Microbubble-based Ultrasound Contrast Agents Research Articles

In vitro comparison of subharmonic-aided pressure estimation sensitivity among microfluidic monodisperse microbubbles, sonazoid, and definity

The subharmonic response of microbubble-based ultrasound contrast agents (UCAs) typically exhibits an inverse linear relationship with the surrounding ambient pressure. Although SHAPE has been clinically successful, the sensitivity of SHAPE at lower pressures is suboptimal. While previous studies optimized SHAPE using commercially available UCAs without considering their size distribution, in this study, two SHAPE-specific monodisperse microbubble (MMB) UCAs with different bubble diameters were compared with commercial polydisperse UCAs as well as their buoyancy-separated subpopulations. UCAs were imaged in a hydrostatic tank containing 350 ml of deionized water. The pressure within the tank was increased from 0–100 mmHg. Using a modified Logiq E10 scanner (GE HealthCare) with acoustic power optimization, SHAPE data was acquired using subharmonic imaging mode with a C1-6 curvilinear probe at 2.5 MHz (receiving at 1.25 MHz). After identification of the optimal acoustic output power, MMB with a mean diameter 1.66 mm demonstrated a mean SHAPE sensitivity of −0.207 dB/mmHg (r2 = 0.94). The second MMB with a mean diameter 3.45 mm demonstrated a mean SHAPE sensitivity of −0.300 dB/mmHg (r2 = 0.92). In contrast, this was 2.5–4 times more sensitive than the two polydisperse UCAs (slope range of −0.081 to −0.074 dB/mmHg) and 1.45–5 times more sensitive than the buoyancy-separated UCAs (ranging −0.061 to −0.142 dB/mmHg). These results suggest that SHAPE-optimized MMB are more sensitive to measuring changes in pressure.

Ultrasound contrast agents: microbubbles made simple for the pediatric radiologist.

The ability to provide prompt, real-time, easily accessible and radiation-free diagnostic assessments makes ultrasound (US) one of the most versatile imaging modalities. The introduction and development of stable microbubble-based ultrasound contrast agents (UCAs) in the early 1990s improved visualization of complex vascular structures, overcoming some of the limitations of B-mode and Doppler imaging. UCAs have been used extensively in the adult population to visualize vasculature and to evaluate perfusion and blood flow dynamics in organs and lesions. Since the first observations that air bubbles within a liquid can generate a strong echogenic effect, to the early makeshift approaches with agitated saline, and later to the development of industrially produced and federally approved UCAs, these agents have evolved to become both clinically and commercially viable. Perhaps the most exciting potential of UCAs is being uncovered by current research that explores the use of these agents for molecular imaging and therapeutic applications. As contrast-enhanced ultrasound (CEUS) becomes more widely available, it is important for pediatric radiologists to understand the physics of the interaction between the US signal and the microbubbles in order to properly utilize them for the highest level of diagnostic imaging and interventions. In this article we introduce the composition of UCAs and the physics of their behavior in US, and we offer a brief history of their development over the last decades.

Size determination of microbubbles in optical microscopy: a best-case scenario

Microbubble-based ultrasound contrast agents are used in clinical settings to enhance backscattered ultrasound signals from blood during perfusion and blood flow measurements. The dynamics of microbubbles contained in ultrasound contrast agents are typically studied with a high-speed camera attached to a microscope. Such microbubbles, with resting diameters between 1 µm and 7 µm, are considered in optical focus if the bubble centers are in the focal plane of the objective lens. Nonetheless, when a three-dimensional object, a stack of infinitely thin two-dimensional layers, is imaged through a microscope, the image formed onto the charge coupled device element consists of contributions from all layers. If a bubble is larger than the depth of focus, the part of the bubble above the focal plane influences the image formation and therefore the bubble size measured. If a bubble is of a size in the order of the wavelengths of the light used, the system resolution and the segmentation method influence the bubble size measured. In this study, the projections of three dimensional microbubbles (hollow spheres) were computed with an ideal, weighted three-dimensional point spread function to find out under which circumstances the optical image formation leads to a significant deviation in measurement of the actual size. The artificial images were subjected to segmentation techniques for objectively comparing original microbubble sizes with measured microbubble sizes. Results showed that a systematic error was observed for objects in focus with radius ≤ 1.65µm. Also it was concluded that even though a three-dimensional object is in focus, there is discrepancy of up to 0.66% in size measurement. In addition, size measurement of an object for the same shift above and below focus could differ by up to 3.6%. Moreover, defocusing above 25% severely deviates size measurements while defocusing up to 90% could result in mean percentage error of up to 67.96.

Nanodroplet-Vaporization-Assisted Sonoporation for Highly Effective Delivery of Photothermal Treatment.

Sonoporation refers to the use of ultrasound and acoustic cavitation to temporarily enhance the permeability of cellular membranes so as to enhance the delivery efficiency of therapeutic agents into cells. Microbubble-based ultrasound contrast agents are often used to facilitate these cavitation effects. This study used nanodroplets to significantly enhance the effectiveness of sonoporation relative to using conventional microbubbles. Significant enhancements were demonstrated both in vitro and in vivo by using gold nanorods encapsulated in nanodroplets for implementing plasmonic photothermal therapy. Combined excitation by ultrasound and laser radiation is used to trigger the gold nanodroplets to induce a liquid-to-gas phase change, which induces cavitation effects that are three-to-fivefold stronger than when using conventional microbubbles. Enhanced cavitation also leads to significant enhancement of the sonoporation effects. Our in vivo results show that nanodroplet-vaporization-assisted sonoporation can increase the treatment temperature by more than 10 °C above that achieved by microbubble-based sonoporation.

Nonthermal ablation in the rat brain using focused ultrasound and an ultrasound contrast agent: long-term effects.

OBJECTIVE Thermal ablation with transcranial MRI-guided focused ultrasound (FUS) is currently under investigation as a less invasive alternative to radiosurgery and resection. A major limitation of the method is that its use is currently restricted to centrally located brain targets. The combination of FUS and a microbubble-based ultrasound contrast agent greatly reduces the ultrasound exposure level needed to ablate brain tissue and could be an effective means to increase the "treatment envelope" for FUS in the brain. This method, however, ablates tissue through a different mechanism: destruction of the microvasculature. It is not known whether nonthermal FUS ablation in substantial volumes of tissue can safely be performed without unexpected effects. The authors investigated this question by ablating volumes in the brains of normal rats. METHODS Overlapping sonications were performed in rats (n = 15) to ablate a volume in 1 hemisphere per animal. The sonications (10-msec bursts at 1 Hz for 60 seconds; peak negative pressure 0.8 MPa) were combined with the ultrasound contrast agent Optison (100 µl/kg). The rats were followed with MRI for 4-9 weeks after FUS, and the brains were examined with histological methods. RESULTS Two weeks after sonication and later, the lesions appeared as cyst-like areas in T2-weighted MR images that were stable over time. Histological examination demonstrated well-defined lesions consisting of a cyst-like cavity that remained lined by astrocytic tissue. Some white matter structures within the sonicated area were partially intact. CONCLUSIONS The results of this study indicate that nonthermal FUS ablation can be used to safely ablate tissue volumes in the brain without unexpected delayed effects. The findings are encouraging for the use of this ablation method in the brain.

Differential Diagnosis of Gallbladder Wall Thickening: The Usefulness of Contrast-Enhanced Ultrasound

The purpose of this study was to evaluate the usefulness of contrast-enhanced ultrasound (CEUS) in the differential diagnosis of gallbladder wall (GBW) thickening and determine the predictors of malignant GBW thickening. One hundred fifty-nine patients with GBW thickening, including 76 men and 83 women, from eight institutions were enrolled. CEUS was performed after injection of a sulfur hexafluoride microbubble-based ultrasound contrast agent. Multiple logistic regression analysis was used to reveal independent predictors associated with malignant GBW thickening. The final diagnoses were 48 gallbladder carcinomas and 111 benign gallbladder diseases. Maximal thicknesses of the GBW in malignant and benign GBW thickening were 17.3 ± 5.2 (6–30) mm and 8.6 ± 5.1 (4–26) mm respectively (p < 0.001). CEUS revealed significant differences in intra-lesional vessels, enhancement homogeneity, time to hypo-enhancement, inner layer discontinuity, outer layer discontinuity and adjacent liver involvement (all p-values < 0.05) between malignant and benign GBW thickening. Patient age > 46.5 y, focal GBW thickening, inner layer discontinuity and outer layer discontinuity were found to be associated with malignancy by multiple logistic regression analysis (all p-values < 0.05). Receiver operating characteristic curve analysis revealed Az values for patient age, focal GBW thickening, inner wall discontinuity and outer wall discontinuity of 0.709 (95% confidence interval [CI]: 0.627–0.790), 0.714 (95% CI: 0.630–0.798), 0.860 (95% CI: 0.791–0.928) and 0.858 (95% CI: 0.783–0.933), respectively. CEUS is useful in the differential diagnosis between malignant and benign GBW thickening. Focal GBW thickening, inner wall discontinuity and outer wall discontinuity observed on CEUS are diagnostic clues for malignant GBW thickening.

Nonthermal ablation with microbubble-enhanced focused ultrasound close to the optic tract without affecting nerve function

Tumors at the skull base are challenging for both resection and radiosurgery given the presence of critical adjacent structures, such as cranial nerves, blood vessels, and brainstem. Magnetic resonance imaging-guided thermal ablation via laser or other methods has been evaluated as a minimally invasive alternative to these techniques in the brain. Focused ultrasound (FUS) offers a noninvasive method of thermal ablation; however, skull heating limits currently available technology to ablation at regions distant from the skull bone. Here, the authors evaluated a method that circumvents this problem by combining the FUS exposures with injected microbubble-based ultrasound contrast agent. These microbubbles concentrate the ultrasound-induced effects on the vasculature, enabling an ablation method that does not cause significant heating of the brain or skull. In 29 rats, a 525-kHz FUS transducer was used to ablate tissue structures at the skull base that were centered on or adjacent to the optic tract or chiasm. Low-intensity, low-duty-cycle ultrasound exposures (sonications) were applied for 5 minutes after intravenous injection of an ultrasound contrast agent (Definity, Lantheus Medical Imaging Inc.). Using histological analysis and visual evoked potential (VEP) measurements, the authors determined whether structural or functional damage was induced in the optic tract or chiasm. Overall, while the sonications produced a well-defined lesion in the gray matter targets, the adjacent tract and chiasm had comparatively little or no damage. No significant changes (p > 0.05) were found in the magnitude or latency of the VEP recordings, either immediately after sonication or at later times up to 4 weeks after sonication, and no delayed effects were evident in the histological features of the optic nerve and retina. This technique, which selectively targets the intravascular microbubbles, appears to be a promising method of noninvasively producing sharply demarcated lesions in deep brain structures while preserving function in adjacent nerves. Because of low vascularity--and thus a low microbubble concentration--some large white matter tracts appear to have some natural resistance to this type of ablation compared with gray matter. While future work is needed to develop methods of monitoring the procedure and establishing its safety at deep brain targets, the technique does appear to be a potential solution that allows FUS ablation of deep brain targets while sparing adjacent nerve structures.

Determination of the Interfacial Rheological Properties of a Poly(DL-lactic acid)-Encapsulated Contrast Agent Using In Vitro Attenuation and Scattering

The stabilizing encapsulation of a microbubble-based ultrasound contrast agent (UCA) critically affects its acoustic properties. Polymers, which behave differently from materials commonly used (i.e., lipids or proteins) for monolayer encapsulation, have the potential for better stability and improved control of encapsulation properties. Air-filled microbubbles coated with poly(DL-lactic acid) (PLA) are characterized here using in vitro acoustic experiments and several models of encapsulation. The interfacial rheological properties of the encapsulation are determined according to each model using attenuation of ultrasound through a suspension of microbubbles. Then the model predictions are compared with scattered non-linear (sub- and second harmonic) responses. For this microbubble population (average diameter, 1.9 μm), the peak in attenuation measurement indicates a weighted-average resonance frequency of 2.5–3 MHz, which, in contrast to other encapsulated microbubbles, is lower than the resonance frequency of a free bubble of similar size (diameter, 1.9 μm). This apparently contradictory result stems from the extremely low surface dilational elasticity (around 0.01–0.07 N/m) and the reduced surface tension of the poly(DL-lactic acid) encapsulation, as well as the polydispersity of the bubble population. All models considered here are shown to behave similarly even in the non-linear regime because of the low surface dilational elasticity value. Pressure-dependent scattering measurements at two different excitation frequencies (2.25 and 3 MHz) revealed strongly non-linear behavior with 25–30 dB and 5–20 dB enhancements in fundamental and second-harmonic responses, respectively, for a contrast agent concentration of 1.33 μg/mL in the suspension. Sub-harmonic responses are registered above a relatively low generation threshold of 100–150 kPa, with up to 20 dB enhancement beyond that pressure. Numerical predictions from all models show good agreement with the experimentally measured fundamental response, but not with the experimental second-harmonic response. The characteristic features of sub-harmonic responses and the steady response beyond the threshold are matched well by model predictions. However, prediction of the threshold value depends on estimated properties and size distribution. The variation in size distribution from sample to sample leads to variation in estimates of encapsulation properties: the lowest estimated value for surface dilational viscosity better predicts the sub-harmonic threshold.

Liquid Flooded Flow-Focusing Microfluidic Device for in situ Generation of Monodisperse Microbubbles.

Current microbubble-based ultrasound contrast agents are administered intravenously resulting in large losses of contrast agent, systemic distribution, and strict requirements for microbubble longevity and diameter size. Instead we propose in situ production of microbubbles directly within the vasculature to avoid these limitations. Flow focusing microfluidic devices (FFMDs) are a promising technology for enabling in situ production as they can produce microbubbles with precisely controlled diameters in real-time. While the microfluidic chips are small, the addition of inlets and interconnects to supply the gas and liquid phase greatly increases the footprint of these devices preventing the miniaturization of FFMDs to sizes compatible with medium and small vessels. To overcome this challenge, we introduce a new method for supplying the liquid (shell) phase to an FFMD that eliminates bulky interconnects. A pressurized liquid-filled chamber is coupled to the liquid inlets of an FFMD, which we term a flooded FFMD. The microbubble diameter and production rate of flooded FFMDs were measured optically over a range of gas pressures and liquid flow rates. The smallest FFMD manufactured measured 14.5 × 2.8 × 2.3 mm. A minimum microbubble diameter of 8.1 ± 0.3 μm was achieved at a production rate of 450,000 microbubbles/s (MB/s). This represents a significant improvement with respect to any previously reported result. The flooded design also simplifies parallelization and production rates of up to 670,000 MB/s were achieved using a parallelized version of the flooded FFMD. In addition, an intravascular ultrasound (IVUS) catheter was coupled to the flooded FFMD to produce an integrated ultrasound contrast imaging device. B-mode and IVUS images of microbubbles produced from a flooded FFMD in a gelatin phantom vessel were acquired to demonstrate the potential of in situ microbubble production and real-time imaging. Microbubble production rates of 222,000 MB/s from a flooded FFMD within the vessel lumen provided a 23 dB increase in B-mode contrast. Overall, the flooded design is a critical contribution towards the long- term goal of utilizing in situ produced microbubbles for contrast enhanced ultrasound imaging of, and drug delivery to, the vasculature.

Microbubbles and ultrasound-mediated thrombolysis: a review of recent in vitro studies

Current treatment of acute ischemic stroke with recombinant tissue plasminogen activator (rtPA) is limited to a small percentage of patients due to exclusion criteria and side effects. Sonothrombolysis (ultrasound-mediated thrombolysis) is a new treatment strategy shown to be effective for clot lysis, particularly when combined with ultrasound contrast agents (UCAs). The present review surveys recent in vitro studies of sonothrombolysis (STL). Different UCAs and STL approaches are presented and, in particular, the involved mechanisms are discussed. Acoustic cavitations generate microstreaming and microjets at the clot surface leading to direct (clot damage) and/or indirect (enhancement of thrombolytic drug effect) clot destruction. Microbubble-based UCAs have been shown to greatly potentiate cavitation effects and reduce the required acoustic pressure. However, STL remains a complex process with multiple interactions between blood clots, ultrasound, UCAs and thrombolytic drugs. All these parameters have to be optimized with regard to efficacy and safety.

Subharmonic Contrast Microbubble Signals for Noninvasive Pressure Estimation under Static and Dynamic Flow Conditions

Our group has proposed the concept of subharmonic aided pressure estimation (SHAPE) utilizing microbubble-based ultrasound contrast agent signals for the noninvasive estimation of hydrostatic blood pressures. An experimental system for in vitro SHAPE was constructed based on two single-element transducers assembled confocally at a 60 degree angle to each other. Changes in the first, second and subharmonic amplitudes of five different ultrasound contrast agents were measured in vitro at static hydrostatic pressures from 0-186 mmHg, acoustic pressures from 0.35-0.60 MPa peak-to-peak and frequencies of 2.5-6.6 MHz. The most sensitive agent and optimal parameters for SHAPE were determined using linear regression analysis and implemented on a Logiq 9 scanner (GE Healthcare, Milwaukee, WI). This implementation of SHAPE was then tested under dynamic-flow conditions and compared to pressure-catheter measurements. Over the pressure range studied, the first and second harmonic amplitudes reduced approximately 2 dB for all contrast agents. Over the same pressure range, the subharmonic amplitudes decreased by 9-14 dB and excellent linear regressions were achieved with the hydrostatic pressure variations (r = 0.98, p < 0.001). Optimal sensitivity was achieved at a transmit frequency of 2.5 MHz and acoustic pressure of 0.35 MPa using Sonazoid (GE Healthcare, Oslo, Norway). A Logiq 9 scanner was modified to implement SHAPE on a convex transducer with a frequency range from 1.5-4.5 MHz and acoustic pressures from 0-3.34 MPa. Results matched the pressure catheter (r2 = 0.87). In conclusion, subharmonic contrast signals are a good indicator of hydrostatic pressure. Out of the five ultrasound contrast agents tested, Sonazoid was the most sensitive for subharmonic pressure estimation. Real-time SHAPE has been implemented on a commercial scanner and offers the possibility of allowing pressures in the heart and elsewhere to be obtained noninvasively.

Blood-Brain Barrier Disruption and Vascular Damage Induced by Ultrasound Bursts Combined with Microbubbles can be Influenced by Choice of Anesthesia Protocol

Numerous animal studies have demonstrated that ultrasound bursts combined with a microbubble-based ultrasound contrast agent can temporarily disrupt the blood-brain barrier (BBB) with little or no other apparent effects to the brain. As the BBB is a primary limitation to the use of most drugs in the brain, this method could enable a noninvasive means for targeted drug delivery in the brain. This work investigated whether BBB disruption and vessel damage when overexposure occurs can be influenced by choice of anesthesia protocol, which have different vasoactive effects. Four locations were sonicated transcranially in each brain of 16 rats using an unfocused 532 kHz piston transducer. Burst sonications (10 ms bursts applied at 1 Hz for 60 s) were combined with intravenous Definity (10 μl/kg) injections. BBB disruption was evaluated using contrast-enhanced MRI. Half of the animals were anesthetized with i.p. ketamine and xylazine, and the other half with inhaled isoflurane and oxygen. Over the range of exposure levels tested, MRI contrast enhancement was significantly higher ( p < 0.05) for animals anesthetized with ketamine/xylazine. Furthermore, the threshold for extensive erythrocyte extravasation was lower with ketamine/xylazine. These results suggest that BBB disruption and/or vascular damage can be affected by vascular or other factors that are influenced by different anesthesia protocol. These experiments may also have been influenced by the recently reported findings that the circulation time for perfluorocarbon microbubbles is substantially reduced when oxygen is used as the carrier gas.

Enhanced Ablation of High Intensity Focused Ultrasound with Microbubbles: An Experimental Study on Rabbit Hepatic VX2 Tumors

This study was designed to assess the enhanced effect of high intensity focused ultrasound (HIFU) ablation with microbubbles on rabbit hepatic VX2 tumors and to compare the detection sensitivity of CEUS and CECT to determine the residual viable tissue after ablation of HIFU. Forty rabbits with hepatic VX2 tumors were randomly separated into two groups (20 animals per group) before HIFU ablation. A bolus of 0.2mL of saline or a microbubble-based ultrasound (US) contrast agent was injected intravenously to group I rabbits and group II rabbits, respectively. The HIFU ablation procedure was started 15s after the injection. Tumors were examined with grayscale contrast-enhanced ultrasound (CEUS) and contrast-enhanced computed tomography (CECT) immediately before and after HIFU ablation. Histopathologic assessment was performed immediately after treatment imaging. Before ablation, intense contrast enhancement during arterial phase was observed at the whole tumors or the periphery of the tumors by CEUS and CECT. Lower HIFU energy was used in group II than in group I (P<0.001). Histopathologic assessment revealed local residual viable tumor tissues due to incomplete ablation in 47.4% (9/19) of tumors in group I and 10% (2/20) of tumors in group II (P<0.05). The concordance rate of CEUS (90.9%) with histopathology on residual tumor detection was higher than that of CECT (27.3%, P<0.05). Introduction of the microbubble agent enhances HIFU therapeutic efficacy. CEUS proves to have high sensitivity in assessment of residual viable rabbit VX2 tumor after HIFU.

Sonografische Leberdiagnostik bei Tumorpatienten ohne und mit Kontrastmittel

In patients with known malignancy, correct detection and characterization of liver lesions has important therapeutic consequences. Conventional sonography is the most commonly used modality for liver imaging in tumor patients. However, it has a lower sensitivity for the detection of liver metastases compared to contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI). The majority of liver metastases are hypoechoic and well defined in baseline ultrasound (US), while detection of isoechoic or small liver metastases <1 cm is difficult and the differentiation of liver metastases from benign liver lesions and other malignant liver tumors can be impossible with baseline US. The use of microbubble-based ultrasound contrast agents and contrast-specific imaging techniques advanced the accuracy of ultrasound in liver imaging. Levovist and SonoVue are the US contrast agents approved for liver imaging in Europe. Compared to Levovist, SonoVue allows continuous imaging of the liver in real-time over a period of up to 5 minutes. As a result, SonoVue became the preferred contrast agent for liver imaging in the recent years, while Levovist became less important. Important for the detection of liver metastases are the portal venous and late phases in which metastases show a wash-out and can be detected as hypoechoic lesions in homogeneous enhanced liver parenchyma. The detection of hepatic metastases is substantially improved by CEUS compared to conventional B-mode sonography. Several studies showed sensitivity in detecting liver metastases comparable to that of contrast-enhanced CT and MRI. Furthermore, the typical enhancement patterns of the different benign and malignant liver lesions allow reliable characterization and differentiation from liver metastases in the majority of cases. This paper provides information about the advantages and expedient application of contrast-enhanced ultrasound (CEUS) in tumor patients.

Enhancing ultrasound-mediated cell membrane permeabilisation (sonoporation) using a high frequency pulse regime and implications for ultrasound-aided cancer chemotherapy

Delivering ultrasound to HeLa cells at 1 MHz using a high frequency pulse regime (40 kHz) and at a maximum energy density of 270 Jcm −2 resulted in significant cell membrane permeabilisation. Using FITC-dextran as a fluorogenic marker, optimally up to 64% of treated populations were permeabilised with cell viability remaining above 80%. Although cell membrane permeabilisation was observed in the presence of the microbubble-based ultrasound contrast agent, SonoVue ®, cell viability was severely compromised. Using the high frequency pulse regime in the absence of microbubbles, the LD 50 of the cancer chemotherapeutic agent, camptothecin, was reduced from 58 to 18 nM.

Hepatocellular cancer response to radiofrequency tumor ablation: contrast-enhanced ultrasound

Radiofrequency ablation (RFA) is increasingly being used as percutaneous treatment of choice for patients with early stage hepatocellular carcinoma (HCC). An accurate assessment of the RFA therapeutic response is of crucial importance, considering that a complete tumor ablation significantly increases patient survival, whereas residual unablated tumor calls for additional treatment. Imaging modalities play a pivotal role in accomplishing this task, but ultrasound (US) is not considered a reliable modality for the evaluation of the real extent of necrosis, even when color/power Doppler techniques are used. Recently, newer microbubble-based US contrast agents used in combination with grey-scale US techniques, which are very sensitive to non-linear behavior of microbubbles, have been introduced. These features have opened new prospects in liver ultrasound and may have a great impact on daily practice, including cost-effective assessment of therapeutic response of percutaneous ablative therapies. Technical evolution of CEUS focusing on findings after RFA are illustrated. These latter are detailed, cross-referenced with the literature and discussed on the basis of our personal experience. Timing of CEUS posttreatment assessment among with advantages and limitations of CEUS are also described with a perspective on further technologic refinement.

Grafting of abciximab to a microbubble-based ultrasound contrast agent for targeting to platelets expressing GP IIb/IIIa – Characterization and in vitro testing

Abciximab-grafted ultrasound sensitive microbubbles were developed for the diagnosis of stroke. The antibody fragment abciximab, which binds to the GP IIb/IIIa and α vβ 3 receptors expressed by activated platelets, was chosen because most ischemic strokes are due to arterial thrombi that are mainly composed of platelets. The abciximab antibody fragment was activated by reduction of the disulfide bond for grafting on the microbubbles. The suspension was freeze-dried after the grafting was performed directly on the formed microbubbles. Quantification of the amounts of abciximab present on the surface of the microbubbles was assessed semi-quantitatively by flow cytometry, and quantitatively using radio-labeled abciximab. A protocol for human and rat platelets deposition and fixation was implemented and the expression of the GP IIb/IIIa receptor was validated by immunostaining. The abciximab-grafted microbubbles showed high static and dynamic binding to fixed platelets. Detection by ultrasonography of microbubbles bound on white and red clots gave higher signals compared to SonoVue microbubbles.

The potential of a new stable ultrasound contrast agent for site-specific targeting. An in vitro experiment

Microbubble-based ultrasound contrast agents can be used for specific site targeting, but demonstrate time-limited opacification. We have previously demonstrated the potential of gold-bound microtubules to provide a stable ultrasound contrast effect. Aim of the present study was to test the feasibility of gold-bound microtubules specifically to bind to human thrombi and to inflammatory activated human umbilical vein endothelial cells (HUVEC) in vitro. HUVEC were incubated with tumor necrosis factor, to induce expression of adhesion molecules. Human clots and HUVEC were incubated with biotinylated monoclonal antifibrin and anti-E-selectin antibodies, respectively. Probes were incubated with excess avidin followed by biotinylated gold-bound microtubules and by secondary Cy3-anti-β-tubulin antibody and processed for immune fluorescence microscopy. Clots were transferred in copolymer foils filled with buffer and were ultrasonographically imaged before and after their treatment with the antifibrin antibody and with biotinylated microtubules, using a broadband harmonic transducer, transmitting and receiving at a mean frequency of 1.7 MHz and 3.2 MHz. The feasibility of specific gold-bound microtubules conjugation to antibody treated clots and HUVEC was confirmed using immune fluorescence analysis. Contrast intensities of the clots significantly increased after their treatment with antifibrin antibody and incubation with gold-bound microtubules (39 ± 2 dB versus 26 ± 2 dB, p < 0.001) and remained high after 20 min of ultrasound exposure (37 ± 2 dB versus 39 ± 2 dB, p = NS). Thus, gold-bound microtubules can specifically bind to human thrombi and to endothelial cells, providing a significant contrast effect which remains stable in the ultrasound field. This may be a promising approach to target thrombi and inflammatory active atherosclerotic plaques. (E-mail: gkorosoglou@hotmail.com)

Vascular effects induced by combined 1-MHz ultrasound and microbubble contrast agent treatments in vivo

Previous in vivo studies have demonstrated that microvessel hemorrhages and alterations of endothelial permeability can be produced in tissues containing microbubble-based ultrasound contrast agents when those tissues are exposed to MHz-frequency pulsed ultrasound of sufficient pressure amplitudes. The general hypothesis guiding this research was that acoustic ( viz., inertial) cavitation, rather than thermal insult, is the dominant mechanism by which such effects arise. We report the results of testing five specific hypotheses in an in vivo rabbit auricular blood vessel model: (1) acoustic cavitation nucleated by microbubble contrast agent can damage the endothelia of veins at relatively low spatial-peak temporal-average intensities, (2) such damage will be proportional to the peak negative pressure amplitude of the insonifying pulses, (3) damage will be confined largely to the intimal surface, with sparing of perivascular tissues, (4) greater damage will occur to the endothelial cells on the side of the vessel distal to the source transducer than on the proximal side and (5) ultrasound/contrast agent-induced endothelial damage can be inherently thrombogenic, or can aid sclerotherapeutic thrombogenesis through the application of otherwise subtherapeutic doses of thrombogenic drugs. Auricular vessels were exposed to 1-MHz focused ultrasound of variable peak pressure amplitude using low duty factor, fixed pulse parameters, with or without infusion of a shelled microbubble contrast agent. Extravasation of Evans blue dye and erythrocytes was assessed at the macroscopic level. Endothelial damage was assessed via scanning electron microscopy (SEM) image analysis. The hypotheses were supported by the data. We discuss potential therapeutic applications of vessel occlusion, e.g., occlusion of at-risk gastric varices. (E-mail: jooha@u.washington.edu)

Characterization of Tumor Imaging with Microbubble-Based Ultrasound Contrast Agent, Sonazoid, in Rabbit Liver

In order to investigate improvement of hepatic tumor detectability by Sonazoid with phase inversion imaging, the contrast effects on the liver of metastatic carcinoma-model rabbits were evaluated by videodensitometry and visual assessment. Correlation between the contrast enhancement of Sonazoid and histopathology was examined using the same animals. Electron microscopy was performed on hepatic tissue from another healthy rabbits to identify the distribution of Sonazoid microbubbles. As a result, all tumors were smaller than 12 mm in diameter, and after intravenous injection of Sonazoid, they were surrounded with a ring of enhanced signal during the early phase (up to 30 s), followed by a clear contrast defect during the delayed phase (after 10 min). Histopathologic observation revealed that the ring-enhancement was caused by neovasculature in the tumor, and the contrast defects corresponded to living and dead parts of tumors, which lack Kupffer cells. Videodensitometric differences between tumor and healthy tissue markedly increased at delayed phase, and visual detectability of tumors was improved when Sonazoid was used. Ultrastructural analysis showed microbubble-like structures in Kupffer cells, which indicated that Sonazoid microbubbles were taken up with these cells. In conclusion, Sonazoid, used with phase inversion imaging, greatly increases the detectability of small hepatic tumors by highlighting neovascularity at early phase and providing clear contrast defects due to absence of Kupffer cells, which take up Sonazoid microbubbles, at delayed phase.