In-vitro sonothrombolysis using thick-shelled polymer microbubbles - a comparison with thin-shelled microbubbles.

BACKGROUND: Vascular thrombosis can be treated pharmacologically, however, serious shortcomings such as bleeding may occur. Several studies suggest that sonothrombolysis can induce lysis of the clots using ultrasound. Moreover, intravenously injected thin-shelled microbubbles (MBs) combined with ultrasound can further improve clot lysis. Thick-shelled MBs have been used for drug delivery, targeting and multimodal imaging. However, their capability to enhance sonothrombolysis is unknown. In this study, using an in-vitro set-up, the enhancement of clot lysis using ultrasound and thick-shelled MBs was investigated. Thin-shelled MBs was used for comparison. METHOD: The main components in the in-vitro set-up was a vessel mimicking phantom, a pressure mearing system and programmable ultrasound machine. Blood clots were injected and entrapped on a pore mesh in the vessel phantom. Four different protocols for ultrasound transmission and MB exposure (7 blood clots/protocol) were considered together with a control test were no MBs and ultrasound were used. For each protocol, ultrasound exposure of 20 min was used. The upstream pressure of the partially occluded mesh was continuously measured to assess clot burden. At the end of each protocol blood clots were removed from the phantom and the clot mass loss was computed. RESULTS: For the thick-shelled MBs no difference in clot mass loss compared with the control tests was found. A 10% increase in the clot mass loss compared with the control tests was found when using thin-shelled MBs and low pressure/long pulses ultrasound exposure. Similarly, in terms of upstream pressure over exposure time, no differences were found when using the thick-shelled MBs, whereas thin-shelled MBs showed a 15% decrease achieved within the first 4 min of ultrasound exposure. CONCLUSION: No increase in clot lysis was achieved using thick-shelled MBs as demonstrated by no significant change in clot mass or upstream pressure. Although thick-shelled MBs are promising for targeting and drug delivery, they do not enhance clot lysis when considering the ultrasound sequences used in this study. On the other hand, ultrasound in combination with thin-shelled MBs can facilitate thrombolysis when applying long ultrasound pulses with low pressure.

Analysis of polyvinyl alcohol microbubbles in human blood plasma using capillary electrophoresis.

Recently, a new type of ultrasound contrast agent that consists of air-filled microbubbles stabilized with a shell of polyvinyl alcohol was developed. When superparamagnetic nanoparticles of iron oxide are incorporated in the polymer shell, a multimodal contrast agent can be obtained. The biodistribution and elimination pathways of the polyvinyl alcohol microbubbles are essential to investigate, which is limited with today's techniques. The aim of the present study was, therefore, to develop a method for qualitative and quantitative analysis of microbubbles in biological samples using capillary electrophoresis with ultraviolet detection. The analysis parameters were optimized to a wavelength at 260 nm and pH of the background electrolyte ranging between 11.9 and 12. Studies with high-intensity ultrasonication degraded microbubbles in water showed that degraded products and intact microbubbles could be distinguished, thus it was possible to quantify the intact microbubbles solely. Analysis of human blood plasma spiked with either plain microbubbles or microbubbles with nanoparticles demonstrated that it is possible to separate them from biological components like proteins in these kinds of samples.

The potential clinical value of contrast-enhanced echocardiography beyond current recommendations.

BACKGROUND: Contrast agents are used in resting echocardiography to opacify the left ventricular (LV) cavity and to improve LV endocardial border delineation in patients with suboptimal image quality. If a wider use of contrast-enhanced echocardiography would be adopted instead of the current selective approach, diagnoses such as myocardial ischemia and LV structural abnormalities could potentially be detected earlier. The aim was therefore to retrospectively investigate if contrast-enhanced echocardiography beyond the current recommendations for contrast agent usage affects assessment of wall motion abnormalities, ejection fraction (EF) and detection of LV structural abnormalities. A secondary aim was to evaluate the user dependency during image analysis. METHODS: Experienced readers (n = 4) evaluated wall motion score index (WMSI) and measured EF on greyscale and contrast-enhanced images from 192 patients without indications for contrast-enhanced echocardiography. Additionally, screening for LV structural abnormalities was performed. Repeated measurements were performed in 20 patients by the experienced as well as by inexperienced (n = 2) readers. RESULTS: Contrast analysis resulted in significantly higher WMSI compared to greyscale analysis (p < 0.003). Of the 83 patients, classified as healthy by greyscale analysis, 55% were re-classified with motion abnormalities by contrast analysis. No significant difference in EF classification (≥55%, 45-54%, 30-44%, < 30%) was observed. LV structural abnormalities, such as increased trabeculation (n = 21), apical aneurysm (n = 4), hypertrophy (n = 1) and thrombus (n = 1) were detected during contrast analysis. Intra- and interobserver variability for experienced readers as well as the variability between inexperienced and experienced readers decreased for WMSI and EF after contrast analysis. CONCLUSIONS: Contrast-enhanced echocardiography beyond current recommendations for contrast agent usage increased the number of detected wall motion and LV structural abnormalities. Moreover, contrast-enhanced echocardiography increased reproducibility for assessment of WMSI and EF.

Fluorescence labeled microbubbles for multimodal imaging.

Air-filled polyvinyl alcohol microbubbles (PVA-MBs) were recently introduced as a contrast agent for ultrasound imaging. In the present study, we explore the possibility of extending their application in multimodal imaging by labeling them with a near infrared (NIR) fluorophore, VivoTag-680. PVA-MBs were injected intravenously into FVB/N female mice and their dynamic biodistribution over 24 h was determined by 3D-fluorescence imaging co-registered with 3D-µCT imaging, to verify the anatomic location. To further confirm the biodistribution results from in vivo imaging, organs were removed and examined histologically using bright field and fluorescence microscopy. Fluorescence imaging detected PVA-MB accumulation in the lungs within the first 30 min post-injection. Redistribution to a low extent was observed in liver and kidneys at 4 h, and to a high extent mainly in the liver and spleen at 24 h. Histology confirmed PVA-MB localization in lung capillaries and macrophages. In the liver, they were associated with Kupffer cells; in the spleen, they were located mostly within the marginal-zone. Occasional MBs were observed in the kidney glomeruli and interstitium. The potential application of PVA-MBs as a contrast agent was also studied using ultrasound (US) imaging in subcutaneous and orthotopic pancreatic cancer mouse models, to visualize blood flow within the tumor mass. In conclusion, this study showed that PVA-MBs are useful as a contrast agent for multimodal imaging.

Endocardial border delineation capability of a novel multimodal polymer-shelled contrast agent.

BACKGROUND: A novel polymer-shelled contrast agent (CA) with multimodal and target-specific potential was developed recently. To determine its ultrasonic diagnostic features, we evaluated the endocardial border delineation as visualized in a porcine model and the concomitant effect on physiological variables. METHODS: Three doses of the novel polymer-shelled CA (1.5 ml, 3 ml, and 5 ml [5 × 10(8) microbubbles (MBs)/ml]) and the commercially available CA SonoVue (1.5 ml [2-5 × 10(8) MBs/ml]) were used. Visual evaluations of ultrasound images of the left ventricle were independently performed by three observers who graded each segment in a 6-segment model as either 0 = not visible, 1 = weakly visible, or 2 = visible. Moreover, the duration of clinically useful contrast enhancement and the left ventricular opacification were determined. During anesthesia, oxygen saturation, heart rate, and arterial pressure were sampled every minute and the effect of injection of CA on these physiological variables was evaluated. RESULTS: The highest dose of the polymer-shelled CA gave results comparable to SonoVue. Thus, no significant difference in the overall segment score distribution (2-47-95 vs. 1-39-104), time for clinically sufficient contrast enhancement (20-40 s for both) and left ventricular overall opacification was found. In contrast, when comparing the endocardial border delineation capacity for different regions SonoVue showed significantly higher segment scores for base and mid, except for the mid region when injecting 1.5 ml of the polymer-shelled CA. Neither high nor low doses of the polymer-shelled CA significantly affected the investigated physiological variables. CONCLUSIONS: This study demonstrated that the novel polymer-shelled CA can be used in contrast-enhanced diagnostic imaging without influence on major physiological variables.