Development of a platform for co-registered ultrasound and MR contrast imaging in vivo.

Imaging of the microvasculature is often performed using contrast agents in combination with either ultrasound (US) or magnetic resonance (MR) imaging. Contrast agents are used to enhance medical imaging by highlighting microvascular properties and function. Dynamic signal changes arising from the passage of contrast agents through the microvasculature can be used to characterize different pathologies; however, comparisons across modalities are difficult due to differences in the interactions of contrast agents with the microvasculature. Better knowledge of the relationship of contrast enhancement patterns with both modalities could enable better characterization of tissue microvasculature. We developed a co-registration platform for multi-modal US and MR imaging using clinical imaging systems in order to study the relationship between US and MR contrast enhancement. A preliminary validation study was performed in phantoms to determine the registration accuracy of the platform. In phantoms, the in-plane registration accuracy was measured to be 0.2 ± 0.2 and 0.3 ± 0.2 mm, in the lateral and axial directions, respectively. The out-of-plane registration accuracy was estimated to be 0.5 mm ±0.1. Co-registered US and MR imaging was performed in a rabbit model to evaluate contrast kinetics in different tissue types after bolus injections of US and MR contrast agents. The arrival time of the contrast agent in the plane of imaging was relatively similar for both modalities. We studied three different tissue types: muscle, large vessels and fat. In US, the temporal kinetics of signal enhancement were not strongly dependent on tissue type. In MR, however, due to the different amounts of agent extravasation in each tissue type, tissue-specific contrast kinetics were observed. This study demonstrates the feasibility of performing in vivo co-registered contrast US and MR imaging to study the relationships of the enhancement patterns with each modality.

Microbubble mediated sonoporation of cells in suspension: clonogenic viability and influence of molecular size on uptake.

This work investigates whether the application of sonoporation is limited by the size of a macromolecule being delivered and by the ability of cells to proliferate following uptake. KHT-C cells in suspension were exposed to variations in ultrasound pressure (0-570 kPa) and microbubble shell-type (lipid and protein) at fixed settings of 500 kHz centre frequency, 32 micros pulse duration, 3 kHz pulse repetition frequency and 2 min insonation. Reversible permeability (P(R)), defined as the number of cells stained with FITC-dextran and unstained with propidium iodide (i.e., PI-viable), was measured with flow cytometry for marker molecules ranging from 10 kDa to 2 MDa in size. Viable permeability (P(V)) defined as the number of permeabilised cells that maintained their ability to proliferate, was measured by clonogenic assay. Comparable intracellular delivery of all sizes of molecules was achieved, indicating that intracellular delivery of common therapeutic drugs may not be limited by molecular size. Maximum P(R)'s of 80% (at 10 kDa) and 55% (at 10 kDa) were achieved with lipid coated bubbles at 3.3% v/v and protein coated bubbles at 6.7% v/v concentrations. The PI-viability was approximately 80% at 570 kPa in both cases. The maximum P(V) achieved with both agents was 22%, while inducing a lower overall clonogenic viability with the lipid (39%) compared to the protein (56%) shelled bubbles. This study demonstrates that large macromolecules, up to 2 MDa in size, can be delivered with high efficiency to cells which undergo reversible permeabilisation, maintaining long-term viability in approximately half of the cells.

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.

The influence of fragmentation on the acoustic response from shrinking bubbles.

Bubble disruption is associated with the response of ultrasound contrast agents (UCAs) exposed to high acoustic pressures. This behavior is important for bubble detection techniques as well as flow quantitation and some proposed therapeutic applications. Previous work has measured acoustically the disruption threshold and postdisruption echo from populations of microbubbles. This suggests a model for UCA disruption whereby ultrasound breaks their shell, leaving free gas bubbles. Diffusion of gas causes the bubbles to shrink and, consequently, reduces the measured backscatter echo over time. In this work, similar bubbles containing three different gases were measured and their echo behavior with time compared with a simple simulation based on diffusion of gas out of the bubble. It was found that, in general, the simulations and experiments compared well at low disruption pressures. Incorporating bubble fragmentation in the simulation model brought its results closer to experiment.

Quantitative measurement of ultrasound disruption of polymer-shelled microbubbles.

The goal of this study was to assess the threshold of disruption and subsequent time-course of acoustic response of four experimental nitrogen-filled polymer-shelled microbubbles. Using an in vitro measurement system, a sequence of low-amplitude detection pulses measured the change in echo caused by a high-amplitude disruption pulse on a dilute suspension of bubbles. Detection pulses were transmitted 0.5 ms before disruption and between 1 and 200 ms after disruption. Separate transducers, aligned confocally and orthogonally, were used to transmit and receive bubble echoes. After disruption, all agents demonstrated a transient increase in scattered power. Above the disruption threshold, highly echogenic, nonlinear scatterers were observed. Their echoes slowly disappeared after disruption with median decay times from 7.4 to 13.6 ms, calculated by fitting to a mono-exponential decay. This is consistent with a process wherein the shell is disrupted, releasing the gas and generating free gas bubbles, which cause high-amplitude nonlinear scattering followed by relatively slow diffusion of the gas into solution. This picture has been observed optically with single bubbles and differs from the concept of "stimulated acoustic emission" from disrupted bubbles.

A model for reflectivity enhancement due to surface bound submicrometer particles.

Submicrometer particles filled with liquid perfluorocarbon have been shown to increase the ultrasound reflectivity of surfaces onto which they bind and, consequently, are seen as potential targeted contrast agents. The objective of this study is to explain the reflectivity enhancement as a result of the presence of randomly distributed particles on a surface. A model is presented where the diffraction-weighted scattering of all particles is summed over the exposed surface. Experiments were performed at frequencies ranging from 15 MHz to 60 MHz, with glass microbeads and perfluorohexane particles deposited on the surface of agar and Aqualene, a rubber closely matched to water, to confirm the validity of the model. Results showed that the model predicts the surface density and the frequency dependence of the reflectivity enhancement up to a density corresponding to twice the maximum packing of spheres on a surface (200% confluence fraction) for glass beads and a fifth (20% confluence fraction) for perfluorohexane particles. This suggests the possibility of predicting signal enhancement due to a bound contrast agent in simple geometries.

Investigating perfluorohexane particles with high-frequency ultrasound.

Submicron particles filled with liquid perfluorocarbon are currently being studied as a potential ultrasound-targeted contrast agent. The objective of this study was to evaluate the scattering properties of these particles. Sets of perfluorohexane-filled particles of different average sizes (300 nm to 1000 nm) were produced with a constant total volume fraction. The attenuation coefficient was measured in the 15- to 50-MHz frequency range and was found to increase smoothly with frequency and to be independent of the amplitude and bandwidth of the transmitted pulse. The values range from 0.31 to 0.64 dB/mm at 30 MHz for mean particle size ranging from 970 to 310 nm, respectively. The backscattering spectra of the particle solutions were measured and showed no sign of nonlinear scattering. The backscattering coefficient increased with the power 3.9 +/- 0.3 of the frequency. These results confirm that liquid perfluorocarbon droplets behave as linear Rayleigh scatterers.