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.
Nonsystemic (ie, focal) drug delivery is desired in many disease conditions, such as cancerous tumors and vascular disease, in which many drugs are too potent to be delivered systemically. Rapamycin is an antiproliferative drug that has been shown to decrease neointima formation, but it is consistent with complications at high doses,1,2 such as decreased immune function and increased risk of cancers.
Microbubbles, which are ultrasound contrast agents, have been shown to increase reagent delivery to cells/tissues in the presence of low-frequency ultrasound.3 By increasing the duration of the ultrasound pulse bursts, microbubbles can be pushed to vessel walls4 before rupture and drug delivery. We attempted to target rapamycin delivery, and thereby reduce neointimal proliferation, by focusing ultrasound to the site of arterial injury. We validated the hypothesis that focused ultrasound-mediated drug delivery from microbubbles can effectively reduce the drug dose necessary for therapeutic effect on neointima formation.
Materials and Methods
All animal experiments were approved by the animal care and use committee of the University of Virginia. Sprague-Dawley rats underwent carotid balloon injury by inserting a 2F balloon catheter 1.5 cm past the bifurcation of the right common carotid. The balloon was inflated with 0.04 mL of saline, pulled toward the bifurcation, deflated, and then withdrawn back into the carotid a total of 3 times. After injury, 2 ultrasound transducers were aligned with the injured vessel—1 for imaging and bursting of microbubbles and 1 for pushing of microbubbles (see Figure 1A). Radiation-force ultrasound was applied to push microbubbles via 135 kPa, 1.2 MHz continuous-wave ultrasound, and burst ultrasound was applied from a linear array (Sequoia 15L8, Siemens Medical Solutions, Malvern, PA) by emitting 5 MHz, 1.5 MPa pulses.
Rapamycin-loaded and fluorescent DiI–loaded microbubbles were prepared as described previously.5 DiI microbubbles were injected (109) in acute studies to visualize delivery along the vessel walls following no ultrasound (n=4), burst ultrasound (n=2), or dual-burst and radiation-force ultrasound applications (n=4). Microbubbles were infused through a left jugular vein catheter over 5 minutes. Ultrasound was applied concurrently with injection and then for another 3 minutes, for a total of 8 minutes. For chronic studies, rapamycin microbubbles (RMBs) at a high (109 RMBs total, n=7) or low (108 RMBs total, n=6) dose were infused through a left jugular vein catheter over 5 minutes. Both ultrasound modes were applied during infusion plus an additional 3 minutes. Control rats received high-dose (n=7) or low-dose (n=6) RMBs without ultrasound. Rats that received RMBs were euthanized 2 weeks after insonation. In each set of animals, left carotids served as contralateral uninjured, noninsonated controls. Arteries were excised and processed for sectioning and histology. Images were traced to find the area of the lumen, neointima, and media. Statistical analysis between groups was performed with a Student t test. For more details on methods, results, and the discussion, see the supplemental material available at http://atvb.ahajournals.org.
DiI delivery was observed in the carotids of injured rats where ultrasound was applied (Figure 1C). DiI delivery was enhanced 3.4-fold in arteries that received burst ultrasound (n=4, P<0.001) compared with arteries not treated with ultrasound (n=2). The addition of radiation-force ultrasound (n=2) enhanced delivery 1.9-fold compared with burst ultrasound alone, but not significantly (P=0.09).
The neointima to media ratio (NI/M) of rats that underwent only balloon injury was 1.54±0.25 (n=11) compared with 0.0 for uninjured controls (n=5). The NI/M of injured carotids treated with high-dose (109) RMBs alone was 1.10±0.16 (n=7) as compared with 1.01±0.33 (n=7) for carotids treated with 109 RMBs and ultrasound (Figure 2A). These results correspond to reductions in NI/M of 34.9% and 28.7% (P<0.001) with and without dual ultrasound application, respectively. Among injured arteries exposed to the low dose (108) of RMBs, NI/M was significantly reduced by 35.9% (n=6, P<0.001) only in those treated with ultrasound. NI/M of 1.54±0.24 (n=6) were observed in injured arteries treated with the low dose of RMBs (108) without ultrasound. Representative images of arteries from each treatment group are shown in Figure 2B to 2F. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining revealed no significant difference in apoptosis rates between arteries treated with or without ultrasound, or RMBs (Figure 2G–2I).
The results demonstrate that dual-frequency ultrasound combined with RMBs significantly reduces neointima formation with 1/10 of the dose necessary to achieve an equal therapeutic effect without ultrasound. Although the high dose of microbubbles may have been sufficient to reduce proliferation on its own, it corresponds to a rapamycin concentration of 7.3 μg/kg less than one third of the dose associated with side effects in humans (2 mg1). The low dose of RMBs is approximately the concentration suggested for contrast imaging with Optison or Definity contrast agents. Not only does this therapeutic tool have promise for reducing neointima formation using only a fraction of the drug dose, but it can also be applied to drug delivery models in which localized drug delivery is desired, particularly with drugs that are very toxic in high levels systemically. This is particularly advantageous in experimental animal models of vascular injury and disease whereby pharmacological intervention (eg, small molecules, antibody, plasmid DNA, RNA interference) to reduce pathology is often achieved by intraperitoneal or intravenous injection of a particular agent, and thus the therapeutic mechanism of action at the vessel wall is difficult to deduce. Currently, the technology is limited to preclinical small animal restenosis models, but translation to an intravascular catheter will allow for larger animal studies in swine, as we showed similarly for intravenous ultrasound-mediated microbubble delivery of DNA to swine coronary arteries in vivo.6
Sources of Funding
This study was supported by the National Institutes of Health, National Institute of Biomedical Imaging and Bioengineering Grants EB002185 and HL090700, and a University of Virginia Coulter Translational Research Grant.
We thank Bobi Thornhill for her surgical expertise.
- Received June 6, 2011.
- Accepted September 14, 2011.
- © 2011 American Heart Association, Inc.
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