Phase-Change Contrast Agents for Imaging and Therapy

Phase-change contrast agents (PCCAs) for ultrasound-based applications have resulted in novel ways of approaching diagnostic and therapeutic techniques beyond what is possible with microbubble contrast agents and liquid emulsions. When subjected to sufficient pressures delivered by an ultrasound transducer, stabilized droplets undergo a phase-transition to the gaseous state and a volumetric expansion occurs. This phenomenon, termed acoustic droplet vaporization, has been proposed as a means to address a number of in vivo applications at the microscale and nanoscale. In this review, the history of PCCAs, physical mechanisms involved, and proposed applications are discussed with a summary of studies demonstrated in vivo. Factors that influence the design of PCCAs are discussed, as well as the need for future studies to characterize potential bioeffects for administration in humans and optimization of ultrasound parameters

Intracellular delivery and ultrasonic activation of folate receptor-targeted phase-change contrast agents in breast cancer cells in vitro

Breast cancer is a diverse and complex disease that remains one of the leading causes of death among women. Novel, outside-of-the-box imaging and treatment methods are needed to supplement currently available technologies. In this study, we present evidence for the intracellular delivery and ultrasound-stimulated activation of folate receptor (FR)-targeted phase-change contrast agents (PCCAs) in MDA-MB-231 and MCF-7 breast cancer cells in vitro. PCCAs are lipid-coated, perfluorocarbon-filled particles formulated as nanoscale liquid droplets capable of vaporization into gaseous microbubbles for imaging or therapy. Cells were incubated with 1:1 decafluorobutane (DFB) / octafluoropropane (OFP) PCCAs for 1 hour, imaged via confocal microscopy, exposed to ultrasound (9 MHz, MI = 1.0 or 1.5), and imaged again after insonation. FR-targeted PCCAs were observed intracellularly in both cell lines, but uptake was significantly greater (p < 0.001) in MDA-MB-231 cells (93.0% internalization at MI = 1.0, 79.5% at MI = 1.5) than MCF-7 cells (42.4% internalization at MI = 1.0, 35.7% at MI = 1.5). Folate incorporation increased the frequency of intracellular PCCA detection 45-fold for MDA-MB-231 cells and 7-fold for MCF-7 cells, relative to untargeted PCCAs. Intracellularly activated PCCAs ranged from 500 nm to 6 microns (IQR = 800 nm – 1.5 microns) with a mean diameter of 1.15 ± 0.59 (SD) microns. The work presented herein demonstrates the feasibility of PCCA intracellular delivery and activation using breast cancer cells, illuminating a new platform toward intracellular imaging or therapeutic delivery with ultrasound

Phase-transition thresholds and vaporization phenomena for ultrasound phase-change nanoemulsions assessed via high-speed optical microscopy

Ultrasonically activated phase-change contrast agents (PCCAs) based on perfluorocarbon droplets have been proposed for a variety of therapeutic and diagnostic clinical applications. When generated at the nanoscale, droplets may be small enough to exit the vascular space and then be induced to vaporize with high spatial and temporal specificity by externally-applied ultrasound. The use of acoustical techniques for optimizing ultrasound parameters for given applications can be a significant challenge for nanoscale PCCAs due to the contributions of larger outlier droplets. Similarly, optical techniques can be a challenge due to the sub-micron size of nanodroplet agents and resolution limits of optical microscopy. In this study, an optical method for determining activation thresholds of nanoscale emulsions based on the in vitro distribution of bubbles resulting from vaporization of PCCAs after single, short (<10 cycles) ultrasound pulses is evaluated. Through ultra-high-speed microscopy it is shown that the bubbles produced early in the pulse from vaporized droplets are strongly affected by subsequent cycles of the vaporization pulse, and these effects increase with pulse length. Results show that decafluorobutane nanoemulsions with peak diameters on the order of 200 nm can be optimally vaporized with short pulses using pressures amenable to clinical diagnostic ultrasound machines

Analysis of Multi-Layer Immiscible Fluid Flow in a Microchannel

The development of microfluidics platforms in recent years has led to an increase in the number of applications involving the flow of multiple immiscible layers of viscous electrolyte fluids. In this study, numerical results as well as analytic equations for velocity and shear stress profiles were derived for N layers with known viscosities, assuming steady laminar flow in a microchannel driven by pressure and/or electro-static (Coulomb) forces. Numerical simulation results, using a commercial software package, match analytical results for fully-developed flow. Entrance flow effects with centered fluid-layer shrinking were studied as well. Specifically, cases with larger viscosities in the inner layers show a very good agreement with experimental correlations for the dimensionless entrance length as a function of inlet Reynolds number. However, significant deviations may occur for multilayer flows with smaller viscosities in the inner layers. A correlation was deduced for the two-layer electroosmotic flow and the pressure driven flow, both being more complex when compared with single-layer flows. The impact of using powerlaw fluids on resulting velocity profiles has also been explored and compared to Newtonian fluid flows. The present model readily allows for an exploration of the impact of design choices on velocity profiles, shear stress, and channel distribution in multilayer microchannel flows as a function of layered viscosity distribution and type of driving force

In vitro parameter optimization for spatial control of focused ultrasound ablation when using low boiling point phase-change nanoemulsions

Abstract Background Phase-shift nanoemulsions (PSNEs) provide cavitation sites when the perfluorocarbon (PFC) nanodroplets (ND) are vaporized to microbubbles by acoustic energy. Their presence lowers the power required to ablate tissue by high-intensity focused ultrasound (HIFU), potentially making it a safer option for a broader range of treatment sites. However, spatial control over the ablation region can be problematic when cavitation is used to enhance heating. This study explored relationships between vaporization, ablation, and the PSNE concentration in vitro to optimize the acoustic intensity and insonation time required for spatially controlled ablation enhancement using a PSNE that included a volatile PFC component. Methods HIFU (continuous wave at 1 MHz; insonation times of 5, 10, 15, and 20 s; cool-down times of 2, 4, and 6 s; peak negative pressures of 2, 3, and 4 MPa) was applied to albumin-acrylamide gels containing PFC agents (1:1 mix of volatile decafluorobutane and more stable dodecafluoropentane at 105 to 108 PFC ND per milliliter) or agent-free controls. Vaporization fields (microbubble clouds) were imaged by conventional ultrasound, and ablation lesions were measured directly by calipers. Controlled ablation was defined as the production of ‘cigar’-shaped lesions corresponding with the acoustic focal zone. This control was considered to be lost when ablation occurred in prefocal vaporization fields having a predominantly ‘tadpole’ or oblong shape. Results Changes in the vaporization field shape and location occurred on a continuum with increasing PSNE concentration and acoustic intensity. Working with the maximum concentration-intensity combinations resulting in controlled ablation demonstrated a dose-responsive relationship between insonation time and volumes of both the vaporization fields (approximately 20 to 240 mm3) and the ablation lesions (1 to 135 mm3) within them. Conclusions HIFU ablation was enhanced by this PSNE and could be achieved using intensities ≤650 W/cm2. Although the ablation lesions were located within much larger microbubble clouds, optimum insonation times and intensities could be selected to achieve an ablation lesion of desired size and location for a given PSNE concentration. This demonstration of controllable enhancement using a PSNE that contained a volatile PFC component is another step toward developing phase-shift nanotechnology as a potential clinical tool to improve HIFU

Methods of Generating Submicrometer Phase-Shift Perfluorocarbon Droplets for Applications in Medical Ultrasonography

Continued advances in the field of ultrasound and ultrasound contrast agents have created new approaches to imaging and medical intervention. Phase-shift perfluorocarbon droplets, which can be vaporized by ultrasound energy to transition from the liquid to the vapor state, are one of the most highly researched alternatives to clinical ultrasound contrast agents (i.e., microbubbles). In this paper, part of a special issue on methods in biomedical ultrasonics, we survey current techniques to prepare ultrasound-activated nanoscale phase-shift perfluorocarbon droplets, including sonication, extrusion, homogenization, microfluidics, and microbubble condensation. We provide example protocols and discuss advantages and limitations of each approach. Finally, we discuss best practice in characterization of this class of contrast agents with respect to size distribution and ultrasound activation

Formulation and Acoustic Studies of a New Phase-Shift Agent for Diagnostic and Therapeutic Ultrasound

Recent efforts in the area of acoustic droplet vaporization with the objective of designing extravascular ultrasound contrast agents has led to the development of stabilized, lipid-encapsulated nanodroplets of the highly volatile compound decafluorobutane (DFB). We have developed two methods of generating DFB droplets, the first of which involves condensing DFB gas (boiling point of −1.1° to −2° C) followed by extrusion with a lipid formulation in HEPES buffer. Acoustic droplet vaporization of micron-sized lipid-coated droplets at diagnostic ultrasound frequencies and mechanical indices were confirmed optically. In our second formulation methodology, we demonstrate the formulation of sub-micron sized lipid-coated nanodroplets based upon condensation of pre-formed microbubbles containing DFB. The droplets are routinely in the 200 – 300 nm range and yield microbubbles on the order of 1 – 5 microns once vaporized, consistent with ideal gas law expansion predictions. The simple and effective nature of this methodology allows for the development of a variety of different formulations that can be used for imaging, drug and gene delivery, and therapy. This study is the first to our knowledge to demonstrate both a method of generating ADV agents by microbubble condensation and formulation of primarily sub-micron droplets of decafluorobutane that remain stable at physiological temperatures. Finally, activation of DFB nanodroplets is demonstrated using pressures within the FDA guidelines for diagnostic imaging, which may minimize the potential for bioeffects in humans. This methodology offers a new means of developing extravascular contrast agents for diagnostic and therapeutic applications

Vaporization dynamics of volatile perfluorocarbon droplets: A theoretical model and in vitro validation

Perfluorocarbon (PFC) microdroplets, called phase-change contrast agents (PCCAs), are a promising tool in ultrasound imaging and therapy. Interest in PCCAs is motivated by the fact that they can be triggered to transition from the liquid state to the gas state by an externally applied acoustic pulse. This property opens up new approaches to applications in ultrasound medicine. Insight into the physics of vaporization of PFC droplets is vital for effective use of PCCAs and for anticipating bioeffects. PCCAs composed of volatile PFCs (with low boiling point) exhibit complex dynamic behavior: after vaporization by a short acoustic pulse, a PFC droplet turns into a vapor bubble which undergoes overexpansion and damped radial oscillation until settling to a final diameter. This behavior has not been well described theoretically so far. The purpose of our study is to develop an improved theoretical model that describes the vaporization dynamics of volatile PFC droplets and to validate this model by comparison with in vitro experimental data

Microfluidic Generation of Acoustically Active Nanodroplets

A microfluidic approach for the generation of perfluorocarbon nanodroplets as the primary emulsion with diameters as small as 300-400 nm is described. The system uses a pressure-controlled delivery of all reagents and increased viscosity in the continuous phase to drive the device into an advanced tip-streaming regime, which results in generation of droplets in the sub-micrometer range. Such nanodroplets may be appropriate for emerging biomedical applications

Precision Manufacture of Phase-Change Perfluorocarbon Droplets Using Microfluidics

Liquid perfluorocarbon droplets have been of interest in the medical acoustics community for use as acoustically activated particles for tissue occlusion, imaging, and therapeutics. To date, methods to produce liquid perfluorocarbon droplets typically result in a polydisperse size distribution. Since the threshold of acoustic activation is a function of diameter, there would be benefit from a monodisperse population to preserve uniformity in acoustic activation parameters. Through the use of a microfluidic device with flow focusing technology, the production of droplets of perfluoropentane with a uniform size distribution is demonstrated. Stability studies indicate that these droplets are stable in storage for at least two weeks. Acoustic studies illustrate the thresholds of vaporization as a function of droplet diameter, and a logarithmic relationship is observed between acoustic pressure and vaporization threshold within the size ranges studied. Droplets of uniform size have very little variability in acoustic vaporization threshold. Results indicate that microfluidic technology can enable greater manufacturing control of phase change perfluorocarbons for acoustic droplet vaporization applications