Perfluorocarbon-exposed Sonicated Dextrose Albumin Microbubbles Research Articles

Early stage atherosclerosis regression using combined sonotherapy and mitoxantrone-mediated sonoporation therapy

Abstract Background In atherosclerosis, local inflammation and associated macrophage activity can lead to foam cell-rich plaque formation, which results to plaque rupture and thrombosis, making inflammation an important therapeutic target in cardiovascular disease. The early stage of atherosclerosis is characterized by the accumulation of low-density lipoprotein (LDL) droplets, leading to the creation of foam cells. Foam cells are generated by uncontrolled uptake of modified LDL, especially oxidized LDL (oxLDL). Purpose In this study, we developed an experimental focused ultrasound-based combined sonotherapy and sonoporation therapy system, and investigated its effectiveness on macrophage-derived foam cells reduction, wherein a high resolution diagnostic ultrasound is adjuncted with combination therapy system, with a goal of increased safety. Methods Briefly, New Zealand white rabbits underwent primary balloon dilatation injury at the right common carotid artery followed by a 1.5% cholesterol-rich diet injury for three weeks. Histopathology results showed the early stage atherosclerosis formation in all of the rabbits' arteries. Then common carotid arteries of the treatment group at the lesion region, treated using extracorporeal low-level pulsed-focused ultrasound (P=11 W, F=1.1 MHz, DF=50%) sonotherapy accompanied by mitoxantrone-loaded PESDA (Perfluorocarbon-Exposed Sonicated Dextrose Albumin) microbubbles (100 ml/kg, 2–5x105 bubbles/ml)-mediated sonoporation therapy. Foam cells density were evaluated in the treatment group compared with the other groups using B-mode ultrasonography and histopathology. Results Results from B-mode ultrasonography and histopathology showed a significant reduction in the mean value for immune cells and foam cells density within the early stage atherosclerotic lesion in the treatment group compared with the other groups (p<0.05). Conclusion Enhanced sonoporation effect of ultrasound, induced by collapsed PESDA microbubbles accompanied by anti-inflammatory effect of sonotherapy and antineoplastic and cytotoxic effect of mitoxantrone can cause to reduce the immune cells and foam cells density within the lesion. These finding provide the basis for developing low-level pulsed-focused ultrasound-based combined sonotherapy and sonoporation therapy for a successful clinical application in the treatment of the early stage atherosclerosis. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Mehrad Research Lab

Sonothrombolysis of embolic carotid artery, using pesda microbubbles- mediated B-mode ultrasound- guided extracorporeal pulsed low- level focused confocal dual frequency ultrasound accompanied by streptokinase administration

Background and Aims: Therapeutic ultrasound was found to selectively lyse thrombi with a wide margin of safety. Ultrasound effects sonothrombolysis by selective disruption of the fibrin matrix of the thrombus. In this study, we developed an experimental sonothrombolysis system, and investigated its effectiveness on carotid artery recanalization, accompanied by intravenous streptokinase and PESDA (perfluorocarbon exposed sonicated dextrose albumin) microbubbles administration, wherein diagnostic ultrasound imaging system is combined with the therapy system, with a goal of increased safety.

A method for the making and utility of gadolinium-labeled albumin microbubbles

Objective Perfluorocarbon-exposed sonicated dextrose albumin microbubbles (PESDA) binds scavenger receptors and can be noninvasively imaged. To enhance imaging, gadolinium (Gd)-labeled PESDA was developed and tested in a model of vascular inflammation by magnetic resonance imaging (MRI). Methods and Results Purified human serum albumin (HSA) (5%) was labeled with Gd via the covalent binding of diethylenetriaminepentacetic acid. Abdominal aortic tissues in Sprague–Dawley rats ( n=5 per group) were analyzed by 7-T MRI and scanning electron microscopy to evaluate PESDA binding. Labeling-purified 5% human albumin resulted in an average of 16.1 Gd atoms per albumin molecule as determined by atomic absorption. Forty-eight hours after balloon angioplasty, aortic tissue was enhanced with Gd-PESDA as compared to control tissue. 7-T MRI of explanted tissues was sensitive to the detection of retained PESDA. Enhancement of aortic tissue in vivo was present albeit to a lesser extent than explanted tissue from the same animals. Conclusions HSA was successfully labeled, and an albumin-based microbubble with Gd was synthesized. This contrast agent, Gd-PESDA, may serve as an additional agent for the MRI evaluation of innate inflammation and used to noninvasively image early vascular pathophysiologic processes. Condensed Abstract In this study, Gd-PESDA microbubbles and were synthesized and shown to detect the binding of these microbubbles using MRI in injured aortic tissue. The method for synthesizing Gd-PESDA is detailed, and the proposed utility of this new contrast agent is discussed.

Albumin-based Microbubbles Bind Up-regulated Scavenger Receptors following Vascular Injury

We have shown previously that perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles bind to injured vascular tissue and can be detected with ultrasound imaging techniques. Prior studies have shown that scavenger receptors (SRs) are regulators of innate and adaptive immune responses and are involved in the progression of vascular disease such as atherosclerosis. In this study, we sought to determine the molecular mechanism of PESDA binding to balloon-injured vasculature. RT-PCR analysis of angioplastied aortas demonstrated a significantly (p ≤ 0.01) increased expression of SRs. Binding to SRs was confirmed using SR-expressing CHO cells, and this binding was blocked by competitive inhibition with the SR-binding ligands oxidized LDL and malondialdehyde-acetaldehyde-modified LDL. Confocal imaging confirmed the co-localization of PESDA microbubbles to CD36, SRB-1, and Toll-like receptor 4, but not to monocytes/macrophages. This study demonstrates that PESDA binds to SRs and that this binding is in major part dependent upon the oxidized nature of PESDA microbubble shell proteins. The extent of SR mRNA expression was increased with injury and associated with microbubble retention as defined by scanning electron microscopy and immunohistochemistry. These findings clarify the mechanisms of how albumin-based microbubbles bind to injured and inflamed vasculature and further support the potential of this imaging technique to detect early vascular innate inflammatory pathophysiologic processes.

Targeted vascular delivery of antisense molecules using intravenous microbubbles

Perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles bind the antisense to the c-myc protooncogene (anti-c-myc) which prevents neointimal hyperplasia following vascular endothelial injury. The microbubbles also adhere to sites of damaged vascular endothelium and thus may be a method of systemically targeting delivery of anti-c-myc. Laser scanning microscopy was performed on the aorta of 10 mice (five which were complement depleted) that received intravenous FITC-PESDA following aortic endothelial injury. C-myc expression was quantified following selective intracoronary injury in nine pigs that received intravenous (IV) anti-c-myc bound to PESDA. Finally, neointimal formation was measured following intracoronary stent deployment in 30 pigs that received either IV anti-c-myc alone or the same dose bound to PESDA. Fluorescent microscopy confirmed selective PESDA microbubble adherence to aortic endothelium in all mice with aortic injury. This binding was nearly abolished when serum complement was depleted prior to injury. C-myc expression at the site of coronary endothelial injury was significantly lower in pigs treated with systemic anti-c-myc bound to PESDA. There was a 33% reduction in % stenosis and a 28% reduction in intimal area at 45 days post-stent deployment in pigs that received IV antisense plus PESDA. The stent margins also had reduced neointimal formation. Systemic administration of anti-c-myc bound to PESDA microbubbles may be a good method for preventing coronary neointimal formation within and around implanted stents.

Microbubble responses to a similar mechanical index with different real-time perfusion imaging techniques.

The purpose of this study was to determine differences in contrast enhancement and microbubble destruction rates with current commercially available low-mechanical index (MI) real-time perfusion imaging modalities. A tissue-mimicking phantom was developed that had vessels at 3 cm (near field) and 9 cm (far field) from a real-time transducer. Perfluorocarbon-exposed sonicated dextrose albumin microbubbles (PESDA) were injected proximal to a mixing chamber, and then passed through these vessels while the region was insonified with either pulses of alternating polarity with pulse inversion Doppler (PID) or pulses of alternating amplitude by power modulation (PM) at MIs of 0.1, 0.2 and 0.3. Effluent microbubble concentration, contrast intensity and the slope of digital contrast intensity vs. time were measured. Our results demonstrated that microbubble destruction already occurs with PID at an MI of 0.1. Contrast intensity seen with PID was less than with PM. Therefore, differences in contrast enhancement and microbubble destruction rates occur at a similar MI setting when using different real-time pulse sequence schemes.

Effectiveness of transcranial and transthoracic ultrasound and microbubbles in dissolving intravascular thrombi.

To examine the effectiveness of 1 -MHz and 40-kHz ultrasound with and without microbubbles in fragmenting thrombi in attenuated conditions. First, an vitro transcranial model was used to examine the ability of these frequencies to fragment thrombi in the presence or absence of perfluorocarbon-exposed sonicated dextrose albumin microbubbles. Second, an in vivo transthoracic model was used to test the effectiveness of these same frequencies with intravenous perfluorocarbon-exposed sonicated dextrose albumin in fragmenting left circumflex coronary thrombotic occlusions. In the in vitro model, both transcranial 1-MHz and 40-kHz ultrasonic frequencies were effective at fragmenting thrombi only in the presence of microbubbles. In the in vivo model, 1-MHz ultrasound with intravenous perfluorocarbon-exposed sonicated dextrose albumin angiographically recanalized only 4 of 14 occlusions but was consistently effective at improving myocardial blood flow to the risk area even in the absence of angiographic recanalization. Both 40-kHz and 1-MHz ultrasound with perfluorocarbon-exposed sonicated dextrose albumin improved regional wall-thickening and electrocardiographic abnormalities (P < .05 compared with control or ultrasound alone). Transcranial and transthoracic ultrasound in the presence of intravenous microbubbles can improve flow to ischemic regions and should be considered as a supplement to current pharmacologic therapy.

The clinical implications of no reflow demonstrated with intravenous perfluorocarbon containing microbubbles following restoration of Thrombolysis in Myocardial Infarction (TIMI) 3 flow in patients with acute myocardial infarction

Intravenous injections or infusions of perfluorocarbon-exposed sonicated dextrose albumin microbubbles were given 2.4 ± 1.6 days following acute myocardial infarction to 45 consecutive patients. Patients were divided into 3 groups: patients with Thrombolysis In Myocardial Infarction (TIMI) grade 3 angiographic flow but persistent myocardial contrast defects by echocardiography (no reflow), patients with TIMI 3 flow and myocardial contrast enhancement (reflow), and patients with TIMI grade 0 to 2 flow in the infarct vessel. Thirty-five patients had TIMI 3 flow at the time of contrast study. Of these, 25 had evidence of reflow with intravenous contrast, whereas 10 (29%) still had contrast defects. At followup, end-systolic volume index decreased significantly in patients who exhibited reflow (21 ± 8 ml/m 2 at baseline to 18 ± 8 ml/m 2 at follow-up; p = 0.04), whereas those with no reflow had a significant increase (26 ± 9 ml/m 2 at baseline to 32 ± 9 ml/m 2 at follow-up; p = 0.006). A persistent contrast defect in the infarct zone demonstrated with intravenous ultrasound contrast following restoration of TIMI grade 3 flow in the infarct vessel identified patients likely to have deterioration in both regional and global systolic function.

The effect of ultrasound frame rate on perfluorocarbon-exposed sonicated dextrose albumin microbubble size and concentration when insonifying at different flow rates, transducer frequencies, and acoustic outputs

The purpose of this article was to compare the effects of 1 and 30 Hz frame rates on perfluorocarbon-exposed sonicated dextrose albumin microbubble size and concentration in a flow cell containing either saline or blood at 37° C. Microbubble size and concentration of perfluorocarbon-exposed sonicated dextrose albumin were measured after insonation at different acoustic outputs, transducer frequencies, and flow rates with the use of the two different frame rates and compared with no ultrasound exposure. At 2.0 MHz insonation frequency, microbubble concentration was significantly reduced with the use of a 30 Hz frame rate and peak negative pressures of 1.1 megaPascal (mPa). This destruction did not occur when using a lower acoustic output, a 1 Hz frame rate, or when flow rate was increased to 100 cc/min. One-hertz frame rates at 2.0 MHz resulted in a significantly larger mean microbubble size than 30 Hz or no ultrasound in both saline and blood, which was in part due to selective destruction of smaller microbubbles. These findings indicate that 30 Hz frame rates destroy perfluorocarbon-exposed sonicated dextrose albumin microbubbles only at higher diagnostic acoustic outputs. A 1 Hz frame rate prevents this destruction, especially destruction of larger (>5.0 μm) microbubbles. (J Am Soc Echocardiogr 1997;10:593-601.)

Thrombolytic enhancement with perfluorocarbon-exposed sonicated dextrose albumin microbubbles

Whereas low-intensity, high-frequency ultrasound (US) alone appears to cause minimal thrombolysis, US combined with air-filled microbubbles does increase the amount of urokinase (UK)-mediated clot lysis (CL). Because this phenomenon may be mediated by cavitation-induced streaming, we hypothesized that perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles, which are more stable than air-filled microbubbles, may also enhance US-induced thrombolysis. We measured the percentage CL of equally sized thrombi (1.0 ± 0.1 mg) made from freshly drawn blood incubated for 2 hours and then exposed to 20 kHz US (0.846 MPa peak negative pressure). The thrombi were bathed in 4 ml of saline solution, UK alone (20,000 U), PESDA alone, or a combination of PESDA with UK. The percentage CL achieved with PESDA and therapeutic US was also compared with the percentage CL achieved with room air—filled sonicated dextrose albumin (RASDA) microbubbles. When compared with US alone (24% ± 13% CL) or UK alone (17% ± 3% CL), PESDA plus US produced significantly better CL (43% ± 17%; p < 0.05). PESDA combined with US also produced significantly greater CL than RASDA combined with US (28% ± 9%; p < 0.05). The optimal CL was achieved with a combination of PESDA with UK with US (60% ± 14% CL). We conclude that PESDA microbubbles alone may be capable of inducing thrombolysis when insonified with a low-frequency transducer.

Increased ultrasound contrast and decreased microbubble destruction rates with triggered ultrasound imaging

Although transient myocardial contrast imaging has been able to produce visually evident myocardial contrast in animals and humans with very low intravenous doses of perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles, the mechanism for improved contrast remains unclear. In this study we devised a flow chamber that measured the concentration of PESDA microbubbles that remained after exposure to diagnostic ultrasound pressures of 0.9 to 1.9 MPa and frequencies of 2.0, 2.5, and 3.5 MHz (first and second harmonic for 2.0 MHz), which were delivered at either 30 Hz (frames per second), 0.5 to 1.0 Hz, or without any ultrasound transmission. The videointensity within the flow chamber was also measured at 0, 20, 40, and 100 ml/min flow rates with the flow loop closed (i.e., constant microbubble concentration) with both triggered (0.5 to 1.0 Hz) and conventional (30 Hz) frame rates. The effluent microbubble concentration was significantly larger when PESDA was exposed to either no ultrasound or 0.5 to 1.0 Hz ultrasound. Furthermore, the videointensity of a constant number of microbubbles was significantly greater with 0.5 to 1.0 Hz (triggered) compared with 30 Hz (conventional) frame rates at each transmit frequency. The greatest difference was noted with the lower 2.0 MHz transmit frequency and the 20 ml/min flow rate, especially when a second harmonic receiving frequency was used. We conclude that the mechanism for improved contrast with triggered ultrasound imaging is because of both less microbubble destruction and increased videointensity from a constant number of microbubbles. Lower transducer frequencies and lower flow rates result in the greatest improvement in videointensity with triggered ultrasound transmission.

Interaction of diagnostic ultrasound with synthetic oligonucleotide-labeled perfluorocarbon-exposed sonicated dextrose albumin microbubbles.

The purpose of this study was to determine, first, whether the albumin on perfluorocarbon-exposed sonicated dextrose albumin microbubbles has retained its known ability to bind antisense oligonucleotides (DNA utilized to inhibit viral replication and intimal hyperplasia). The binding kinetics of synthetic antisense oligonucleotides to perfluorocarbon-exposed sonicated dextrose albumin microbubbles as well as to room air-containing sonicated dextrose albumin microbubbles were compared. Second, the effect of diagnostic ultrasound on this binding is unknown. The ability of diagnostic ultrasound to release synthetic antisense oligonucleotides from perfluorocarbon-exposed sonicated dextrose albumin microbubbles also was tested in vitro and in vivo in three dogs. Synthetic antisense oligonucleotides exhibited binding to perfluorocarbon-exposed sonicated dextrose albumin microbubbles much like that of native albumin but did not bind uniformly with room air-containing sonicated dextrose albumin microbubbles. Diagnostic ultrasound resulted in significant partitioning of synthetic antisense oligonucleotides into non-bubble containing regions after insonation in vitro as well as deposition of greater amounts of synthetic antisense oligonucleotides in the insonated kidney after intravenous injections of synthetic anti-sense oligonucleotide-labeled perfluorocarbon-exposed sonicated dextrose albumin microbubbles. We conclude that perfluorocarbon-exposed sonicated dextrose albumin microbubbles, unlike room air-containing sonicated dextrose albumin microbubbles, have bioactive albumin on their surface that can bind synthetic antisense oligonucleotides and then release them in the presence of diagnostic ultrasound.

Significant enhancement of therapeutic ultrasound-induced thrombolysic using perfluorocarbon-exposed sonicated dextrose albumin microbubbles

Significant enhancement of therapeutic ultrasound-induced thrombolysic using perfluorocarbon-exposed sonicated dextrose albumin microbubbles