Ross Williams

Sonoporation by ultrasound-activated microbubble contrast agents: effect of acoustic exposure parameters on cell membrane permeability and cell viability.

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

Dynamic Microbubble Contrast-enhanced US to Measure Tumor Response to Targeted Therapy: A Proposed Clinical Protocol with Results from Renal Cell Carcinoma Patients Receiving Antiangiogenic Therapy

PURPOSE To develop and implement an evidence-based protocol for characterizing vascular response of renal cell carcinoma (RCC) to targeted therapy by using dynamic contrast material-enhanced (DCE) ultrasonography (US). MATERIALS AND METHODS The study was approved by the institutional research ethics board; written informed consent was obtained from all patients. Seventeen patients (four women; median age, 58 years; range, 42-72 years; 13 men, median age, 62 years; range, 45-81 years) with metastatic RCC were examined by using DCE US before and after 2 weeks of treatment with sunitinib (May 2007 to October 2009). Two contrast agent techniques--bolus injection and disruption-replenishment infusion of microbubbles--were compared. Changes in tumor blood velocity and fractional blood volume were measured with both methods, together with reproducibility and effect of compensation for respiratory motion. Tumor changes were assessed with computed tomography, by using the best response with the Response Evaluation Criteria in Solid Tumors (RECIST) and progression-free survival (PFS). Follow-up RECIST measurements were performed at 6-week intervals until progressive disease was detected. RESULTS In response to treatment, median tumor fractional blood volume measured with the disruption-replenishment infusion method decreased by 73.2% (interquartile range, 46%-87%) (P < .002), with repeated-measure reproducibility of 9%-15%. Significant decreases were also seen with the bolus method, but with poor correlation of changes in bolus peak (r = 0.46, P = .066) and area under the curve (r = 0.47, P = .058), compared with infusion measurements. Changes in DCE US parameters over 2 weeks did not correlate with PFS and could not be used to predict long-term assessment of best response by using RECIST. Follow-up times ranged 28-501 days; the median was 164 days. CONCLUSION DCE US provides reproducible and sensitive assessment of vascular changes in response to antiangiogenic therapy. The disruption-replenishment infusion protocol is a flexible method suitable for many tumor types, but further studies are needed to assess whether this protocol may be predictive of patient outcome.

Microfluidic Assembly of Monodisperse, Nanoparticle-Incorporated Perfluorocarbon Microbubbles for Medical Imaging and Therapy

New medical imaging contrast agents that permit multiple imaging and therapy applications using a single agent can result in more accurate diagnosis and local treatment of diseased tissue. Solid nanoparticles (NPs) (5-150 nm in size) have emerged as promising imaging and therapy agents, as have micrometer-scale, perfluorocarbon gas-filled microbubbles (MBs) used in patients as intravascular ultrasound contrast agents. We propose that the modular combination of small, solid NPs and larger, highly compressible MBs into a single agent is an effective way to attain the desired complementary and hybrid properties of two very different agents. Presented here is a new strategy for the simple and robust incorporation of various medical NPs with monodisperse MBs based upon the controlled pH-based regulation of the electrostatic attraction between NPs and the MB shell. Using this simple approach, microfluidic-generated, protein-lipid-coated, perfluorobutane MBs (with size control down to 3 microm) were incorporated with silica-coated NPs, including CdSe/ZnS quantum dots, gold nanorods, iron oxide NPs, and Gd-loaded mesoporous silica NPs. The silica interface permits NP inclusion within MBs to be independent of NP composition, morphology, and size. Significantly, the NP-incorporated MBs (NP-MBs) diluted in saline were detectable using low-pressure ultrasound, and the monodisperse MB platform can be produced at high-throughput, sufficient for in vivo usage (10(6) MB/sec). The modular synthesis of a variety of NP-MBs can facilitate flexible, user-defined, multifunctional imaging and therapy agents tailored for specific applications and disease types.

The efficiency and stability of bubble formation by acoustic vaporization of submicron perfluorocarbon droplets

Submicron droplets of liquid perfluorocarbon converted into microbubbles with applied ultrasound have been studied, for a number of years, as potential next generation extravascular ultrasound contrast agents. In this work, we conduct an initial ultra-high-speed optical imaging study to examine the vaporization of submicron droplets and observe the newly created microbubbles in the first microseconds after vaporization. It was estimated that single pulses of ultrasound at 10 MHz with pressures within the diagnostic range are able to vaporize on the order of at least 10% of the exposed droplets. However, only part of the newly created microbubbles survives immediately following vaporization - the bubbles may recondense back into the liquid droplet state within microseconds of nucleation. The probability of bubble survival within the first microseconds of vaporization was shown to depend on ultrasound excitation pressure as well as on bubble coalescence during vaporization, a behavior influenced by the presence of coating material on the newly created bubbles. The results of this study show for the first time that although initial vaporization of droplets is necessary to create echogenic bubbles, additional factors, such as coalescence and bubble shell properties, are important and should be carefully considered for the production of microbubbles for use in medical imaging.

Optical studies of vaporization and stability of fluorescently labelled perfluorocarbon droplets

Droplets of liquid perfluorocarbon (PFC) are under study as the next generation of contrast agents for ultrasound (US). These droplets can be selectively vaporized into echogenic gas bubbles in situ by externally applied US, with numerous applications to diagnosis and therapy. However, little is known about the mechanisms of droplet vaporization and the stability of the bubbles so produced. Here we observe optically the vaporization of fluorescent PFC droplets and the stability of the newly created bubbles. Fluorescent markers were used to label selectively either the liquid PFC core or the shell of the droplets. It was found that, following vaporization, the fluorescent marker is quickly expelled from the core of the newly created bubble and is retained on the gas-liquid interface. At the same time, it was shown that bubbles retain the original shells encapsulating their droplet precursors. The efficiency of encapsulation was found to depend strongly on the nature of the stabilizing material itself. These results provide direct evidence of droplet encapsulation post-vaporization, and suggest that the behaviour of the vaporized droplets is strongly dependent on the choice of the stabilizing material for the emulsion.

In vitro characterization of the subharmonic ultrasound signal from Definity microbubbles at high frequencies

Ultrasound microbubble contrast agents have been demonstrated to scatter subharmonic energy at one-half the driving frequency. At ultrasound frequencies in the 20-40 MHz range, the subharmonic offers the potential to differentiate the blood in the microcirculation from the surrounding tissue. It is unknown whether current contrast agents, manufactured to be resonant between 2 and 12 MHz, are ideal for subharmonic imaging at higher frequencies. We performed numerical simulations of the Keller-Miksis model for the behavior of a single bubble and experimental investigations of Definity microbubbles in water. The results supported the hypothesis that off-resonant bubbles, excited at their second harmonic, may be primarily responsible for the observed subharmonic energy. For frequencies between 20 and 32 MHz and 32 and 40 MHz, the optimal bubble diameters for the generation of subharmonics in vitro were determined experimentally to be 1.2-5 microm and less than 1.2 microm, respectively. Definity may be a suitable ultrasound contrast agent for subharmonic imaging at 20 MHz with peak-negative pressures between 380 and 590 kPa and pulses greater than or equal to four cycles in duration.

Golay Pulse Encoding for Microbubble Contrast Imaging in Ultrasound

We present a technique that uses Golay phase encoding, pulse inversion, and amplitude modulation (GPIAM) for microbubble contrast agent imaging with ultrasound. This technique improves the contrast-to-tissue ratio (CTR) by increasing the time-bandwidth product of the insonating waveforms. A nonlinear pulse compression algorithm is used to compress the signal energy upon receive. A 6.5-dB improvement in CTR was observed using an 8-chip GPIAM sequence compared to a conventional pulse-inversion amplitude-modulation sequence. The CTR improvement comes at the cost of a reduction in frame rate: GPIAM coding uses four input pulses whereas most contrast imaging sequences require two or three pulses. Our results showed that the microbubble response can be phase encoded and subsequently compressed using a nonlinear matched-filtering algorithm, in order to enhance the signal from the contrast agent, while maintaining resolution and suppressing the tissue signal.

Combined perfusion and doppler imaging using plane-wave nonlinear detection and microbubble contrast agents.

Plane-wave imaging offers image acquisition rates at the pulse repetition frequency, effectively increasing the imaging frame rates by up to two orders of magnitude over conventional line-by-line imaging. This form of acquisition can be used to achieve very long ensemble lengths in nonlinear modes such as pulse inversion Doppler, which enables new imaging trade-offs that were previously unattainable. We first demonstrate in this paper that the coherence of microbubble signals under repeated exposure to acoustic pulses of low mechanical index can be as high as 204 ± 5 pulses, which is long enough to allow an accurate power Doppler measurement. We then show that external factors, such as tissue acceleration, restrict the detection of perfusion at the capillary level with linear Doppler, even if long Doppler ensembles are considered. Hence, perfusion at the capillary level can only be detected with ultrasound through combined microbubbles and Doppler imaging. Finally, plane-wave contrast-enhanced power and color Doppler are performed on a rabbit kidney in vivo as a proof of principle. We establish that long pulse-inversion Doppler sequences and conventional wall-filters can create an image that simultaneously resolves both the vascular morphology of veins and arteries, and perfusion at the capillary level with frame rates above 100 Hz.

Radial Modulation Imaging of Microbubble Contrast Agents at High Frequency

In this paper, radial modulation imaging of microbubbles is investigated at high frequency. A modulation pulse frequency of 3.7 MHz with an amplitude ranging from 0 to 250 kPa, and a 1.3-MPa 20-MHz broadband imaging pulse were used. Radial modulation effects were observed on a population of flowing microbubbles and quantified using a Doppler-type processing technique. Artifact signals related to the generation of harmonics by bubbles strongly resonating at the modulation frequency were observed. The bubble response to simultaneous modulation and imaging excitations was simulated for different combinations of bubble sizes and modulation amplitudes. Simulation results confirm the hypothesis that the generation of harmonics of the modulation frequency can be detected by the imaging transducer. Simulations indicate that the modulation frequency should be chosen lower than the resonant frequency of the biggest bubbles present in the population. The simulation also suggests that a 10% variation of bubble diameter induced by the modulation excitation is sufficient for radial modulation imaging. In conclusion, the effects of radial modulation are detectable at a high frequency. Therefore, radial modulation imaging has potential for high-resolution imaging of microbubbles in the microvasculature.

Pseudoenhancement Within the Local Ablation Zone of Hepatic Tumors Due to a Nonlinear Artifact on Contrast-Enhanced Ultrasound

OBJECTIVE Pseudoenhancement of an avascular region on contrast-enhanced ultrasound often occurs within an echogenic region of a radiofrequency ablation zone due to nonlinear ultrasound propagation through intervening microbubble-perfused tissue. The purpose of this study was to describe the imaging features of this artifact. MATERIALS AND METHODS Twenty-six patients with no tumor recurrence within ablation zones were included. Two radiologists assessed contrast-enhanced ultrasound pseudoenhancement in the arterial (< 30 seconds), portal (30-90 seconds), and late (> 90 seconds) phases. If pseudoenhancement was present, the following information was recorded: the degree, time to first appearance, progression over time, and location. The corresponding gray-scale echogenicity (hypo-, iso-, or hyperechoic) and lesion depth were also noted. RESULTS Fourteen lesions (14/26, 54%) showed pseudoenhancement on contrast-enhanced ultrasound. Fourteen (100%) corresponded to the hyperechoic area within the ablation zone on gray-scale ultrasound and were nonmarginal in location. Pseudoenhancement occurred more frequently in deep lesions (> or = 5 cm) than in superficial lesions (< 5 cm) (p = 0.002). Pseudoenhancement was initiated most frequently in the portal phase (9/14, 64%), followed by the arterial phase (4/14, 29%) and late phase (1/14, 7%). Progression in the degree of pseudoenhancement was shown in most cases (12/14, 86%) and no washout was seen. CONCLUSION Pseudoenhancement is frequently seen within ablation zones on contrast-enhanced ultrasound, particularly in deep echogenic lesions. However, pseudoenhancement follows enhancement of the parenchyma between the transducer and target. This observation is consistent with nonlinear propagation of the ultrasound beam, which increases with bubble concentration. Pseudoenhancement shows relatively late initiation, progression over time, and nonmarginal location; these findings are different from those seen in typical tumor recurrence, which shows early enhancement and washout at the margin of the ablation zone.