Microbubble-mediated intravascular ultrasound imaging and drug delivery.

Intravascular ultrasound (IVUS) provides radiation-free, real-time imaging and assessment of atherosclerotic disease in terms of anatomical, functional, and molecular composition. The primary clinical applications of IVUS imaging include assessment of luminal plaque volume and real-time image guidance for stent placement. When paired with microbubble contrast agents, IVUS technology may be extended to provide nonlinear imaging, molecular imaging, and therapeutic delivery modes. In this review, we discuss the development of emerging imaging and therapeutic applications that are enabled by the combination of IVUS imaging technology and microbubble contrast agents.

Phase-shift perfluorocarbon agents enhance high intensity focused ultrasound thermal delivery with reduced near-field heating.

Ultrasound contrast agents are known to enhance high intensity focused ultrasound (HIFU) ablation, but these perfluorocarbon microbubbles are limited to the vasculature, have a short half-life in vivo, and may result in unintended heating away from the target site. Herein, a nano-sized (100-300 nm), dual perfluorocarbon (decafluorobutane/dodecafluoropentane) droplet that is stable, is sufficiently small to extravasate, and is convertible to micron-sized bubbles upon acoustic activation was investigated. Microbubbles and nanodroplets were incorporated into tissue-mimicking acrylamide-albumin phantoms. Microbubbles or nanodroplets at 0.1 × 10(6) per cm(3) resulted in mean lesion volumes of 80.4 ± 33.1 mm(3) and 52.8 ± 14.2 mm(3) (mean ± s.e.), respectively, after 20 s of continuous 1 MHz HIFU at a peak negative pressure of 4 MPa, compared to a lesion volume of 1.0 ± 0.8 mm(3) in agent-free control phantoms. Magnetic resonance thermometry mapping during HIFU confirmed undesired surface heating in phantoms containing microbubbles, whereas heating occurred at the acoustic focus of phantoms containing the nanodroplets. Maximal change in temperature at the target site was enhanced by 16.9% and 37.0% by microbubbles and nanodroplets, respectively. This perfluorocarbon nanodroplet has the potential to reduce the time to ablate tumors by one-third during focused ultrasound surgery while also safely enhancing thermal deposition at the target site.

Nanoparticle delivery enhancement with acoustically activated microbubbles.

The application of microbubbles and ultrasound to deliver nanoparticle carriers for drug and gene delivery is an area that has expanded greatly in recent years. Under ultrasound exposure, microbubbles can enhance nanoparticle delivery by increasing cellular and vascular permeability. In this review, the underlying mechanisms of enhanced nanoparticle delivery with ultrasound and microbubbles and various proposed delivery techniques are discussed. Additionally, types of nanoparticles currently being investigated in preclinical studies, as well as the general limitations and benefits of a microbubble- based approach to nanoparticle delivery, are reviewed.

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

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 10(5) to 10(8) 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 mm(3)) and the ablation lesions (1 to 135 mm(3)) within them. CONCLUSIONS: HIFU ablation was enhanced by this PSNE and could be achieved using intensities ≤650 W/cm(2). 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.

Intravascular ultrasound detection and delivery of molecularly targeted microbubbles for gene delivery.

We are investigating the combination of microbubble-based targeted drug delivery and intravascular ultrasound (IVUS) imaging as a potential therapy to reduce incidence of restenosis following stent placement in atherosclerotic coronary arteries. The goal of these studies was to determine whether IVUS could be used to detect targeted microbubbles and enhance drug/gene delivery through targeting. Quiescent vascular smooth muscle cells (SMCs) were stimulated with cytokine IL-1ß to induce the inflammatory cell surface marker vascular cell adhesion molecule 1 (VCAM-1). Molecular-targeted (VCAM-1 Ab or IgG control Ab), fluorescent-labeled microbubbles were conjugated with plasmid DNA expressing green fluorescent protein (GFP, pMax-GFP) and exposed to the inflamed SMCs under flow to measure adhesion compared with control microbubbles. Gene delivery was performed using a modified IVUS catheter to generate 1.5-MHz ultrasound at 200 kPa. Detection of adherent microbubbles to inflamed SMCs in culture and flow chambers was measured using an IVUS catheter and scanner. VCAM-1-targeted microbubbles enhanced adhesion to inflamed SMCs 100-fold over nontargeted microbubbles. Compared with noninflamed SMCs, VCAM-1-targeted microbubbles exhibited a 7.9-fold increase in adhesion to IL-1ß-treated cells. Targeted microbubbles resulted in a 5.5-fold increase in plasmid DNA transfection over nontargeted microbubbles in conjunction with a focused 2.54-cm (1-in) diameter 1-MHz transducer and also enhanced transfection by the modified IVUS transducer at 1.5 MHz. Targeted microbubbles (at a density of 3 × 104 microbubbles/mm²) increased IVUS image intensity 13.2 dB over non-microbubble-coated surfaces. Rupture of microbubbles from the modified IVUS transducer resulted in a 53% reduction in image intensity. Taken together, these results indicate that IVUS may be used to detect targeted microbubbles to inflamed vasculature and subsequently deliver a gene/drug locally.

Focused ultrasound-mediated drug delivery from microbubbles reduces drug dose necessary for therapeutic effect on neointima formation--brief report.

OBJECTIVE: We hypothesized that (1) neointima formation in a rat carotid balloon injury model could be reduced in vivo following targeted ultrasound delivery of rapamycin microbubbles (RMBs), and (2) the addition of dual-mode ultrasound decreases the total amount of drug needed to reduce neointima formation. METHODS AND RESULTS: Balloon injury was performed in rat carotids to induce neointima formation. High or low doses of RMBs were injected intravenously and ruptured at the site of injury with ultrasound. Compared with nontreated injured arteries, neointima formation was reduced by 0% and 35.9% with 10(8) RMBs and by 28.7% and 34.9% in arteries treated with 10(9) RMBs with and without ultrasound, respectively. CONCLUSIONS: Without ultrasound, 10-fold higher concentrations of RMBs were needed to reduce neointima formation by at least 28%, whereas 10(8) RMBs combined with ultrasound were sufficient to achieve the same therapeutic effect, demonstrating that this technology may have promise for localized potent drug therapy.

Localized ultrasound enhances delivery of rapamycin from microbubbles to prevent smooth muscle proliferation.

Microbubble contrast agents have been shown to enhance reagent delivery when activated by ultrasound. We hypothesized that ultrasound would enhance delivery of rapamycin, an antiproliferative agent, from the shell of microbubbles, thus reducing proliferation of vascular smooth muscle cells. Our objective was to determine optimal ultrasound parameters that maximized therapeutic efficacy, maintained cell adherence, and minimized the drug exposure time. In vitro assays determined that ultrasound (1 MHz, 0.5% duty cycle) is required to successfully deliver rapamycin from microbubbles and reduce proliferation. Co-injection of rapamycin with control microbubbles did not result in a reduction in proliferation. Successful reduction in proliferation (>50%) required pulses at least 10 cycles in length and at least 300 kPa peak negative pressure at which point 90% of cells remained adherent. The anti-proliferative effect was also localized within a 6mm wide zone by focusing the ultrasound beam.

Targeted gene transfection from microbubbles into vascular smooth muscle cells using focused, ultrasound-mediated delivery.

We investigated a method for gene delivery to vascular smooth muscle cells using ultrasound triggered delivery of plasmid DNA from electrostatically coupled cationic microbubbles. Microbubbles carrying reporter plasmid DNA were acoustically ruptured in the vicinity of smooth muscle cells in vitro under a range of acoustic pressures (0 to 950 kPa) and pulse durations (0 to 100 cycles). No effect on gene transfection or viability was observed from application of microbubbles, DNA or ultrasound alone. Microbubbles in combination with ultrasound (500-kPa, 1-MHz, 50-cycle bursts at a pulse repetition frequency [PRF] of 100 Hz) significantly reduced viability both with DNA (53 +/- 27%) and without (19 +/- 8%). Maximal gene transfection ( approximately 1% of cells) occurred using 50-cycle, 1-MHz pulses at 300 kPa, which resulted in 40% viability of cells. We demonstrated that we can locally deliver DNA to vascular smooth muscle cells in vitro using microbubble carriers and focused ultrasound.

Focused in vivo delivery of plasmid DNA to the porcine vascular wall via intravascular ultrasound destruction of microbubbles.

BACKGROUND: Safety concerns associated with drug-eluting stents have spurred interest in alternative vessel therapeutics following angioplasty. Microbubble contrast agents have been shown to increase gene transfection in vivo in the presence of ultrasound. OBJECTIVES/METHODS: The purpose of this study was to determine whether an intravascular ultrasound (IVUS) catheter could mediate plasmid DNA transfection from microbubble carriers to the porcine coronary artery wall following balloon angioplasty. RESULTS: In the presence of plasmid-coupled microbubbles in vitro only cells exposed to ultrasound from the modified IVUS catheter significantly expressed the transgene. A porcine left anterior descending coronary artery underwent balloon angioplasty followed by injection and insonation of microbubbles from the IVUS catheter at the site of angioplasty. After 3 days, an approximately 6.5-fold increase in transgene expression was observed in arteries that received microbubbles and IVUS compared to those that received microbubbles with no IVUS. CONCLUSIONS: The results of this study demonstrate for the first time that IVUS is required to enhance gene transfection from microbubble carriers to the vessel wall in vivo. This technology may be applied to both drug and gene therapy to reduce vessel restenosis.