Jonathan T. Sutton

ULTRASOUND-ENHANCED rt-PA THROMBOLYSIS IN AN EX VIVO PORCINE CAROTID ARTERY MODEL

Ultrasound is known to enhance recombinant tissue plasminogen activator (rt-PA) thrombolysis. In this study, occlusive porcine whole blood clots were placed in flowing plasma within living porcine carotid arteries. Ultrasonically induced stable cavitation was investigated as an adjuvant to rt-PA thrombolysis. Aged, retracted clots were exposed to plasma alone, plasma containing rt-PA (7.1 ± 3.8 μg/mL) or plasma with rt-PA and Definity® ultrasound contrast agent (0.79 ± 0.47 μL/mL) with and without 120-kHz continuous wave ultrasound at a peak-to-peak pressure amplitude of 0.44 MPa. An insonation scheme was formulated to promote and maximize stable cavitation activity by incorporating ultrasound quiescent periods that allowed for the inflow of Definity®-rich plasma. Cavitation was measured with a passive acoustic detector throughout thrombolytic treatment. Thrombolytic efficacy was measured by comparing clot mass before and after treatment. Average mass loss for clots exposed to rt-PA and Definity® without ultrasound (n = 7) was 34%, and with ultrasound (n = 6) was 83%, which constituted a significant difference (p < 0.0001). Without Definity® there was no thrombolytic enhancement by ultrasound exposure alone at this pressure amplitude (n = 5, p < 0.0001). In the low-oxygen environment of the ischemic artery, significant loss of endothelium occurred but no correlation was observed between arterial tissue damage and treatment type. Acoustic stable cavitation nucleated by an infusion of Definity® enhances rt-PA thrombolysis without apparent treatment-related damage in this ex vivo porcine carotid artery model.

Ultrasound-enhanced delivery of targeted echogenic liposomes in a novel ex vivo mouse aorta model

The goal of this study was to determine whether targeted, Rhodamine-labeled echogenic liposomes (Rh-ELIP) containing nanobubbles could be delivered to the arterial wall, and whether 1-MHz continuous wave ultrasound would enhance this delivery profile. Aortae excised from apolipoprotein-E-deficient (n=8) and wild-type (n=8) mice were mounted in a pulsatile flow system through which Rh-ELIP were delivered in a stream of bovine serum albumin. Half the aortae from each group were treated with 1-MHz continuous wave ultrasound at 0.49 MPa peak-to-peak pressure, and half underwent sham exposure. Ultrasound parameters were chosen to promote stable cavitation and avoid inertial cavitation. A broadband hydrophone was used to monitor cavitation activity. After treatment, aortic sections were prepared for histology and analyzed by an individual blinded to treatment conditions. Delivery of Rh-ELIP to the vascular endothelium was observed, and sub-endothelial penetration of Rh-ELIP was present in five of five ultrasound-treated aortae and was absent in those not exposed to ultrasound. However, the degree of penetration in the ultrasound-exposed aortae was variable. There was no evidence of ultrasound-mediated tissue damage in any specimen. Ultrasound-enhanced delivery within the arterial wall was demonstrated in this novel model, which allows quantitative evaluation of therapeutic delivery.

Ultrasound-mediated drug delivery for cardiovascular disease.

Introduction: Ultrasound (US) has been developed as both a valuable diagnostic tool and a potent promoter of beneficial tissue bioeffects for the treatment of cardiovascular disease. These effects can be mediated by mechanical oscillations of circulating microbubbles, or US contrast agents, which may also encapsulate and shield a therapeutic agent in the bloodstream. Oscillating microbubbles can create stresses directly on nearby tissue or induce fluid effects that effect drug penetration into vascular tissue, lyse thrombi or direct drugs to optimal locations for delivery. Areas covered: The present review summarizes investigations that have provided evidence for US-mediated drug delivery as a potent method to deliver therapeutics to diseased tissue for cardiovascular treatment. In particular, the focus will be on investigations of specific aspects relating to US-mediated drug delivery, such as delivery vehicles, drug transport routes, biochemical mechanisms and molecular targeting strategies. Expert opinion: These investigations have spurred continued research into alternative therapeutic applications, such as bioactive gas delivery and new US technologies. Successful implementation of US-mediated drug delivery has the potential to change the way many drugs are administered systemically, resulting in more effective and economical therapeutics, and less-invasive treatments.

Pulsed ultrasound enhances the delivery of nitric oxide from bubble liposomes to ex vivo porcine carotid tissue

Ultrasound-mediated drug delivery is a novel technique for enhancing the penetration of drugs into diseased tissue beds noninvasively. By encapsulating drugs into microsized and nanosized liposomes, the therapeutic can be shielded from degradation within the vasculature until delivery to a target site by ultrasound exposure. Traditional in vitro or ex vivo techniques to quantify this delivery profile include optical approaches, cell culture, and electrophysiology. Here, we demonstrate an approach to characterize the degree of nitric oxide (NO) delivery to porcine carotid tissue by direct measurement of ex vivo vascular tone. An ex vivo perfusion model was adapted to assess ultrasound-mediated delivery of NO. This potent vasodilator was coencapsulated with inert octafluoropropane gas to produce acoustically active bubble liposomes. Porcine carotid arteries were excised post mortem and mounted in a physiologic buffer solution. Vascular tone was assessed in real time by coupling the artery to an isometric force transducer. NO-loaded bubble liposomes were infused into the lumen of the artery, which was exposed to 1 MHz pulsed ultrasound at a peak-to-peak acoustic pressure amplitude of 0.34 MPa. Acoustic cavitation emissions were monitored passively. Changes in vascular tone were measured and compared with control and sham NO bubble liposome exposures. Our results demonstrate that ultrasound-triggered NO release from bubble liposomes induces potent vasorelaxation within porcine carotid arteries (maximal relaxation 31%±8%), which was significantly stronger than vasorelaxation due to NO release from bubble liposomes in the absence of ultrasound (maximal relaxation 7%±3%), and comparable with relaxation due to 12 μM sodium nitroprusside infusions (maximal relaxation 32%±3%). This approach is a valuable mechanistic tool for assessing the extent of drug release and delivery to the vasculature caused by ultrasound.

Delivery of bevacizumab to atheromatous porcine carotid tissue using echogenic liposomes

Abstract Ultrasound is both a valuable diagnostic tool and a promoter of beneficial tissue bioeffects for the treatment of cardiovascular disease. Vascular effects can be mediated by mechanical oscillations of circulating microbubbles that may also encapsulate and shield therapeutic agents in the bloodstream. Here, the effect of color-Doppler ultrasound exposure on bevacizumab-loaded liposome delivery into the vascular bed was assessed in atheromatous porcine carotids. Bevacizumab, an anti-angiogenic antibody to vascular endothelial growth factor (VEGF-A), was loaded into echogenic liposomes (BEV-ELIP) and confirmed to be immunoreactive. BEV-ELIP flowing within the lumen were exposed to color-Doppler ultrasound at three acoustic pressures for 3.5 min during treatment at physiologic temperature and fluid pressure. To confirm the presence of bubble activity, cavitation was detected within the lumen by a single-element passive cavitation detector. After treatment, the artery was fixed at physiologic pressure and subjected to immunohistochemical analysis to assess the penetration of bevacizumab within the carotid wall. The results suggest that other factors may more strongly influence the deposition of bevacizumab into carotid tissue than color-Doppler ultrasound and cavitation. In both sets of arteries, preferential accumulation of bevacizumab occurred in locations associated with atheroma progression and neointimal thickening: fibrous tissue, necrotic plaque and areas near macrophage infiltration. The delivery of bevacizumab to carotid vascular tissue correlated with the properties of the tissue bed, such as permeability, or affinity for growth-factor binding. Future investigations using this novel therapeutic strategy may focus on characterizing the spatial extent of delivery and bevacizumab colocalization with biochemical markers of atheroma.

Delivery of targeted echogenic liposomes in an ex vivo mouse aorta model.

Optimal ultrasound parameters to enhance delivery of therapeutic‐loaded echogenic immunoliposomes (ELIP) into the arterial wall are being developed for the treatment of atherosclerosis. The aim of this work was to determine whether anti‐ICAM‐targeted, rhodamine‐labeled ELIP (Rh‐ELIP) would adhere to and penetrate the vascular endothelium in atheromatous murine arterial segments with intravascular flow treated with 1‐MHz continuous wave ultrasound (CW US). A broadband focused hydrophone, confocally aligned with the artery and 1‐MHz transducer field was used as a passive cavitation detector (PCD). Arteries were insonified with 1‐MHz CW US (0.49 MPa peak‐to‐peak pressure), and the PCD was used to verify the duration of the resulting stable cavitation. Perivascular saline was collected and analyzed spectrofluorometrically for the presence of Rh‐ELIP. Arteries were prepared for histological analysis by a pathologist blinded to the exposure conditions. Arteries exposed to Rh‐labeled ELIP and 1‐MHz US exhibited ...

Ultrasound-mediated delivery of bioactive nanobubbles to vascular tissue

Bubble liposomes (BLs) are under development for ultrasound-triggered release of a potent vasodilator within the vasculature. Nano-sized vesicles facilitate this process by enclosing bubbles to enhance ultrasound image contrast, and deliver therapeutic agents. Assessment of drug delivery in living tissue allows for mechanistic pathways to be revealed. In this study, we used a novel ex vivo model to assess the vascular effects of ultrasound-mediated delivery of a bioactive gas-nitric oxide (NO)-from nanobubble liposomes. Porcine carotid arteries were excised post-mortem and mounted in physiologic buffer. Vascular tone was assessed in real time by coupling the artery to an isometric force transducer. NO-loaded BLs were infused into the lumen of the artery, which was exposed to 1-MHz pulsed ultrasound, while acoustic cavitation emissions were monitored. Changes in vascular tone were concurrently measured and compared to control and sham NO exposures. Our results demonstrate that ultrasound-triggered NO release from BLs induces potent vasorelaxation within porcine carotid arteries. This approach is a valuable mechanistic tool to assess the bioeffects that NO elicits within the vasculature upon release from BLs exposed to 1-MHz ultrasound.

Transcranial thermal ablation with a 230 kHz MRI-guided focused ultrasound system in a large animal mode

We evaluated the feasibility of thermal ablation in using a 230 kHz transcranial MRI-guided Focused Ultrasound (TcMRgFUS) system in three rhesus macaques. Thalamic targets were sonicated at 40–50s at 90–560 acoustic Watts. Focal heating sufficient to create an MRI-evident lesion was achieved in 4/6 targets where thermal dose exceeded 240 CEM43°C. Focal heating increased linearly as a function of the applied energy at a rate of 3.2±0.4°C/kJ (R²:0.81). Lesion sizes were consistent with 240 CEM43°C contours. The findings suggest that the lesions were consistent with thermal mechanisms. No evidence of cavitation-related petechiae were evident after sonication. Similar tests in macaques with a version of this system operating at 670 kHz (Hynynen et al., Eur. J. Radiol. 2006) measured skull-induced heating of 130°C/kJ/cm² of outer skull surface, more than twice of that measured here (63°C per kJ/cm²). While no or minimal focal heating was observed at 670 kHz, we reached ablation-level thermal dose values at 230...

Design, characterization, and performance of a dual aperture, focused ultrasound system for microbubble-mediated, non-thermal ablation in rat brain

Submegahertz ultrasound (US) with microbubbles (µB) can ablate brain tissue: a low amplitude (< 1 MPa) alternative to thermal ablation. Single element transducers at transcranial frequencies have broad axial profiles compared to the size of targets in small animals. Thus, we sought a system to ablate millimeter volumes in normal and tumorous rat brain non-thermally using US and µB. The system consisted of two transducers oriented at 120°, driven at different frequencies (F = 837 kHz, ΔF = 30 Hz) to reduce the depth-of-field by 78%. To monitor cavitation, a passive detector (650 kHz) was confocally aligned with the therapy field. Targets were registered stereotactically. µB injections (100, 200, and 400 µl/kg) with 5-minute sonications proceeded at acoustic pressures relative to the in vivo cavitation threshold (0.3–0.6 MPa) determined a priori. Following MRI and sacrifice (1 h, 4 days, 10 days), tissue was fixed and stained. At 1 h, small lesions (<2 mm) were selectively comprised of stenotic capillaries ...

Ultrasound‐enhanced thrombolysis in an ex vivo porcine carotid artery model.

Ultrasound enhances recombinant tissue plasminogen activator (rt‐PA) thrombolysis via a cavitational mechanism. An ex vivo porcine carotid arterial model incorporating physiologic flow and pressure was developed and stable cavitation promoted for thrombolysis. Aged, retracted whole blood clots were exposed to plasma alone, plasma containing rt‐PA (3.15 μg/ml), or plasma with rt‐PA and the Definity ultrasound contrast agent (0.31 μl/ml), with and without 120‐kHz continuous wave ultrasound at a peak‐to‐peak pressure amplitude of 0.44 MPa. An insonation scheme was formulated to promote and maximize stable cavitation activity by incorporating ultrasound quiescent periods that allowed for the inflow of Definity‐rich plasma. Cavitation was measured with a passive acoustic detector throughout thrombolytic treatment. Thrombolytic efficacy was measured by comparing clot mass before and after treatment. Average mass loss for clots exposed to rt‐PA and Definity without ultrasound was 34%, and with ultrasound was 83%...