resulted in faster clearance. This was evidenced by a 1.6-fold reduction in half-life and area under the curve (AUC) (p < 0.05) in the central compartment. Conversely, the AUC in the peripheral compartment increased with PEG density, suggesting enhanced MB trapping by the mononuclear phagocyte system. This was supported by an in vitro assay, which showed a significant rise in complement C3a activation with a higher PEG content. In conclusion, a minimal PEG concentration on the MB shell (mushroom configuration) was found to prolong circulation and mitigate immunogenicity.

Sensitivity improvement of subharmonic-based pressure measurement using phospholipid-coated monodisperse microbubbles

The use of the subharmonic signal from microbubbles exposed to ultrasound is a promising safe and cost-effective approach for the non-invasive measurement of blood pressure. Achieving a high sensitivity of the subharmonic amplitude to the ambient overpressure is crucial for clinical applications. However, currently used microbubbles have a wide size distribution and diverse shell properties. This causes uncertainty in the response of the subharmonic amplitude to changes in ambient pressure, which limits the sensitivity. The aim of this study was to use monodisperse microbubbles to improve the sensitivity of subharmonic-based pressure measurements. With the same shell materials and gas core, we used a flow-focusing microfluidic chip and a mechanical agitation method to fabricate monodisperse (∼2.45-µm mean radius and 4.7 % polydisperse index) and polydisperse microbubbles (∼1.51-µm mean radius and 48.4 % polydisperse index), respectively. We varied the ultrasound parameters (i.e., the frequency, peak negative pressure (PNP) and pulse length), and found that there was an optimal excitation frequency (2.8 MHz) for achieving maximal subharmonic emission for monodisperse microbubbles, but not for polydisperse microbubbles. Three distinct regimes (occurrence, growth, and saturation) were identified in the response of the subharmonic amplitude to increasing PNP for both monodisperse and polydisperse microbubbles. For the polydisperse microbubbles, the subharmonic amplitude decreased either monotonically or non-monotonically with ambient overpressure, depending on the PNP. By contrast, for the monodisperse microbubbles, there was only a monotonic decrease at all PNPs. The maximum sensitivity (1.18 dB/kPa, R2 = 0.97) of the subharmonic amplitude to ambient overpressure for the monodisperse microbubbles was ∼6.5 times higher than that for the polydisperse microbubbles (0.18 dB/kPa, R2 = 0.88). These results show that monodisperse microbubbles can achieve a more consistent response of the subharmonic signal to changes in ambient overpressure and greatly improve the measurement sensitivity.

Dynamics and frequency response analysis of encapsulated microbubble under nonlinear ultrasound

Bubble dynamic behavior and frequency response of encapsulated microbubbles in nonlinear acoustic field is significant in applications such as tumor therapy, thrombolysis, tissue destruction, and ultrasonic lithotripsy. The acoustic cavitation effect includes stable cavitation and transient cavitation. The transformation from stable cavitation to transient cavitation requires a certain threshold, which is also called the transient cavitation threshold. Phospholipid-coated microbubbles are commonly used to enhance acoustic cavitation. However, the acoustic effects of different coating materials are not very clear, especially when considering the nonlinear effects caused by diffraction, scattering, and reflection during ultrasonic propagation. In this paper, the bubble dynamic behaviors and frequency responses of microbubbles under different frequencies, acoustic pressures, and viscoelastic properties of different shell materials are analyzed by coupling the Gilmore-Akulichev-Zener model with the nonlinear model of a lipid envelope and using the KZK equation to simulate the nonlinear acoustic field. At the same time, the influence of the coated material and nonlinear acoustic effects are considered. The bubble dynamic behavior and frequency response under the actually measured sound field are compared with those simulated by the KZK equation. The results show that the nonlinearity will lead the velocity of the microbubble wall to decrease, and when the pressure of ultrasound increases, the main frequency component of the microbubble oscillation increases, making the radial motion of the microbubble more violent. When the frequency changes, the closer the oscillation frequency of the microbubble is to the resonant frequency, the stronger the radial motion of the microbubble is. The coating material can change the harmonic component in the oscillation frequency. When the harmonic is close to the resonance frequency, the radial motion of the microbubble is enhanced. The elasticity of the coated material has almost no effect on the microbubble's frequency response, and the initial viscosity and surface tension of encapsulated microbubble will change the oscillation frequency distribution of encapsulated microbubble. When the initial viscosity of the coated microbubble is smaller, the subharmonic component of the microbubble oscillation increases. When the frequency of the subharmonic is closer to the resonance frequency than the main frequency, the acoustic cavitation effect is significantly enhanced. On the other hand, when the initial surface tension of the encapsulated microbubble increases, the main frequency and subharmonic component of the microbubble oscillation are enhanced, so that the acoustic cavitation effect is also enhanced. Therefore, this study can further elucidate the bubble dynamics of encapsulated microbubbles, stimulated by nonlinear ultrasound, benefiting the frequency response analysis of coated microbubbles under nonlinear acoustic fields.

Protein-Decorated Microbubbles for Ultrasound-Mediated Cell Surface Manipulation.

Delivering cargo to the cell membranes of specific cell types in the body is a major challenge for a range of treatments, including immunotherapy. This study investigates employing protein-decorated microbubbles (MBs) and ultrasound (US) to "tag" cellular membranes of interest with a specific protein. Phospholipid-coated MBs were produced and functionalized with a model protein using a metallochelating complex through an NTA(Ni) and histidine residue interaction. Successful "tagging" of the cellular membrane was observed using microscopy in adherent cells and was promoted by US exposure. Further modification of the MB surface to enable selective binding to target cells was then achieved by functionalizing the MBs with a targeting protein (transferrin) that specifically binds to a receptor on the target cell membrane. Attachment and subsequent transfer of material from MBs functionalized with transferrin to the target cells significantly increased, even in the absence of US. This work demonstrates the potential of these MBs as a platform for the noninvasive delivery of proteins to the surface of specific cell types.

Bubbles as high-frequency rheometers for soft matter characterization

Precise knowledge of bubble dynamics in tissue is essential for optimizing both ultrasound imaging and ultrasound therapy. Great efforts have therefore been made, over the past decade, to model the behavior of (micro)bubbles in viscoelastic materials. This has led to a series of models based on modified Rayleigh–Plesset equations. Last to date, we propose a novel generalized model build on finite strain theory and relaxation functions. At this stage however, the bottle neck remains our limited knowledge of tissue viscoelasticity at high strain rates, which is a necessary input to any theoretical model. Indeed, traditional rheometers cannot reach frequencies above 1kHz, which is 3 orders of magnitude below the desired MHz frequencies. We will show how ultrasound-driven bubbles can be used as microscale rheometers. More specifically, we use the volumetric oscillations of (monodisperse) phospholipid-coated microbubbles embedded in hydrogels to measure the storage and loss moduli in the MHz frequency range. In addition, we can bridge the gap between kHz and MHz using the surface modes of cylindrical microbubbles trapped in micropits. These oscillations can be both modelled and simulated, and provide access to the effective surface tension and viscosity in the range of 50 to 500 kHz.

Ultrasound sensitive O2 microbubbles radiosensitize murine breast cancer but lead to higher metastatic spread.

The inadequate level of oxygenation in tumors has been shown to correlate not only with greater invasiveness of cancer cells, but also with a reduction in their sensitivity to anticancer therapies. Over the years, many attempts have been made to increase the oxygenation level of cancer, but most of them have been ineffective. We investigated the heterogeneous response of tumor tissue to phospholipid-coated oxygen microbubbles (OMB) in murine tumors in vivo using oxygen and hemoglobin saturation mapping and the influence of OMB treatment on microvasculature, perfusion, and radiotherapy effectiveness. Intravenous administration of OMB followed by ultrasound pulse leads to increased oxygenation of a tumor, found mainly in the vicinity of tumor vessels, while intratumoral delivery resulted in areas of increased pO2 more evenly distributed within the tumor. Furthermore, hemoglobin contributes little to the increase in tumor oxygenation caused by oxygen microbubbles. Extensive vasculature disruption was observed in the groups treated with both oxygen/nitrogen microbubbles and ultrasound pulse. This therapy also led to a reduction in the coverage of the vessels by pericytes, while the density of the microvessels was unchanged. Radiotherapy with a single dose of 12Gy reduced tumor growth by 50% in all treated groups. Unfortunately, at the same time, the number of macroscopic metastases in the lungs increased significantly after intravenous administration of oxygen/nitrogen microbubbles and the application of an ultrasound pulse. In conclusion, ultrasound-sensitive oxygen microbubbles are effective in delivering oxygen to tumor tissue, thus increasing the effectiveness of radiotherapy. However, cavitation effects and destruction of the integrity of tumor vessels result in greater spread of cancer cells in the host organism.

Tuning microbubble-mediated endothelial drug delivery outcome with monodisperse microbubbles of different sizes

Microbubble-mediated drug delivery using polydisperse microbubbles has shown differences in drug delivery outcome due to the microbubble’s differences in size and, thus, acoustic response. The aim of this study was to investigate whether monodisperse microbubbles can achieve controllable sonoporation and tunnel formation. Using the Horizon microfluidics platform, monodisperse phospholipid-coated microbubbles were produced with radii of 1.25–3.5 μm. Single microbubble-endothelial cell interactions (n = 82) upon insonification at 2 MHz (200 kPa PNP, 10 cycles) were investigated in vitro using confocal microscopy and ultra-high-speed imaging (10Mfps). No cellular response was observed for the 3.5 μm microbubbles having excursion amplitudes (Rmax-R0) ranging from 0.3 to 0.6 μm. PI uptake and resealing pores were observed more often for the 1.5 μm microbubbles (50%) with excursion amplitudes ranging from 0.5 to 0.9 μm than for the other microbubble sizes. PI uptake and tunnel formation was most observed for the 1.25 μm microbubbles (46.6%; 0.4–0.6 μm excursion amplitudes) and least for the 3 μm microbubbles (4.2%; 1.0 μm excursion amplitude). No other cellular responses were observed for the 3 μm microbubbles. The excursion ratio (Rmax-Rmin/R0) better separated the tunnel formation events (&amp;gt;0.6) from other cellular responses (0.1–0.6). This study shows the importance of monodisperse microbubbles for tuning drug delivery.

Coupling Two Ultra-high-Speed Cameras to Elucidate Ultrasound Contrast-Mediated Imaging and Therapy.

Ultrasound contrast-mediated medical imaging and therapy both rely on the dynamics of micron- and nanometer-sized ultrasound cavitation nuclei, such as phospholipid-coated microbubbles and phase-change droplets. Ultrasound cavitation nuclei respond non-linearly to ultrasound on a nanosecond time scale that necessitates the use of ultra-high-speed imaging to fully visualize these dynamics in detail. In this study, we developed an ultra-high-speed optical imaging system that can record up to 20 million frames per second (Mfps) by coupling two small-sized, commercially available, 10-Mfps cameras. The timing and reliability of the interleaved cameras needed to achieve 20 Mfps was validated using two synchronized light-emitting diode strobe lights. Once verified, ultrasound-activated microbubble responses were recorded and analyzed. A unique characteristic of this coupled system is its ability to be reconfigured to provide orthogonal observations at 10 Mfps. Acoustic droplet vaporization was imaged from two orthogonal views, by which the 3-D dynamics of the phase transition could be visualized. This optical imaging system provides the temporal resolution and experimental flexibility needed to further elucidate the dynamics of ultrasound cavitation nuclei to potentiate the clinical translation of ultrasound-mediated imaging and therapy developments.

Comparison of Acoustofluidic and Static Systems for Ultrasound-Mediated Molecular Delivery to T Lymphocytes.

Continuous-flow acoustofluidic technologies can potentially improve processing of T lymphocytes for cell therapies by addressing the limitations with viral and non-viral delivery methods. The objective of this study was to assess the intracellular delivery efficiency with acoustofluidic treatment compared with that of static ultrasound treatment. Optimization of parameters in acoustofluidic and static configurations was performed by assessing intracellular delivery of a fluorescent compound (calcein) in viable human Jurkat T lymphocytes. Ultrasound pressure and the concentration of cationic phospholipid-coated microbubbles influenced calcein delivery in both systems. In the static system, a treatment time of 45 s increased molecular delivery compared with 0-30 s (p < 0.01). Refined parameters were used to assess molecular delivery of small and large compounds (0.6-kDa calcein and 150-kDa fluorescein isothiocyanate-dextran, respectively) after ultrasound treatment with the acoustofluidic or static systems. Molecular delivery was similar with refined parameters for acoustofluidic treatment and static treatment (p > 0.05), even though acoustofluidic treatment had lower microbubble concentration (24 μg/mL vs. 94 μg/mL) and shorter treatment time (∼2-3 s vs. 45 s). This study indicates that the acoustofluidic system can significantly enhance intracellular molecular delivery, which could potentially enable acoustofluidic cell transfection during continuous flow processing for manufacture of cell therapies or other applications.

Peritoneal Oxygenation as a Novel Technique for Extrapulmonary Ventilation; A Review and Discussion of the Literature.

The COVID-19 crisis has highlighted the difficulties that might occur when attempting to oxygenate patients who have suffered a severe pulmonary insult, including in the development of acute respiratory distress syndrome (ARDS). Traditional mechanical ventilation (MV) is effective; however, in severe cases of hypoxia, the use of rescue therapy, such as extracorporeal membrane oxygenation (ECMO), may be required but is also associated with significant complexity and complications. In this review, we describe peritoneal oxygenation; a method of oxygenation that exploits the peritoneum's gas exchange properties in a fashion that is similar to peritoneal dialysis and has shown considerable promise in animal models. We have conducted a review of the available literature and techniques, including intraperitoneal perfluorocarbons, intraperitoneal jet ventilation, a continuous low-pressure oxygen system (PEROX) and the use of phospholipid-coated oxygen microbubbles (OMBs) through peritoneal microbubble oxygenation (PMO). We conclude that peritoneal oxygenation is a promising technique that warrants further investigation and might be used in clinical settings in the future.

Theranostic Microbubbles with Homogeneous Ligand Distribution for Higher Binding Efficacy.

Phospholipid-coated targeted microbubbles are used for ultrasound molecular imaging and locally enhanced drug delivery, with the binding efficacy being an important trait. The use of organic solvent in microbubble production makes the difference between a heterogeneous or homogeneous ligand distribution. This study demonstrates the effect of ligand distribution on the binding efficacy of phospholipid-coated ανβ3-targeted microbubbles in vitro using a monolayer of human umbilical-vein endothelial cells and in vivo using chicken embryos. Microbubbles with a homogeneous ligand distribution had a higher binding efficacy than those with a heterogeneous ligand distribution both in vitro and in vivo. In vitro, 1.55× more microbubbles with a homogeneous ligand distribution bound under static conditions, while this was 1.49× more under flow with 1.25 dyn/cm2, 1.56× more under flow with 2.22 dyn/cm2, and 1.25× more in vivo. The in vitro dissociation rate of bound microbubbles with homogeneous ligand distribution was lower at low shear stresses (1–5 dyn/cm2). The internalized depth of bound microbubbles was influenced by microbubble size, not by ligand distribution. In conclusion, for optimal binding the use of organic solvent in targeted microbubble production is preferable over directly dispersing phospholipids in aqueous medium.

The Impact of Lipid Handling and Phase Distribution on the Acoustic Behavior of Microbubbles.

Phospholipid-coated microbubbles are ultrasound contrast agents that can be employed for ultrasound molecular imaging and drug delivery. For safe and effective implementation, microbubbles must respond uniformly and predictably to ultrasound. Therefore, we investigated how lipid handling and phase distribution affected the variability in the acoustic behavior of microbubbles. Cholesterol was used to modify the lateral molecular packing of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)-based microbubbles. To assess the effect of lipid handling, microbubbles were produced by a direct method, i.e., lipids directly dispersed in an aqueous medium or indirect method, i.e., lipids first dissolved in an organic solvent. The lipid phase and ligand distribution in the microbubble coating were investigated using confocal microscopy, and the acoustic response was recorded with the Brandaris 128 ultra-high-speed camera. In microbubbles with 12 mol% cholesterol, the lipids were miscible and all in the same phase, which resulted in more buckle formation, lower shell elasticity and higher shell viscosity. Indirect DSPC microbubbles had a more uniform response to ultrasound than direct DSPC and indirect DSPC-cholesterol microbubbles. The difference in lipid handling between direct and indirect DSPC microbubbles significantly affected the acoustic behavior. Indirect DSPC microbubbles are the most promising candidate for ultrasound molecular imaging and drug delivery applications.

Microstreaming inside Model Cells Induced by Ultrasound and Microbubbles.

Studies on the bioeffects produced by ultrasound and microbubbles have focused primarily on transport in bulk tissue, drug uptake by individual cells, and disruption of biological membranes. Relatively little is known about the physical perturbations and fluid dynamics of the intracellular environment during ultrasound exposure. To investigate this, a custom acoustofluidic chamber was designed to expose model cells, in the form of giant unilamellar vesicles, to ultrasound and microbubbles. The motion of fluorescent tracer beads within the lumen of the vesicles was tracked during exposure to laminar flow (∼1 mm s-1), ultrasound (1 MHz, ∼150 kPa, 60 s), and phospholipid-coated microbubbles, alone and in combination. To decouple the effects of fluid flow and ultrasound exposure, the system was also modeled numerically by using boundary-driven streaming field equations. Both the experimental and numerical results indicate that all conditions produced internal streaming within the vesicles. Ultrasound alone produced an average bead velocity of 6.5 ± 1.3 μm/s, which increased to 8.5 ± 3.8 μm/s in the presence of microbubbles compared to 12 ± 0.12 μm/s under laminar flow. Further research on intracellular forces in mammalian cells and the associated biological effects in vitro and in vivo are required to fully determine the implications for safety and/or therapy.

Ultrasound Responsive Noble Gas Microbubbles for Applications in Image-Guided Gas Delivery.

Noble gases, especially xenon (Xe), have been shown to have antiapoptotic effects in treating hypoxia ischemia related injuries. Currently, in vivo gas delivery is systemic and performed through inhalation, leading to reduced efficacy at the injury site. This report provides a first demonstration of the encapsulation of pure Xe, Ar, or He in phospholipid-coated sub-10 µm microbubbles, without the necessity of stabilizing perfluorocarbon additives. Optimization of shell compositions and preparation techniques show that distearoylphosphatidylcholine (DSPC) with DSPE-PEG5000 can produce stable microbubbles upon shaking, while dibehenoylphosphatidylcholine (DBPC) blended with either DSPE-PEG2000 or DSPE-PEG5000 produces a high yield of microbubbles via a sonication/centrifugation method. Xe and Ar concentrations released into the microbubble suspension headspace are measured using GC-MS, while Xe released directly in solution is detected by the fluorescence quenching of a Xe-sensitive cryptophane molecule. Bubble production is found to be amenable to scale-up while maintaining their size distribution and stability. Excellent ultrasound contrast is observed in a phantom for several minutes under physiological conditions, while an intravenous administration of a bolus of pure Xe microbubbles provides significant contrast in a mouse in pre- and post-lung settings (heart and kidney, respectively), paving the way for image-guided, localized gas delivery for theranostic applications.

Ligand Distribution and Lipid Phase Behavior in Phospholipid-Coated Microbubbles and Monolayers.

Phospholipid-coated targeted microbubbles are ultrasound contrast agents that can be used for molecular imaging and enhanced drug delivery. However, a better understanding is needed of their targeting capabilities and how they relate to microstructures in the microbubble coating. Here, we investigated the ligand distribution, lipid phase behavior, and their correlation in targeted microbubbles of clinically relevant sizes, coated with a ternary mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), with PEG40-stearate and DSPE-PEG2000. To investigate the effect of lipid handling prior to microbubble production in DSPC-based microbubbles, the components were either dispersed in aqueous medium (direct method) or first dissolved and mixed in an organic solvent (indirect method). To determine the lipid-phase behavior of all components, experiments were conducted on monolayers at the air/water interface. In comparison to pure DSPC and DPPC, the ternary mixtures had an additional transition plateau around 10–12 mN/m. As confirmed by infrared reflection absorption spectroscopy (IRRAS), this plateau was due to a transition in the conformation of the PEGylated components (mushroom to brush). While the condensed phase domains had a different morphology in the ternary DPPC and DSPC monolayers on the Langmuir trough, the domain morphology was similar in the coating of both ternary DPPC and DSPC microbubbles (1.5–8 μm diameter). The ternary DPPC microbubbles had a homogenous ligand distribution and significantly less liquid condensed (LC) phase area in their coating than the DSPC-based microbubbles. For ternary DSPC microbubbles, the ligand distribution and LC phase area in the coating depended on the lipid handling. The direct method resulted in a heterogeneous ligand distribution, less LC phase area than the indirect method, and the ligand colocalizing with the liquid expanded (LE) phase area. The indirect method resulted in a homogenous ligand distribution with the largest LC phase area. In conclusion, lipid handling prior to microbubble production is of importance for a ternary mixture of DSPC, PEG40-stearate, and DSPE-PEG2000.

Vibration Characteristics and Persistence of Poloxamer- or Phospholipid-Coated Single Microbubbles under Ultrasound Irradiation.

Microbubbles shelled with soft materials are expected to find applications as ultrasound-sensitive drug delivery systems, including through sonoporation. Microbubbles with specific vibrational characteristics and long intravascular persistence are required for clinical uses. To achieve this aim, the kinetics of the microbubble shell components at the gas/liquid interface while being subjected to ultrasound need to be better understood. This paper investigates the vibration characteristics and lifetime of single microbubbles coated with a poloxamer surfactant, Pluronic F-68, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) under ultrasound irradiation. Air- and perfluorohexane (PFH)-filled microbubbles coated with Pluronic F-68 and DMPC at several concentrations (0 to 10-2 mol L-1) were produced. An optical measurement system using a laser Doppler vibrometer and microscope was used to observe the radial vibration mode of single microbubbles. The vibrational displacement amplitude and resonance radius of Pluronic- or DMPC-coated microbubbles were found to depend very little on the concentrations. The resonance radius was around 65 μm at 38.8 kHz under all the experimental conditions investigated. The lifetime of the microbubbles was investigated simultaneously by measuring their temporal change in volume, and it was increased with Pluronic concentration. Remarkably, the oscillation amplitude of the bubble has an effect on the bubble lifetime. In other words, larger oscillation under the resonance condition accelerates the diffusion of the inner gas.

Detection of nucleic acid-loaded microbubbles in mouse hearts during ultrasound-mediated delivery

Heart disease is a leading cause of death worldwide. Recently, there has been a growing interest in nucleic acid-based therapeutics, such as microRNAs (miRs) or microRNA inhibitors (antimiRs), for cardiac repair. Previous studies have shown that regulating microRNA levels in the heart can promote proliferation of cardiac cells, decrease fibrosis, and improve cardiac function. Ultrasound targeted microbubble destruction (UTMD) is in development to focus the delivery of therapeutic nucleic acids to the heart and reduce adverse systemic effects. Microbubbles can be loaded with nucleic acids and exposed to ultrasound in order to induce cavitation and enhance delivery at the target site. In this study phospholipid-coated microbubbles were loaded with therapeutic miR mimics or antimiRs and injected intravenously in mice. Ultrasound pulses (2.5 MHz, 0.9 MPa peak negative pressure) were applied to the heart using a P4-1 array on a Verasonics Vantage ultrasound system. The ultrasound images were analyzed to detect microbubbles in the heart during treatment. Increased mean intensity during infusion was associated with increased delivery of nucleic acids to the heart as assessed with qPCR. The results suggest that quantitative analysis of ultrasound images to detect microbubbles in vivo may aid in monitoring UTMD treatment for improved cardiac health.

Improved coalescence stability of monodisperse phospholipid-coated microbubbles formed by flow-focusing at elevated temperatures.

Monodisperse phospholipid-coated ultrasound contrast agent (UCA) microbubbles can be directly synthesized in a lab-on-a-chip flow-focusing device. However, high total lipid concentrations are required to minimize on-chip bubble coalescence. Here, we characterize the coalescence probability and the long-term size stability of microbubbles formed using DPPC and DSPC based lipid mixtures as a function of temperature. We show that the coalescence probability can be dramatically reduced by increasing the temperature during bubble formation. Moreover, it is shown that the increased coalescence stability can be explained from an exponential increase of the relative viscosity in the thin liquid film between the colliding bubbles. Furthermore, it was found that the relative viscosity of a DPPC lipid mixture is 7.6 times higher than that of a DSPC mixture and that it can be explained solely from the higher DPPC liposome concentration. Regarding long-term bubble stability, the ratio of the initial on-chip bubble size to the final stable bubble size was always found to be 2.2 for DPPC and DSPC coated bubbles with 10 mol% DPPE-PEG5000, independent of the temperature. Moreover, it was demonstrated that the microbubble suspensions formed at elevated temperatures are highly stable over a time window of 2 to 4 days when collected in a vial. All in all, this work shows that, by increasing the temperature during bubble formation from room temperature to 70 °C, the efficiency of the use of phospholipids in microbubble formation by flow-focusing can be increased by 5 times.