Lipid-coated Microbubbles Research Articles

Behavior of Microbubbles on Air-Aqueous Interfaces.

Animal-derived lung surfactants have saved millions of lives of preterm neonates with neonatal Respiratory Distress Syndrome (nRDS). However, a replacement for animal-derived lung surfactants has been sought for decades due to its high manufacturing cost, inaccessibility in low-income countries, and failure to show efficacy when nebulized. This study investigated the use of lipid-coated microbubbles as potential replacements for exogenous lung surfactants. Three different formulations of microbubbles (DPPC with/out PEG40-stearate and poractant alfa) were prepared, and their equilibrium and dynamic surface tensions were tested on a clean air-saline interface or a simulated air-lung fluid interface using a Langmuir-Blodgett trough. In dynamic surface measurements, microbubbles reduced the minimum surface tension compared with the equivalent composition lipid suspension: e.g., PEG-free microbubbles had a minimum surface tension of 4.3 mN/m while the corresponding lipid suspension and poractant alfa had 20.4 (p ≤ 0.0001) and 21.8 mN/m (p ≤ 0.0001), respectively. Two potential mechanisms for the reduction of surface tension were found: Fragmentation of the foams created by microbubble coalescence; and clustering of microbubbles in the aqueous subphase disrupting the interfacial phospholipid monolayer. The predominant mechanism appears to depend on the formulation and/or the environment. The use of microbubbles as a replacement for exogenous lung surfactant products thus shows promise and further work is needed to evaluate efficacy in vivo.

Deciphering the hydrodynamics of lipid-coated microbubble sonoluminescence for sonodynamic therapy

Sonodynamic therapy (SDT) is a minimally invasive targeted cancer therapy that uses focused low-intensity ultrasound (<10 MPa, <10 W/cm2) to activate sonosensitizer drugs. Once activated, these chemical compounds generate reactive oxygen species (ROS) to damage and kill cancer cells. A Phase I clinical trial has shown promising results for treating glioblastoma with SDT. We hypothesize that the efficacy of SDT can be improved by introducing lipid-coated microbubbles that produce a sonochemical effect that enhances ROS production. We investigate the hydrodynamics of a U.S. Food and Drug Administration (FDA)-approved microbubble, Lumason®, and a phospholipid-coated oxygen microbubble to predict the ultrasound parameters that induce sonoluminescence onset in biophysically relevant medium (e.g., water and blood) under clinical SDT conditions. The threshold pressures and frequencies for sonoluminescence with these therapeutic agents lie between 20 kHz – 1 MHz and 0.05 MPa – 1 MPa, respectively. The lipid-coated oxygen microbubble exhibits stronger sonoluminescence than the Lumason® microbubble, suggesting its use for improving SDT efficacy.

Influence of the liquid ionic strength on the resonance frequency and estimated shell parameters of lipid-coated microbubbles

Measurement of the resonance frequency and shell properties of coated microbubbles (MBs) is important in understanding and optimizing their response to ultrasound. In many applications MBs are in ionic liquids; however, the influence of the medium charges on the MB behavior is not well investigated. This study aims to measure the medium charge interactions with MBs by measuring the frequency-dependent attenuation of the same size MBs in mediums of varying charge density. In-house lipid-coated MBs were size isolated to a mean size of 2.35 μm using differential centrifugation. MBs were suspended in distilled water (DW), Phosphate-Buffered Saline solution (PBS1×) and PBS10×. The frequency-dependent attenuation of the MBs solutions was measured using a system of two aligned 100% bandwidth transducers with 10 MHz center frequency. The MB shell properties were estimated by fitting the linear equation to experiments. With increasing salinity, the frequency of the peak attenuation decreased (13, 7.5 and 6.25 MHz in DW, PBS1× and PBS-10×, respectively) and attenuation peak increased (∼140%). Estimated MBs shell elasticity decreased by 64% between DW and PBS-1× and 36% between PBS-1x and PBS-10x. The estimated shell viscosity reduced by ∼40% between DW and PBS-1× and 42% between PBS-1× and PBS-10×. The significant reduction in the fitted stiffness and viscosity is possibly due to the formation of a densely charged layer around the shell and requires proper inclusion in the related MB models.

Controlling the stability of monodisperse lipid-coated microbubbles by tuning their buckling pressure

Through recent years, major hurdles associated with the microfluidic production of phospholipid-coated monodisperse microbubble have been overcome which has granted them a long shelf life. This has heaved the hope of using their unique properties in vivo for improved contrast, controlled therapy, or noninvasive pressure measurement. However, these bubbles are also very sensitive to small changes in ambient pressure which compromises their clinical translation: upon intravenous injection, physiological pressures will cause bubble dissolution, degrading their uniformity. Here, we demonstrate the direct relation between shell buckling and bubble dissolution by acoustically measuring the buckling pressure and the response of bubbles to controlled pressure changes. We show that the concentration of PEGylated lipid, necessary for microfluidic operation, can be used to tune the buckling pressure, and thereby increase bubble stability. We show that this concentration can be changed either directly during bubble production or, more conveniently, by heating the microbubbles after production. The proposed heating step exploits the phase-change of the phospholipids within the shell to selectively expel the PEGylated lipids. Doing so, the bubble buckling pressure can be increased from 0 kPa to 27 kPa which is above physiological pressures.

Microstreaming profile of a phospholipid-coated wall-attached microbubble undergoing shape oscillation

Ultrasound-activated microbubbles induce shape oscillation and microstreaming. However, a thorough understanding remains elusive. This study investigated the 3D microstreaming profile of clinically relevant lipid-coated microbubbles undergoing shape oscillation when bound to a wall. Size-controlled biotinylated microbubbles (radius 3–8 μm) were produced by a flow-focusing device and bound to a streptavidin-coated glass. Insonification spanned 85–425 kPa at 25,000 cycles and 1.25 MHz. Two cameras, operating at 5 Mfps and 10 kfps, coupled to a microscope, captured microbubble shape oscillation and cavitation microstreaming, respectively. Astigmatic particle tracking velocimetry measured 3D particle trajectories of 500-nm beads. In the dataset (n = 79), four microstreaming profiles were observed: quadrupole, dipole, radial, and patternless. Modal decomposition revealed that quadrupole patterns resulted from self-interaction of the predominant shape mode, while dipole patterns arose from strong interactions of two nearby modes at the same oscillation frequency. Microstreaming reached 0.002–0.01 m/s at varying acoustic pressures. Quadrupole microstreaming generally induced higher shear stresses than dipole and radial patterns at identical acoustic pressure. A lower shape mode induced higher shear stresses than a higher mode at the same acoustic pressure. The unique microstreaming characteristics yield diverse outcomes in mechanical impact, which could aid efficient therapeutic applications of ultrasound-activated microbubbles.

Influence of the liquid ionic strength on the resonance frequency and shell parameters of lipid-coated microbubbles

The correct measurement of the resonance frequency and shell properties of coated microbubbles (MBs) is essential in understanding and optimizing their response to ultrasound (US) exposure parameters. In diagnostic and therapeutic ultrasound, MBs are typically surrounded by blood; however, the influence of the medium charges on the MB resonance frequency has not been systematically studied using controlled measurements. This study aims to measure the medium charge interactions on MB behavior by measuring the frequency-dependent attenuation of the same size MBs in mediums with different charge densities. In-house lipid-coated MBs with C3F8 gas core were formulated. The MBs were isolated to a mean size of 2.35 μm using differential centrifugation. MBs were diluted to ≈8×105 MBs/mL in distilled water (DW), Phosphate-Buffered Saline solution (PBS1x) and PBS10x. The frequency-dependent attenuation of the MBs solutions was measured using an aligned pair of PVDF transducers with a center frequency of 10MHz and 100% bandwidth in the linear oscillation regime (7 kPa pressure amplitude). The MB shell properties were estimated by fitting the linear equation to experiments. Using a pendant drop tension meter, the surface tension at the equilibrium of ≈6 mm diameter size drops of the same MB shell was measured inside DW, PBS1x and PBS10x. The surface tension at the C3F8/solution interface was estimated by fitting the Young-Laplace equation from the recorded images. The frequency of the peak attenuation at different salinity levels was 13, 7.5 and 6.25 MHz in DW, PBS1x and PBS-10x, respectively. The attenuation peak increased by ≈140% with increasing ion density. MBs' estimated shell elasticity decreased by 64% between DW and PBS-1x and 36% between PBS-1x and PBS-10x. The drop surface tension reduced by 10.5% between DW and PBS-1x and by 5% between PBS-1x and PBS-10x, respectively. Reduction in the shell stiffness is consistent with the drop surface tension measurements. The shell viscosity was reduced by ≈40% between DW and PBS-1x and 42% between PBS-1x and PBS-10x. The reduction in the fitted stiffness and viscosity is possibly due to the formation of a densely charged layer around the shell, further reducing the effective surface tension on the MBs. The changes in the resonance frequency and estimated shell parameters were significant and may potentially help to better understand and explain bubble behavior in applications.

MRNA Sonotransfection of Tumors with Polymeric Microbubbles: Co-Formulation versus Co-Administration.

Despite its high potential, non-viral gene therapy of cancer remains challenging due to inefficient nucleic acid delivery. Ultrasound (US) with microbubbles (MB) can open biological barriers and thus improve DNA and mRNA passage. Polymeric MB are an interesting alternative to clinically used lipid-coated MB because of their high stability, narrow size distribution, and easy functionalization. However, besides choosing the ideal MB, it remains unclear whether nanocarrier-encapsulated mRNA should be administered separately (co-administration) or conjugated to MB (co-formulation). Therefore, the impact of poly(n-butyl cyanoacrylate) MB co-administration with mRNA-DOTAP/DOPE lipoplexes or their co-formulation on the transfection of cancer cells in vitro and in vivo is analyzed. Sonotransfection improved mRNA delivery into 4T1 breast cancer cells in vitro with co-administration being more efficient than co-formulation. In vivo, the co-administration sonotransfection approach also resulted in higher transfection efficiency and reached deeper into the tumor tissue. On the contrary, co-formulation mainly promoted transfection of endothelial and perivascular cells. Furthermore, the co-formulation approach is much more dependent on the US trigger, resulting in significantly lower off-site transfection. Thus, the findings indicate that the choice of co-administration or co-formulation in sonotransfection should depend on the targeted cell population, tolerable off-site transfection, and the therapeutic purpose.

Development of exosome membrane materials-fused microbubbles for enhanced stability and efficient drug delivery of ultrasound contrast agent

Lipid-coated microbubbles are widely used as an ultrasound contrast agent, as well as drug delivery carriers. However, the two main limitations in ultrasound diagnosis and drug delivery using microbubbles are the short half-life in the blood system, and the difficulty of surface modification of microbubbles for active targeting. The exosome, a type of extracellular vesicle, has a preferentially targeting ability for its original cell. In this study, exosome-fused microbubbles (Exo-MBs) were developed by embedding the exosome membrane proteins into microbubbles. As a result, the stability of Exo-MBs is improved over the conventional microbubbles. On the same principle that under the exposure of ultrasound, microbubbles are cavitated and self-assembled into nano-sized particles, and Exo-MBs are self-assembled into exosome membrane proteins-embedded nanoparticles (Exo-NPs). The Exo-NPs showed favorable targeting properties to their original cells. A photosensitizer, chlorin e6, was loaded into Exo-MBs to evaluate therapeutic efficacy as a drug carrier. Much higher therapeutic efficacy of photodynamic therapy was confirmed, followed by cancer immunotherapy from immunogenic cell death. We have therefore developed a novel ultrasound image-guided drug delivery platform that overcomes the shortcomings of the conventional ultrasound contrast agent and is capable of simultaneous photodynamic therapy and cancer immunotherapy.

Resonance behaviors of encapsulated microbubbles oscillating nonlinearly with ultrasonic excitation

The resonance behaviors of a few lipid-coated microbubbles acoustically activated in viscoelastic media were comprehensively examined via radius response analysis. The size polydispersity and random spatial distribution of the interacting microbubbles, the rheological properties of the lipid shell and the viscoelasticity of the surrounding medium were considered simultaneously. The obtained radius response curves present a successive occurrence of linear resonances, nonlinear harmonic and sub-harmonic resonances with the acoustic pressure increasing. The microbubble resonance is radius-, pressure- and frequency-dependent. Specifically, the maximum bubble expansion ratio at the main resonance peak increases but the resonant radius decreases as the ultrasound pressure increases, while both of them decrease with the ultrasound frequency increasing. Moreover, compared to an isolated microbubble case, it is found that large microbubbles in close proximity prominently suppress the resonant oscillations while slightly increase the resonant radii for both harmonic and subharmonic resonances, even leading to the disappearance of the subharmonic resonance with the influences increasing to a certain degree. In addition, the results also suggest that both the encapsulating shell and surrounding medium can substantially dampen the harmonic and subharmonic resonances while increase the resonant radii, which seem to be affected by the medium viscoelasticity to a greater degree rather than the shell properties. This work offers valuable insights into the resonance behaviors of microbubbles oscillating in viscoelastic biological media, greatly contributing to further optimizing their biomedical applications.

Super-Resolved Microbubble Localization in Single-Channel Ultrasound RF Signals Using Deep Learning.

Recently, super-resolution ultrasound imaging with ultrasound localization microscopy (ULM) has received much attention. However, ULM relies on low concentrations of microbubbles in the blood vessels, ultimately resulting in long acquisition times. Here, we present an alternative super-resolution approach, based on direct deconvolution of single-channel ultrasound radio-frequency (RF) signals with a one-dimensional dilated convolutional neural network (CNN). This work focuses on low-frequency ultrasound (1.7 MHz) for deep imaging (10 cm) of a dense cloud of monodisperse microbubbles (up to 1000 microbubbles in the measurement volume, corresponding to an average echo overlap of 94%). Data are generated with a simulator that uses a large range of acoustic pressures (5-250 kPa) and captures the full, nonlinear response of resonant, lipid-coated microbubbles. The network is trained with a novel dual-loss function, which features elements of both a classification loss and a regression loss and improves the detection-localization characteristics of the output. Whereas imposing a localization tolerance of 0 yields poor detection metrics, imposing a localization tolerance corresponding to 4% of the wavelength yields a precision and recall of both 0.90. Furthermore, the detection improves with increasing acoustic pressure and deteriorates with increasing microbubble density. The potential of the presented approach to super-resolution ultrasound imaging is demonstrated with a delay-and-sum reconstruction with deconvolved element data. The resulting image shows an order-of-magnitude gain in axial resolution compared to a delay-and-sum reconstruction with unprocessed element data.

Combining Ultrasound and Capillary-Embedded T-Junction Microfluidic Devices to Scale Up the Production of Narrow-Sized Microbubbles through Acoustic Fragmentation.

Microbubbles are tiny gas-filled bubbles that have a variety of applications in ultrasound imaging and therapeutic drug delivery. Microbubbles can be synthesized using a number of techniques including sonication, amalgamation, and saline shaking. These approaches can produce highly concentrated microbubble suspensions but offer minimal control over the size and polydispersity of the microbubbles. One of the simplest and effective methods for producing monodisperse microbubbles is capillary-embedded T-junction microfluidic devices, which offer great control over the microbubble size. However, lower production rates (∼200 bubbles/s) and large microbubble sizes (∼300 μm) limit the applicability of such devices for biomedical applications. To overcome the limitations of these technologies, we demonstrate in this work an alternative approach to combine a capillary-embedded T-junction device with ultrasound to enhance the generation of narrow-sized microbubbles in aqueous suspensions. Two T-junction microfluidic devices were connected in parallel and combined with an ultrasonic horn to produce lipid-coated SF6 core microbubbles in the size range of 1-8 μm. The rate of microbubble production was found to increase from 180 microbubbles/s in the absence of ultrasound to (6.5 ± 1.2) × 106 bubble/s in the presence of ultrasound (100% ultrasound amplitude). When stored in a closed environment, the microbubbles were observed to be stable for up to 30 days, with the concentration of the microbubble suspension decreasing from ∼2.81 × 109/mL to ∼2.3 × 106/mL and the size changing from 1.73 ± 0.2 to 1.45 ± 0.3 μm at the end of 30 days. The acoustic response of these microbubbles was examined using broadband attenuation spectroscopy, and flow phantom imaging was performed to determine the ability of these microbubble suspensions to enhance the contrast relative to the surrounding tissue. Overall, this approach of coupling ultrasound with microfluidic parallelization enabled the continuous production of stable microbubbles at high production rates and low polydispersity using simple T-junction devices.

Enhancing cavitation dynamics and its mechanical effects with dual-frequency ultrasound

Objective. Acoustic cavitation and its mechanical effects (e.g. stress and strain) play a primary role in ultrasound applications. Introducing encapsulated microbubbles as cavitation nuclei and utilizing dual-frequency ultrasound excitation are highly effective approaches to reduce cavitation thresholds and enhance cavitation effects. However, the cavitation dynamics of encapsulated microbubbles and the resultant stress/strain in viscoelastic tissues under dual-frequency excitation are poorly understood, especially for the enhancement effects caused by a dual-frequency approach. The goal of this study was to numerically investigate the dynamics of a lipid-coated microbubble and the spatiotemporal distributions of the stress and strain under dual-frequency excitation. Approach. The Gilmore–Zener bubble model was coupled with a shell model for the nonlinear changes of both shell elasticity and viscosity to accurately simulate the cavitation dynamics of lipid-coated microbubbles in viscoelastic tissues. Then, the spatiotemporal evolutions of the cavitation-induced stress and strain in the surrounding tissues were characterized quantitatively. Finally, the influences of some paramount parameters were examined to optimize the outcomes. Main results. We demonstrated that the cavitation dynamics and associated stress/strain were prominently enhanced by a dual-frequency excitation, highlighting positive correlations between the maximum bubble expansion and the maximum stress/strain. Moreover, the results showed that the dual-frequency ultrasound with smaller differences in its frequencies and pressure amplitudes could enhance the bubble oscillations and stress/strain more efficiently, whereas the phase difference manifested small influences under these conditions. Additionally, the dual-frequency approach seemed to show a stronger enhancement effect with the shell/tissue viscoelasticity increasing to a certain extent. Significance. This study might contribute to optimizing the dual-frequency operation in terms of cavitation dynamics and its mechanical effects for high-efficient ultrasound applications.

Predicting acoustic emissions of ultrasound-driven lipid-coated microbubbles

Lipid-coated microbubbles excited by ultrasound are utilized in an increasing number of diagnostic and therapeutic medical applications, in which the acoustic waves emitted by the oscillating or collapsing bubbles are the main protagonist. The emitted acoustic waves cause bioeffects, enable contrast enhancement for ultrasonic imaging, and serve as a reference to monitor cavitation activity in situ. However, quantifying the acoustic waves emitted by cavitation bubbles turns out to be difficult: the small and extremely transient phenomenon is challenging to measure in experiments with sufficient accuracy and state-of-the-art computational methods are limited in their ability to predict acoustic emissions reliably. In this contribution, we present our recent work on predicting acoustic emissions of microbubbles based on Rayleigh–Plesset models and, more generally, reduced-order models. Using the nonlinear Westervelt equation including the motion of the bubble wall and the background medium as a result of the bubble oscillations, we investigate the amplitude and frequency modulation (e.g., due to nonlinear Doppler effects, thermoviscous attenuation or geometric distortion) of acoustic waves emitted by lipid-coated microbubbles. Additionally, we explore the capabilities of a reduced-order modeling approach that drastically simplifies the computational complexity of simulating these acoustic emissions.

Relationship between salinity of the liquid and shell composition on the resonance frequency of the C3F8 lipid coated microbubbles

Lipid-coated microbubbles (MBs) are used in contrast-enhanced ultrasound (CEUS) imaging and MB enhanced therapeutic ultrasound (US). Understanding the MB behavior and the influence of the surrounding medium on its response to the US is necessary to select the suitable US exposure parameters. The MB lipid coating is often charged, however the influence of the ions in the medium on MB behavior is not fully understood. In this work, the influence of the medium salinity on the pressure-dependent MB behavior is investigated for the first time. MBs of different lipid shell compositions are size isolated to achieve the same size distribution. The MBs linear and pressure-dependent attenuation are measured in deionized water, PBS 1×, PBS 2×, and PBS 10× using a system of aligned PVDF 100% bandwidth transducers with a center frequency of 10 MHz and exposures with peak to peak pressure range of 3–140 kPa. With increasing salinity, the linear resonance frequency decreases up to 50% for conventional lipid shell compositions, and the pressure dependence of the resonance frequency is inhibited. By modifying the shell PEG ratio, the salinity effects can significantly be altered. Moreover, the nonlinear pressure-dependent resonance frequency is restored with applications to increased CEUS.

Membrane effects from ultrasound interactions with microbubbles—From drug delivery to mechanotransduction

Ultrasound is an ideal modality to interact noninvasively with tissues deep within the body because of its significant penetration depth. However, noninvasive low intensity ultrasound on its own has difficulty creating mechanical forces necessary for localized activation of cells through mechanotransduction or rupture of non-echogenic drug delivery vehicles. Lipid-coated microbubbles are particles that provide a high-level of impedance contrast with the surrounding tissue and undergo significant size oscillations when exposed to ultrasound. This imparts mechanical forces on nearby lipid membranes which can be used for a variety of applications. Here, I will discuss the mechanical forces that are created by ultrasound/microbubble interactions that can be used to transduce mechanical forces across intact membranes for cellular activation, and to rupture membranes of drug delivery vehicles. I will also discuss the interaction of lipid debris left over from by microbubble destruction with cell membranes. The cell activation and vehicle release show promise for clinical applications including neuromodulation and cancer chemotherapy delivery.

Quantitative evaluation of the mortality rate of HeLa cells induced by microbubble vibration and collapse under pulsed ultrasound irradiation

Drug delivery systems (DDS) using ultrasound and microbubbles have been proposed to enhance drug efficacy and reduce undesirable side effects. While several researchers have reported the vibrational characteristics of lipid-coated microbubbles under ultrasound irradiation and the effects on cell damage, it is necessary to quantitatively evaluate the effects to establish the safety criteria of ultrasound DDS. In this paper, the effects of the microbubble behavior on adhesive HeLa cells under ultrasound irradiation were evaluated. Microbubbles coated with a phospholipid shell were fabricated and two size distributions of bubbles were prepared. The HeLa cells were exposed to pulsed ultrasound at 1 MHz with microbubbles in a water tank, and the cell mortality rate was measured quantitatively under fluorescent observation. The collapse of the microbubbles with a resonance size of approximately 2 μm enhanced the cell mortality rate. Greater sound pressure amplitude produced a higher collapsing rate of the microbubble and cell mortality rate, implying the destruction of microbubbles with resonance size generated a shock wave or microjet, inducing cell damage. While the cell mortality rate decreased exponentially with the increase in distance between cells and microbubbles, a smaller distance induced a higher cell mortality rate: 84% of the HeLa cells died within 2.5 μm of microbubbles produced by a pulsed ultrasound with 1.0 MPa at 1 MHz.

Modelling Lipid-Coated Microbubbles in Focused Ultrasound Applications at Subresonance Frequencies

We present a computational study of the behaviour of a lipid-coated SonoVue microbubble with initial radius 1 µm ≤ R0 ≤ 2 µm, excited at frequencies (200–1500 kHz) significantly below the linear resonance frequency and pressure amplitudes of up to 1500 kPa—an excitation regime used in many applications of focused ultrasound. The bubble dynamics are simulated using the Rayleigh–Plesset equation and the Gilmore equation, in conjunction with the Marmottant model for the lipid monolayer coating. Also, a new continuously differentiable variant of the Marmottant model is introduced. Below the onset of inertial cavitation, a linear regime is identified in which the maximum pressure at the bubble wall is linearly proportional to the excitation pressure amplitude and the mechanical index. This linear regime is bounded by the Blake pressure, and, in line with recent in vitro experiments, the onset of inertial cavitation is found to occur at an excitation pressure amplitude of approximately 130–190 kPa, depending on the initial bubble size. In the nonlinear regime the maximum pressure at the bubble wall is found to be readily predicted by the maximum bubble radius, and both the Rayleigh–Plesset and Gilmore equations are shown to predict the onset of sub- and ultraharmonic frequencies of the acoustic emissions compared with in vitro experiments. Neither the surface dilational viscosity of the lipid monolayer nor the compressibility of the liquid has a discernible influence on the quantities studied, but accounting for the lipid coating is critical for accurate prediction of the bubble behaviour. The Gilmore equation is shown to be valid for the bubbles and excitation regime considered, and the Rayleigh–Plesset equation also provides accurate qualitative predictions, even though it is outside its range of validity for many of the cases considered.

DSPC or DPPC As Main Shell Component Influences Ligand Distribution and Binding Area of Lipid‐Coated Targeted Microbubbles

Eur. J. Lipid Sci. Technol. 2014, 116, 1217 https://doi.org/10.1002/ejlt.201300434 The authors regret that there was a mistake in calculating the mol% of the microbubble coating composition used. For all experiments the unit in mg mL−1 was utilized and the conversion mistake only came when converting to mol% in order to define the ratio between the coating formulation components. The correct molecular weight of PEG-40 stearate is 2046.54 g mol−1,[1, 2] not 328.53 g mol−1. On page 1219, Section 2.2.1, it should read: “Biotinylated lipid-coated microbubbles (composition in mol %: DSPC or DPPC 84.8; PEG-40 stearate 8.2; DSPE-PEG2000 5.9, DSPE-PEG2000-biotin 1.1) with a C4F10 gas core were made by sonication for 10 s using the method described by Klibanov et al.[30]” This correction does not change the conclusions published in this work. The authors apologize for any inconvenience caused.

Corrections to "Vibrational Responses of Bound and Nonbound Targeted Lipid-Coated Single Microbubbles".

In the above article [1], the authors regret that there was a mistake in calculating the mol% of the microbubble coating composition. For all experiments, the unit in mg/mL was utilized and the conversion mistake only came when converting to mol% in order to define the ratio between the coating formulation components. The correct molecular weight of PEG-40 stearate is 2046.54 g/mol [2], [3], not 328.53 g/mol. On page 786, paragraph II-A, it should read "The coating was composed of 84.8 mol% DSPC (P6517, Sigma-Aldrich, Zwijndrecht, The Netherlands) or DPPC (850355, Avanti Polar Lipids, Alabaster, AL, USA); 8.2 mol% polyoxyethylene-40-stearate (PEG-40 stearate, P3440, Sigma-Aldrich); 5.9 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG(2000), 880125, Avanti Polar Lipids); and 1.1 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG(2000)-biotin, 880129, Avanti Polar Lipids)."

Photoacoustic Impulse Response of Lipid-Coated Ultrasound Contrast Agents.

The utility of ultrasound imaging and therapy with microbubbles may be greatly enhanced by determining their impulse-response dynamics as a function of size and composition. Prior methods for microbubble characterization utilizing high-speed cameras, acoustic transducers and laser-based techniques typically scan a limited frequency range. Here, we report on the use of a novel photoacoustic technique to measure the impulse response of single microbubbles. Individual microbubbles are driven with a broadband photoacoustic wave generated by a nanosecond-pulse laser illuminating an optical absorber. The resulting microbubble oscillations were detected by following transmission of a second laser as it passes twice through the microbubble. The system could even resolve oscillations resulting from a single-shot. As a proof-of-concept study, the size-dependent, linear impulse response of lipid-coated microbubbles was characterized using this technique. This unique method of microbubble characterization with exceptional spatiotemporal resolution opens new avenues for capturing and analyzing microbubble system dynamics.