Stability testing of gadoteridol and gadobenate dimeglumine formulations under exposure to high-intensity focused ultrasound.

OBJECTIVE: Contrast-enhanced MRI could be useful to guide high-intensity focused ultrasound treatment (HIFU), but the effects of HIFU on gadolinium-based agents is not known. Here, we tested in vitro the stability of gadoteridol and gadobenate dimeglumine, two widely used MR contrast agents, after exposure to HIFU at power levels typically applied in the clinical practice. METHODS: 0.5 M (gadoteridol and gadobenate dimeglumine) and diluted formulations (1:10 gadoteridol in saline) were exposed to different HIFU sequences. Unexposed and exposed solutions were characterized by high-performance liquid chromatography in terms of concentration of gadolinium complex, free gadolinium and free ligand. RESULTS: Gadoteridol formulation after treatment showed concentrations of the complex not significantly different from control. Free Gd and/or free ligand concentrations in the order of 0.002/0.004% w/w, were observed occasionally without significant correlation with intensity and duration of exposure to HIFU. Gadobenate dimeglumine formulation after treatment showed complex assay content values, by-products (0.24-0.26%) and free BOPTA levels (0.07%) comparable to control sample within the experimental error. CONCLUSION: In the range of conditions explored, HIFU exposure did not induce significant dissociations of gadoteridol and gadobenate dimeglumine, nor a detectable increase in the concentration of free species. ADVANCES IN KNOWLEDGE: Our study strengthens the hypothesis that gadolinium-based contrast agents are stable during HIFU treatment for body applications (e.g. thermal ablation of uterine fibroids).

Microbubble formulation influences inflammatory response to focused ultrasound exposure in the brain.

Focused ultrasound and microbubble (FUS + MB)-mediated blood-brain barrier (BBB) permeability enhancement can facilitate targeted brain-drug delivery. While controlling the magnitude of BBB permeability enhancement is necessary to limit tissue damage, little work has attempted to decouple these concepts. This work investigated the relationship between BBB permeability enhancement and the relative transcription of inflammatory mediators 4 h following sonication. Three microbubble formulations, Definity, BG8774, and MSB4, were compared, with the dose of each formulation normalized to gas volume. While changes in the transcription of key proinflammatory mediators, such as Il1b, Ccl2, and Tnf, were correlated to the magnitude of BBB permeability enhancement, these correlations were not independent of microbubble formulation; microbubble size distribution may play an important role, as linear regression analyses of BBB permeability magnitude versus differential gene expression for these proinflammatory mediators revealed significantly greater slopes for MSB4, a monodisperse microbubble with mean diameter of 4 µm, compared to Definity or BG8774, both polydisperse microbubbles with mean diameters below 2 µm. Additionally, the function of an acoustic feedback control algorithm, based on the detection threshold of ultraharmonic emissions, was assessed. While this control strategy was effective in limiting both wideband emissions and red blood cell extravasation, microbubble formulation was found to influence the magnitude of BBB leakage and correlations to acoustic emissions. This work demonstrates that while the initial magnitude of FUS + MB-mediated BBB permeability enhancement has a clear influence on the subsequent inflammatory responses, microbubble characteristics influence these relationships and must also be considered.

Monodisperse versus Polydisperse Ultrasound Contrast Agents: In Vivo Sensitivity and safety in Rat and Pig.

Recent advances in the field of monodisperse microbubble synthesis by flow focusing allow for the production of foam-free, highly concentrated and monodisperse lipid-coated microbubble suspensions. It has been found that in vitro, such monodisperse ultrasound contrast agents (UCAs) improve the sensitivity of contrast-enhanced ultrasound imaging. Here, we present the first in vivo study in the left ventricle of rat and pig with this new monodisperse bubble agent. We systematically characterize the acoustic sensitivity and safety of the agent at an imaging frequency of 2.5 MHz as compared with three commercial polydisperse UCAs (SonoVue/Lumason, Definity/Luminity and Optison) and one research-grade polydisperse agent with the same shell composition as the monodisperse bubbles. The monodisperse microbubbles, which had a diameter of 4.2 µm, crossed the pulmonary vasculature, and their echo signal could be measured at least as long as that of the polydisperse UCAs, indicating that microfluidically formed monodisperse microbubbles are stable in vivo. Furthermore, it was found that the sensitivity of the monodisperse agent, expressed as the mean echo power per injected bubble, was at least 10 times higher than that of the polydisperse UCAs. Finally, the safety profile of the monodisperse microbubble suspension was evaluated by injecting 400 and 2000 times the imaging dose, and neither physiologic nor pathologic changes were found, which is a first indication that monodisperse lipid-coated microbubbles formed by flow focusing are safe for in vivo use. The more uniform acoustic response and corresponding increased imaging sensitivity of the monodisperse agent may boost emerging applications of microbubbles and ultrasound such as molecular imaging and therapy.

Microfluidic preparation of various perfluorocarbon nanodroplets: Characterization and determination of acoustic droplet vaporization (ADV) threshold.

Over the last two decades, liquid perfluorocarbon nanodroplets (PFC-NDs), also known as Phase Change Contrast Agents (PCCAs), that are capable of vaporizing into gaseous echogenic microbubbles via an external stimulus, have gained much attention for diagnostic and therapeutic applications. In the present work, a microfluidic platform is evaluated for the preparation of various size-controlled nanodroplets. Here, two major lines of investigations were carried out. The first was to define the microfluidic device settings for the preparation of nanodroplets depending on the nature of the encapsulating shell such as lipids, fluorinated surfactants and PLGA biopolymers as well as the liquid perfluorocarbon core (perfluoropentane, perfluorohexane). Specifically, the effect of the microfluidic system parameters, such as total flow rate and flow rate ratio on PFC-NDs attributes including size and uniformity was assessed. Secondly, a custom-made set-up, based on echogenicity signals from produced bubbles, was designed and successfully applied to determine the Acoustic Droplet Vaporization (ADV) threshold of PFC-NDs. Finally, the influence of various formulation parameters on the vaporization outcome was investigated depending on the PFC type and the encapsulating shell composition (soft versus hard shells). This study indicates the usefulness of this novel formulation platform enabling the rapid design and optimization of narrowly dispersed nanodroplets at a reliable yield and ultimately accelerate nanomedicines development.

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.

High-precision acoustic measurements of the nonlinear dilatational elasticity of phospholipid coated monodisperse microbubbles.

The acoustic response of phospholipid-coated microbubble ultrasound contrast agents (UCA) is dramatically affected by their stabilizing shell. The interfacial shell elasticity increases the resonance frequency, the shell viscosity increases damping, and the nonlinear shell viscoelasticity increases the generation of harmonic echoes that are routinely used in contrast-enhanced ultrasound imaging. To date, the surface area-dependent interfacial properties of the phospholipid coating have never been measured due to the extremely short time scales of the MHz frequencies at which the microscopic bubbles are driven. Here, we present high-precision acoustic measurements of the dilatational nonlinear viscoelastic shell properties of phospholipid-coated microbubbles. These highly accurate measurements are now accessible for the first time by tuning the surface dilatation, that is, the lipid packing density, of well-controlled monodisperse bubble suspensions through the ambient pressure. Upon compression, the shell elasticity of bubbles coated with DPPC and DPPE-PEG5000 was found to increase up to an elasticity of 0.6 N m-1 after which the monolayer collapses and the elasticity vanishes. During bubble expansion, the elasticity drops monotonically in two stages, first to an elasticity of 0.35 N m-1, and then more rapidly to zero. Integration of the elasticity vs. surface area curves showed that, indeed, a phospholipid-coated microbubble is in a tensionless state upon compression, and that it reaches the interfacial tension of the surrounding medium upon expansion. The measurements presented in this work reveal the detailed features of the nonlinear dilatational shell behavior of micron-sized lipid-coated bubbles.

Characteristics and Echogenicity of Clinical Ultrasound Contrast Agents: An In Vitro and In Vivo Comparison Study.

OBJECTIVES: To compare physicochemical characteristics and in vitro and in vivo contrast-enhanced ultrasound imaging performance of 3 commercially available ultrasound contrast agents: SonoVue (Bracco Imaging SpA, Colleretto Giacosa, Italy; also marketed as Lumason in the USA), Definity (Lantheus Medical Imaging, North Billerica, MA) and Optison (GE Healthcare AS, Oslo, Norway). METHODS: Physicochemical characteristics were measured with a Multisizer Coulter Counter (Beckman Coulter, Fullerton, CA). Two ultrasound systems (Aplio 500; Toshiba Medical Systems Corp, Tochigi-ken, Japan; and Logiq E9; GE Healthcare, Little Chalfont, England) were used with different transducers. Contrast enhancement was measured in vitro by dose-ranging measurements using a custom-built beaker setup; in vivo imaging performances were compared in pigs (heart and liver) and rabbits (liver). Quantitative analyses were performed with VueBox quantification software (Bracco Suisse SA, Plan-les-Ouates, Switzerland). RESULTS: Measured physicochemical characteristics were in agreement with those provided by the manufacturers. In vitro data demonstrated that the performance of SonoVue was similar to or better than that of Definity but superior to Optison (normalized scattered power 2- to 10-fold higher with SonoVue). Similar results were obtained in vivo, although the duration of enhancement in the pig heart was longer for SonoVue compared to Definity, and quantitative analysis revealed higher enhancement for SonoVue (1.5-fold increase). For liver imaging, SonoVue and Definity showed similar contrast enhancement and duration of enhancement, but compared to Optison, both peak enhancement and duration of enhancement were superior for SonoVue (up to 2-fold increase). CONCLUSIONS: Imaging performance of SonoVue was similar to or slightly better than that of Definity, but it was superior to Optison for the conditions used in this study.

Echo-power estimation from log-compressed video data in dynamic contrast-enhanced ultrasound imaging.

Ultrasound (US) scanners typically apply lossy, non-linear modifications to the US data for visualization purposes. The resulting images are then stored as compressed video data. Some system manufacturers provide dedicated software for quantification purposes to eliminate such processing distortions, at least partially. This is currently the recommended approach for quantitatively assessing changes in contrast-agent concentration from clinical data. However, the machine-specific access to US data and the limited set of analysis functionalities offered by each dedicated-software package make it difficult to perform comparable analyses with different US systems. The objective of this work was to establish if linearization of compressed video images obtained with an arbitrary US system can provide an alternative to dedicated-software analysis of machine-specific files for the estimation of echo-power. For this purpose, an Aplio 50 system (Toshiba Medical Systems, Tochigi, Japan), coupled with dedicated CHI-Q (Contrast Harmonic Imaging Quantification) software by Toshiba Medical Systems, was used. Results were compared with two approaches that apply algorithms to estimate relative echo-power from compressed video images: commercially available VueBox software by Bracco Suisse SA (Geneva, Switzerland) and in-laboratory software called PixPower. The echo-power estimated by CHI-Q analysis indicated a strong linear relationship versus agent concentration in vitro (R(2) ≥ 0.9996) for dynamic range (DR) settings of DR60 and DR80, with slopes between 9.22 and 9.57 dB/decade (p = 0.05). These values approach the theoretically predicted dependence of 10.0 dB/decade (equivalent to 3 dB for each concentration doubling). Echo-power estimations obtained from compressed video images with VueBox and PixPower also exhibited strong linear proportionality with concentration (R(2) ≥ 0.9996), with slopes between 9.30 and 9.68 dB/decade (p = 0.05). On an independent in vivo data set (N = 24), the difference in echo-power estimation between CHI-Q and each of the other two approaches was calculated after excluding regions that contain pixels affected by saturated or thresholded pixel values. The mean difference in estimates (expressed in decibels) was -0.25 dB between VueBox and CHI-Q (95% confidence interval: -0.75 to 0.26 dB) and -0.17 dB between PixPower and CHI-Q (95% confidence interval: -0.67 to 0.13 dB). To achieve linearization of data, one of the approaches (VueBox) requires calibration files provided by the software manufacturer for each machine type and setting. The other (PixPower) requires empirical correction of the imaging dynamic range based on ground truth data. These requirements could potentially be removed if US system manufacturers were willing to make relevant information on the applied processing publically available. Reliable echo-power estimation from linearized data would facilitate inclusion of different US systems in multicentric studies and more widespread implementation of emerging techniques for quantitative analysis of contrast ultrasound.

In vitro sonothrombolysis of human blood clots with BR38 microbubbles.

Microbubble-mediated sonothrombolysis is a promising approach for ischemic stroke treatment. The aim of this in vitro study was to evaluate a new microbubble (MB) formulation (BR38) for sonothrombolysis and to investigate the involved mechanisms. Human whole-blood clots were exposed to different combinations of recombinant tissue plasminogen activator (rtPA), ultrasound (US) and MB. Ultrasound at 1.6 MHz was used at 150, 300, 600 and 1000 kPa (peak-negative pressure). Thrombolysis efficacy was assessed by measuring clot diameter changes during 60-min US exposure. The rate of clot diameter loss (RDL) in µm/min was determined and clot lysis profiles were analyzed. The most efficient clot lysis (5.9 µm/min) was obtained at acoustic pressures of 600 and 1000 kPa in combination with MB and a low concentration of rtPA (0.3 µg/mL). This is comparable with the rate obtained with rtPA at 3 µg/mL alone (6.6 µm/min, p > 0.05). Clot lysis profiles were shown to be related to US beam profiles and microbubble cavitation.

Acoustic characterization of single ultrasound contrast agent microbubbles.

Individual ultrasound contrast agent microbubbles (BR14) were characterized acoustically. The bubbles were excited at a frequency of 2 MHz and at peak-negative pressure amplitudes of 60 and 100 kPa. By measuring the transmit and receive transfer functions of both the transmit and receive transducers, echoes of individual bubbles were recorded quantitatively and compared to simulated data. At 100 kPa driving pressure, a second harmonic response was observed for bubbles with a size close to their resonance size. Power spectra were derived from the echo waveforms of bubbles of different sizes. These spectra were in good agreement with those calculated from a Rayleigh-Plesset-type model, incorporating the viscoelastic properties of the phospholipid shell. Small bubbles excited below their resonance frequency have a response dominated by the characteristics of their phospholipid shell, whereas larger bubbles, excited above resonance, have a response identical to those of uncoated bubbles of similar size.

Effects of simultaneous application of ultrasound and microbubbles on intracerebral hemorrhage in an animal model.

Microbubble-enhanced sonothrombolysis (MEST) may be an alternative therapeutic option in ischemic stroke. Clinical study of the efficacy of MEST as an adjunct stroke therapy, before imaging with CT or MRI, requires experimental data on the safety of this approach in the presence of hemorrhagic stroke. We, therefore, investigated the effect of diagnostic transcranial ultrasound combined with microbubbles (US + MB) in an experimental animal model of intracerebral hemorrhage (ICH). ICH was induced in anesthetized rats by intracerebral collagenase injection. Transcranial ultrasound (2 MHz, mechanical index 1.3, 1051 kPa) was applied 3 h after ICH induction to rat brains for 30 min during a continuous IV infusion of sulfur hexafluoride microbubbles (SonoVue). The size of cerebral hemorrhage, the extent of brain edema, and the amount of apoptosis were compared with those from control rats with ICH but without US + MB. Results showed no significant effect of US + MB on hemorrhage size (control 23.3 +/- 10.7 mm(3), US + MB 20.3 +/- 5.8 mm(3)), on the extent of brain edema (control 3.3 +/- 2.0%, US +MB 3.5 +/- 1.9%), or on the rate of apoptosis (control 5.2 +/- 1.5%, US + MB 5.2 +/- 1.0%). We conclude that diagnostic ultrasound in combination with microbubbles does not cause additional damage to the rat brain during ICH in our experimental set-up. This finding provides support for the use of MEST as an early stroke therapy.