Linsey C. Phillips

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.

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.

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 108 RMBs and by 28.7% and 34.9% in arteries treated with 109 RMBs with and without ultrasound, respectively. Conclusion—Without ultrasound, 10-fold higher concentrations of RMBs were needed to reduce neointima formation by at least 28%, whereas 108 RMBs combined with ultrasound were sufficient to achieve the same therapeutic effect, demonstrating that this technology may have promise for localized potent drug therapy.

Intravascular ultrasound mediated delivery of DNA via microbubble carriers to an injured porcine artery in vivo

Millions of stents are implanted worldwide each year in patients with atherosclerotic arteries. Safety concerns relating to drug eluting stents have spurred interest in alternative vessel therapies. We hypothesized that a reporter gene could be delivered to a porcine coronary artery via intravascular ultrasound (IVUS) and plasmid-coupled microbubbles. In vitro delivery resulted in 0.17% of cells exhibiting successful transfection. An anesthetized porcine underwent balloon angioplasty on a coronary artery. Microbubble injection and rupture were controlled at the injury site using a modified intravascular ultrasound catheter. At 3 days post-insonation, gene expression was localized at injured vessel sites in (23.3% of luminal cells) with minimal expression in control arteries (3.6% of luminal cells). Our results demonstrate that IVUS has promise for localized intra-vascular gene therapy and may potentially offer a novel method for preventing restenosis.

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.

Dual perfluorocarbon nanodroplets enhance high intensity focused ultrasound heating and extend therapeutic window in vivo

Perfluorocarbon microbubbles are known to enhance high intensity focused ultrasound (HIFU) ablation by cavitation. However, they can result in superficial skin heating, minimizing their clinical translation. Perfluorocarbon nanodroplets activate only at the higher pressures present at the acoustic focus. We hypothesized that a mixed perfluorocarbon nanodroplet formulation would minimize surface heating while still enhancing ablation. Tissue-mimicking phantoms containing microbubbles or nanodroplets were sonicated (1 MHz, 15 W, 60 s) to assess heating and lesion formation in vitro. Microbubbles or nanodroplets were injected into rats (n = 3) and HIFU (1 MHz, 15 W, 15 s) was focused into each liver while under MRI guidance. Temperature throughout the liver was tracked by MR thermometry. In vitro, microbubbles caused excess surface heating during HIFU, whereas nanodroplets did not. In vivo, microbubbles typically circulate for less than 15 min. In comparison, the nanodroplets remained viable in circulation f...

9B-6 Inhibition of Smooth Muscle Proliferation by Ultrasound-Triggered Release of Rapamycin from Microbubbles

Vascular smooth muscle cell (SMC) proliferation plays a critical role in blood vessel narrowing associated with stent implantation, i.e. in-stent restenosis. Microbubble contrast agents can be modified to carry therapeutic reagents and modulate cell membrane permeability to drugs in the presence of ultrasound. A focused 1 MHz transducer was used to insonate cultured SMC cells. Nine minute application of 35% -6dB BW ultrasound pulses (PRF=1.0 kHz) at 1 MHz, 600 kPa, triggered delivery of rapamycin from a microbubble carrier to smooth muscle cells. Proliferation was decreased by 65% compared to treatment with control (Dil) bubbles. Two minute application of the same ultrasound pulses (except, PRF=5 kHz), within 6 minutes of presence of rapamycin microbubbles resulted in a decrease in proliferation of 72% compared to cells which received neither ultrasound nor microbubbles. Focusing of the drug delivery effect was achieved by narrowing the beam width of the applied ultrasound field which was controlled by axial distance of the transducer with respect to cells. For -6dB beam widths of 11.0, 3.0, and 2.5 mm, a >50% decrease in proliferation was induced in regions of 14, 8, and 6 mm respectively. Results suggest that ultrasound-triggered release of rapamycin from microbubble carriers may ultimately prove to be an effective way to localize treatment and prevention of SMC proliferation.

Ultrasound-microbubble-mediated drug delivery efficacy and cell viability depend on microbubble radius and ultrasound frequency

We have been investigating ultrasound-mediated drug delivery from microbubbles (MBs) as an alternative therapy to reduce hyper-proliferation of smooth muscle cells. We sought to determine if MB size affects drug delivery, cell viability, or cell adherence. Control, DiI, and drug-incorporated-rapamycin-microbubbles (R-MBs) were size sorted into “small” and “large” sub-populations with number-average mean diameters of 1.7µm and 3.7µm respectively. Drug dose was maintained by keeping the surface area between MB sub-populations constant. Vascular smooth muscle cells (SMCs) were exposed to size-sorted R-MBs and insonated at 1MHz, 300 kPa, with 50 cycle sinusoids at a PRF of 100Hz for 8s. Insonation with large MBs reduced the proliferation rate of cells by 66.0% vs. 56.1% (n=8, p=0.46) with small MBs. The LIVE/DEAD® Viability/Cytotoxicity assay revealed a significant reduction in live cells (61.% live vs. 77.6% live), n=4, p=0.41), and a greater, but not statistically significant, increase in the permeabilized cells after insonation (1MHz) with large MBs (15.6%) versus small MBs (11.1%). We also investigated the effect of ultrasound frequency (1, 2.25, 5, and 10MHz) on MB on rupture and viability following 20 ultrasound pulses (20 cycle-sinusoids) at a peak negative pressure 600 kPa. 1MHz insonation ruptured the most microbubbles of either size, permeabilized the most cells (50% & 43% for small and large MBs respectively) and killed (33% & 31%) the most cells. More small (d=1.8 µm) MBs were ruptured at every frequency (p<0.001, see Fig. 4). Permeabilization of cells was approximately inversely related to frequency of insonation. 1MHz ultrasound permeabilized the most cells, but also killed the most cells. The permeable/dead cell ratio was 64% for large MBs and 98% for small MBs indicating a better therapeutic ratio when using small MBs.1

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

We have investigated microbubble based targeted delivery and combined intravascular ultrasound (IVUS) imaging as 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. Fluorescently labeled microbubbles targeted to the inflammatory cell surface marker VCAM-1 were combined with cells under flow to measure adhesion compared to control bubbles. Gene delivery was performed using targeted bubble constructs and 1MHz ultrasound at 200 and 300 kPa - acoustic pressures which can be generated by a modified commercial IVUS catheter. Detection of adherent microbubbles to inflamed cells in culture and flow chambers was measured using a clinical IVUS catheter. VCAM-1 targeted microbubbles enhanced adhesion to inflamed cells up to 100 fold over non-targeted microbubbles. Compared to non-inflamed cells VCAM-1 targeted bubbles exhibited a 7.9 fold increase in adhesion to IL- 1beta treated cells. Targeted microbubbles resulted in a 5.5 fold increase in plasmid DNA transfection over non targeted bubbles in conjunction with a focused 1-inch diameter 1MHz transducer and 1.5 fold increase following insonation from a fabricated IVUS transducer at 1.5 MHz. At an equivalent density of 3×104 bubbles/mm2, IVUS image intensity increased 4.3 fold over that of non-bubble-coated surfaces. Rupture of microbubbles from the modified IVUS transducer resulted in a 53% reduction in image intensity. Taken together, these results indicate IVUS may be used to detect targeted microbubbles to inflamed vasculature and subsequently deliver a gene/drug locally.