Targeted-microbubble binding selectively to GPIIb IIIa receptors of platelet thrombi

RATIONALE AND OBJECTIVES: New targeted microbubbles directed to the GPIIb IIIa receptor have been developed. The objective was to determine whether targeting microbubbles to clots would enhance ultrasound imaging. Systematic studies were designed to determine whether in vitro methodology is an acceptable predictor of in vivo efficacy. MATERIALS AND METHODS: Bioconjugate ligands were inserted into lipid-coated membranes of perfluorocarbon gas microbubbles and binding studies performed on activated platelets immobilized on cell culture plates. Targeted microbubble binding to clots in a flow through chamber was also assessed. Finally, microbubble binding studies on arteriolar and venular clots in a mouse cremasteric muscle model were conducted. RESULTS: Binding studies on platelet-immobilized plates demonstrated an affinity for targeted microbubbles versus untargeted microbubbles. Semiquantitative light obscuration techniques helped to measure extent of targeted microbubble binding. Targeted microbubbles similarly bound to platelet clots in the flow model. Finally, studies in the mouse model confirmed binding of targeted microbubbles in both venules and arterioles. CONCLUSION: The use of receptor selective targeted microbubbles improved binding to vascular thrombi in both in vitro and in vivo settings.     

Ultrasound microbubbles for molecular diagnosis, therapy, and theranostics

Ultrasound imaging is clinically established for routine screening examinations of breast, abdomen, neck, and other soft tissues, as well as for therapy monitoring. Microbubbles as vascular contrast agents improve the detection and characterization of cancerous lesions, inflammatory processes, and cardiovascular pathologies. Taking advantage of the excellent sensitivity and specificity of ultrasound for microbubble detection, molecular imaging can be realized by binding antibodies, peptides, and other targeting moieties to microbubble surfaces. Molecular microbubbles directed against various targets such as vascular endothelial growth factor receptor-2, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, selectins, and integrins were developed and were shown in preclinical studies to be able to selectively bind to tumor blood vessels and atherosclerotic plaques. Currently, the first microbubble formulations targeted to angiogenic vessels in prostate cancers are being evaluated clinically. However, microbubbles can be used for more than diagnosis: disintegrating microbubbles emit acoustic forces that are strong enough to induce thrombolysis, and they can also be used for facilitating drug and gene delivery across biologic barriers. This review on the use of microbubbles for ultrasound-based molecular imaging, therapy, and theranostics addresses innovative concepts and identifies areas in which clinical translation is foreseeable in the near future.     

Detection of individual microbubbles of ultrasound contrast agents: imaging of free-floating and targeted bubbles

RATIONALE AND OBJECTIVES: During echo examinations with microbubble contrast, individual "dots" of ultrasound reflection can be visualized. To address the question whether these signals represent individual microbubbles, very dilute suspensions of ultrasound contrast agents or individual microbubbles attached to Petri dishes were prepared and studied by ultrasound imaging. METHODS: Microbubble suspensions were diluted in saline and evaluated by a clinical ultrasound imaging system. Microbubble concentration was verified by Coulter counter. Single microbubble preparation on a Petri dish was established by streptavidin-biotin interaction under microscopy control and subjected to ultrasound imaging. RESULTS: Ultrasound of dilute microbubble dispersions demonstrated distinct white foci; concentration of these sites was consistent with signals from individual microbubbles as determined by Coulter. Individual microbubbles immobilized on polystyrene were also visualized by ultrasound. CONCLUSION: Ultrasound medical systems can resolve backscatter signals from individual microbubbles of ultrasound contrast, both in solution and in the targeted immobilized state, implying picogram sensitivity.     

Echo enhancers and ultrasound imaging

The quality of diagnostic ultrasound images is sometimes limited by excessive acoustic attenuation within the organs and tissues. Ultrasound echo-enhancers help to overcome that limitation by increasing the intensity of the reflected signal. Acoustic principles dictate that the most effective enhancers are gas-filled microbubbles. The problem of producing a microbubble suspension stable enough for routine clinical use has been overcome in several ways. Newer developments are leading to enhancers with an active acoustic response and that have affinities for specific organs and tissues     

脂质囊泡超声造影剂的质量评价及猪肝彩色多普勒显影研究

目的 对自制的脂质囊泡超声造影剂进行质量评价,研究不同剂量与正常猪肝彩色多普勒显影效果间的相互关系.方法 显微镜观察脂质囊泡的形态、激光粒度测定仪测定粒径及粒度分布,用血细胞分析仪统计不同放置时间条件下和配制浓度粒径在2-8μm范围内的囊泡浓度以及光照和温度对囊泡浓度和平均粒径的影响.进行造影剂剂量与正常猪肝实质彩色多...     

Molecular imaging of cardiovascular disease using ultrasound

Molecular imaging using probes that specifically home to function- or disease-specific targets is a promising tool for both basic research investigations as well as clinical diagnostics. Ultrasound-based molecular imaging utilizes acoustically active particles (contrast agents) bearing targeting ligands that specifically bind to a molecule of interest. In the presence of an ultrasound field, the bound particles are detectable as a persistent contrast effect during ultrasound imaging. Different types of targeted contrast agents have been reported, most of which share in common the presence of a gas encapsulated by a shell of varying chemical formulation. These agents, or “microbubbles,” are typically 2 to 4 μm in diameter, and have a natural resonance frequency that corresponds to the frequencies used in diagnostic echocardiography. This attribute makes it possible to induce microbubble resonance and non-linear oscillation at diagnostic ultrasound frequencies, leading to acoustic emissions from the microbubbles that can be detected as specific signals during two dimensional ultrasound imaging. Targeting ligands that have been attached to microbubbles include monoclonal antibodies, peptides, and the naturally occurring ligands for the receptor of interest, such as vascular endothelial growth factor. Because the contrast agents stay within the intravascular space, they are ideally suited for detection of endothelial epitopes, such as leukocyte adhesion molecules or angiogenesis receptors. Ultrasound molecular imaging with targeted contrast agents has been used to detect inflammation association with ischemia/reperfusion (ischemic memory), cardiac transplant rejection, early atherosclerosis, and angiogenesis. Application to tumor angiogenesis has also been reported using peptides that specifically bind to angiogenic tumor endothelium. Translation of ultrasound molecular imaging to the clinical arena will require optimization of contrast agent design to maximize specific binding, and customization of imaging systems to sensitively detect the binding events Dr Villanueva is supported in part by a grant (RO1HL077534) from the National Institutes of Health.     

Microbubble contrast agent with focused ultrasound to create brain lesions at low power levels: MR imaging and histologic study in rabbits

PURPOSE: To evaluate magnetic resonance (MR) imaging-based thermometry for predicting the onset and spatial extent of lesions produced by focused ultrasound combined with a microbubble contrast agent (Optison; GE Healthcare, Milwaukee, Wis) and to compare the resulting induced temperature increase and threshold for damage with those in studies performed without the agent. MATERIALS AND METHODS: The experiments were approved by the animal care committee. Fifty-three locations in the brains of 15 rabbits were sonicated with various exposure parameters by using a 1.5-MHz focused ultrasound transducer. MR imaging was used to map the temperature rise and, along with light microscopy, to examine the lesions. Diameters of isotherms created from thermometry were compared with the resulting lesions by using Bland-Altman analysis and linear regression. The minimum acoustic power necessary for lesion creation was determined, and the apparent temperature threshold for damage was calculated with probit analysis. These thresholds were compared with prior work performed without the contrast agent. The heating induced with the microbubbles was compared with that in sonications performed without them by using a t test. RESULTS: The MR imaging-mapped temperature distributions matched the shape of the lesions. The diameters of isotherms correlated well with diameters measured at contrast material-enhanced MR imaging (mean difference between measurements, 0.0 mm +/- 0.5; R = 0.93). The temperature increase with microbubbles was statistically larger (P < .01) than for sonications performed without microbubbles. In some locations (mostly continuous wave exposures), damage was observed along the ultrasound beam path. The time-averaged acoustic power damage threshold was reduced by 91% for 10-second exposures when compared with earlier studies performed without microbubbles. The probability of producing lesions was 50% at a temperature increase of 5.9 degrees C, 5.5 degrees C lower than was observed earlier without the agent. CONCLUSION: MR imaging-based temperature measurements appeared to correlate with focused ultrasound-induced lesions in the brain when microbubbles were present, even though the temperature appeared to be below the threshold for thermal damage.     

Contrast-specific ultrasound techniques

The advent of microbubble contrast agents has determined an important evolution of ultrasound (US) technology due to the introduction of contrast-specific US techniques. This was due to the fact that neither colour or power Doppler are suitable for correct management of the signals produced by microbubble insonation, as they are limited by the heavy presence of artefacts. Microbubbles may be insonated by a characteristic frequency named resonance or fundamental frequency (f (0)) by using a high or low transmit power. If insonated by a high transmit power, microbubbles produce a wideband harmonic signal due to microbubble destruction. If insonated by a low transmit power, microbubbles produce harmonic frequencies (2f, 3f, 4f) due to their nonlinear physical behaviour. Contrast-specific US techniques have recently undergone an important technical development with the introduction of innovative algorithms able to register selectively the harmonic signals produced by microbubbles and to suppress the signal produced by stationary tissues. The different contrast-specific US techniques may be distinguished by their basic principle into pseudo-Doppler, harmonic, phase-modulation, amplitude-modulation and phase-and amplitude-modulation techniques.     

Mechanism of hepatic parenchyma-specific contrast of microbubble-based contrast agent for ultrasonography: microscopic studies in rat liver

OBJECTIVE: The objective of this study was to elucidate the mechanism of hepatic parenchyma-specific contrast of Sonazoid (microbubble contrast agent) using microscopic techniques. MATERIALS AND METHODS: Sonazoid was intravenously injected into rats to investigate the microbubble dynamics and distribution within hepatic microcirculation in exteriorized liver using intravital microscopy and to observe dose dependency of ultrasound hepatic contrast effect. In vitro and in vivo uptake of microbubbles by Kupffer cells was examined using confocal laser scanning microscopy. RESULTS: Intravital observation demonstrated freely flowing microbubbles in the sinusoid and some microbubbles co-localized with Kupffer cells. The microbubbles internalized in Kupffer cells were identified with reflected light by confocal laser scanning microscopy. The percentage of Kupffer cells taking up microbubbles was about 1% at clinical dose at which the homogeneous hepatic contrast was observed. CONCLUSIONS: The hepatic parenchyma-specific contrast by Sonazoid is due to distribution of the microbubbles in Kupffer cells.     

Enhanced gene delivery into skeletal muscles with ultrasound and microbubble techniques

RATIONALE AND OBJECTIVES: This experiment was directed to explore the effects of ultrasound microbubbles on gene structure in vitro and green fluorescent protein (GFP) plasmid transfer into skeletal muscles in vivo. By establishing a rat ischemic hind limb model, the effects of ultrasound-mediated microbubble destruction on vascular endothelial growth factor (VEGF) gene transfection to skeletal muscles were also studied in vivo. MATERIALS AND METHODS: Ultrasound irradiation was applied on the mixture of microbubbles and GFP plasmid in vitro. Gel electrophoresis was used to detect the effects of ultrasound and microbubbles on GFP plasmid. For in vivo experiments, ultrasound irradiation was applied on the hind limb after directly injecting microbubbles into the hind limb of Wistar rats. Directly after treatment, the skeletal muscles were harvested to observe the microstructure. We also studied the transfer rate of GFP plasmid DNA into the skeletal muscles of rats by applying ultrasound and microbubble technique. Furthermore, a naked VEGF plasmid was applied to study the feasibility of angiogenesis by using rats ischemia models. RESULTS: Gel electrophoresis of plasmid DNA showed that there was no difference between the groups. By studying the hematoxylin and eosin stained pictures of the skeletal muscles, we found that ultrasound irradiation of skeletal muscle after injection of microbubbles could cause the exudation of the red blood cells, whereas it had no effects on the microstructure of muscle fibers. In vivo experiments showed that an ultrasound microbubble could enhance the transfer of plasmid DNA to the skeletal muscles. CONCLUSIONS: The ultrasound-mediated microbubble technique provides an effective noninvasive method for gene therapy.     

骨髓基质抗原蛋白2靶向微泡的制备及小鼠肿瘤新生血管超声分子成像研究

目的 研究制备针对骨髓基质抗原蛋白2(BST2)的TMBs造影剂(BST2-TMBs),通过超声分子成像技术对小鼠肿瘤血管内皮细胞进行检测,为肿瘤的发生、发展及早期诊断提供实验依据.方法 将抗BST2的抗体通过生物素-亲和素桥接的方式连接于微泡(MBs)表面,获得BST2-TMBs,在光学显微镜下观察TMBs的形态,用粒径分析仪测定其粒径及其分布;通过体外细胞黏附实验研究TMBs与血管内皮细胞的结合性能,并对小鼠前列腺癌肿瘤血管内皮细胞行超声分子成像,用免疫组织化学染色分析BST2在肿瘤血管内皮细胞的表达.采用SPSS 19.0软件行统计学分析,对独立样本行t检验.结果 制备的BST2-TMBs的平均粒径为1.61μm,其中95％的微泡在1～5 μm之间.BST2 -TMBs能够与血管内皮细胞结合,平均每个视野有(165±25)个TMBs结合在内皮细胞表面,远高于非靶向微泡(IgG-MBs)对照组的(10±3)个微泡(t=10.662,P＜0.01).黏附的TMBs能够明显增强内皮细胞的超声信号强度,TMBs为27.93±5.14(灰度值),非靶向微泡为3.61±1.67(灰度值)(t=7.239,P＜0.01).小鼠在体肿瘤超声分子成像表明:BST2-TMBs处理组在微泡注射7min时信号强度(扣除微泡击碎后的信号强度)为38.79±0.29(灰度值),能保持47.65％的微泡注射30 s时的信号强度(灰度值81.40±0.37),而IgG-MBs处理组在微泡注射7 min时的信号强度(扣除微泡击碎后的信号强度)是9.46 ±0.17(灰度值),仅能保持11.39％的微泡注射30 s时的信号强度(灰度值83.01±0.60).相比之下,TMBs在肿瘤部位的超声信号强度较非靶向微泡提高4.27倍(t=65.587,P＜0.01).免疫组织化学证实BST2蛋白在小鼠前列腺癌肿瘤血管内皮细胞上有表达.结论BST2 -TMBs可以用于小鼠前列腺癌血管内皮细胞的超声分子成像,这为肿瘤的发生发展以及早期诊断提供了实验依据.     

A novel technique to measure splanchnic transit time using microbubble ultrasound

OBJECTIVE: The objective of this study was to measure splanchnic transit time by intravenous injection of a microbubble. MATERIALS AND METHODS: Ten volunteers were examined before and after eating. After Doppler indices of splanchnic circulation were obtained, the superior mesenteric artery (SMA) and vein (SMV) were simultaneously interrogated using power Doppler ultrasound after intravenous injection of a microbubble. Contrast arrival in the SMA and subsequently the SMV was recorded and splanchnic transit time calculated from differences in the time-intensity curves. RESULTS: Splanchnic transit time decreased significantly after eating (mean 11 vs. 6.9 seconds; P = 0.007), reflecting splanchnic hemodynamics. Between-subject variability attributable to repeated measurements was least for the SMA resistive index (17%) but 56% for the new index, suggesting poor reproducibility. CONCLUSION: Splanchnic transit time may be measured by microbubble injection but is subject to considerable measurement error. Newer microbubbles and imaging methods may allow more reproducible measurements.     

Assessment of tissue perfusion by contrast-enhanced ultrasound

Contrast-enhanced ultrasound (CEUS) with microbubble contrast agents is a new imaging technique for quantifying tissue perfusion. CEUS presents several advantages over other imaging techniques in assessing tissue perfusion, including the use of microbubbles as blood-pool agents, portability, availability and absence of exposure to radiation or nuclear tracers. Dedicated software packages are necessary to quantify the echo-signal intensity and allow the calculation of the degree of tissue contrast enhancement based on the accurate distinction between microbubble backscatter signals and native tissue background. The measurement of organ transit time after microbubble injection and the analysis of tissue reperfusion kinetics represent the two fundamental methods for the assessment of tissue perfusion by CEUS. Transit time measurement has been shown to be feasible and has started to become accepted as a clinical tool, especially in the liver. The loudness of audio signals from spectral Doppler analysis is used to generate time-intensity curves to follow the wash-in and wash-out of the microbubble bolus. Tissue perfusion may be quantified also by analysing the replenishment kinetics of the volume of microbubbles after their destruction in the imaged slice. This allows to obtain semiquantitative parameters related to local tissue perfusion, especially in the heart, brain, and kidneys.     

Prolonging the ultrasound signal enhancement from thrombi using targeted microbubbles based on sulfur-hexafluoride-filled gas

RATIONALE AND OBJECTIVES: The objective of this study is to develop and characterize new microbubbles based on lipids and sulfur hexafluoride (SF6) for targeting thrombi as an improved ultrasound contrast agent. MATERIALS AND METHODS: Bioconjugate ligands were inserted into the lipid-coated membranes of SF6 gas microbubbles, and their physicochemical properties were determined. Diagnostic efficacies of SF6-filled microbubbles and the contrast agent SonoVue (Bracco Imaging, Geneve, Switzerland) were compared in dogs. RESULTS: Suspensions of lyophilized powder were reconstituted by injecting saline containing 3.1 x 10(8) SF6 microbubbles/mL with a mean diameter of 4.4 microm. More than 90% of microbubbles had diameters between 1 and 10 microm. After reconstitution, echogenicity and microbubble characteristics were unchanged for 8 hours. Targeted microbubbles increased the echogenicity of thrombi significantly and provided a longer period of optimal signal enhancement compared with nontargeted microbubbles. CONCLUSIONS: Our thrombus-targeting microbubble contrast agent shows high echogenicity and stability and thereby enhances the visualization of intravascular thrombi and prolongs the duration of the diagnostic window.     

Characterization of focal liver lesions with a new ultrasound contrast agent using continuous low acoustic power imaging: comparison with contrast enhanced spiral CT

PURPOSE: To evaluate the concordance of the enhancement patterns of a new ultrasound contrast agent (SonoVue) with those obtained with dual-phase contrast-enhanced spiral CT (CE-CT) in the characterization of focal liver lesions (FLLs). MATERIALS AND METHODS: Sixty-two patients with focal liver lesions discovered at ultrasound and also studied with CECT underwent contrast-enhanced ultrasound using continuous low acoustic power imaging after receiving a 2.4 ml bolus of the new US contrast agent SonoVue, consisting of a dispersion of sulphur hexafluoride microbubbles. The examinations were made using ATL HDI-5000, Acuson SEQUOIA and Aloka 5500 Prosound ultrasound systems with 5.2 MHz curved-array probes. The concordance between US and CE-CT images was evaluated on site by two radiologists blinded to CT RESULTS: The FLLs were assessed in the arterial (20 s after CM injection), portal (after 45-60 s) and late (after 120 s) phases for: 1) presence/absence of enhancement 2) distribution of enhancement (homogenous or target distribution, centripetal or centrifugal flow, and other), 3) qualitative enhancement pattern (hyperechoic, hypoechoic, or isoechoic) versus normal liver parenchyma. RESULTS: The concordance between SonoVue-enhanced US and CE-CT was 85%. Moreover during portal venous phase with CEUS it was possible to differentiate between malignancy or benignity of 91% of lesions. CONCLUSIONS: The preliminary data obtained in this study suggest that continuous low acoustic power imaging and contrast-enhanced US show similar results to CT in contrast distribution and contrast enhancement patterns.     

Intraperitoneal distribution of ultrasound contrast medium imaged with B-mode ultrasound and colour-stimulated acoustic emission imaging

Intraperitoneal port catheter systems for local delivery of cytotoxic drugs require imaging prior to chemotherapy to confirm homogenous distribution of an injected fluid in the entire peritoneal cavity. This study was performed to assess whether contrast-enhanced ultrasound (US) is a suitable imaging modality for this task. Twelve patients with peritoneal carcinosis and an implanted intraperitoneal port catheter system were studied before chemotherapy. Ultrasound examinations were performed after bolus injections of the microbubble contrast medium Levovist. Distribution of the contrast medium in the peritoneal cavity was imaged using B-mode US and colour-stimulated acoustic emission imaging (SAE). Contrast-enhanced CT imaging was used as term of reference for evaluating the US results. Distribution of the microbubbles in the peritoneal cavity was easily detected by both US methods. In 10 of 12 patients a free distribution in all abdominal quadrants was seen with both US techniques. In 2 of 12 patients, CT and US showed contrast medium limited to the perihepatic area. Therapy was stopped and surgical repositioning of the catheter was performed. Ultrasound after intraperitoneal injection of a microbubble contrast agent provides reliable information about the distribution of intraperitoneally injected fluid in the peritoneal cavity. This method is therefore well suited for imaging port catheter systems prior to chemotherapy. Electronic Publication     

Enhancement of gas‐filled microbubble R2* by iron oxide nanoparticles for MRI

Gas‐filled microbubbles have the potential to become a unique intravascular MR contrast agent due to their magnetic susceptibility effect, biocompatibility, and localized manipulation via ultrasound cavitation. However, microbubble susceptibility effect is relatively weak when compared with other intravascular MR susceptibility contrast agents. In this study, enhancement of microbubble susceptibility effect by entrapping monocrystalline iron oxide nanoparticles (MIONs) into polymeric microbubbles was investigated at 7 T in vitro. Apparent T₂ enhancement (ΔR₂*) induced by microbubbles was measured to be 79.2 ± 17.5 sec^?1 and 301.2 ± 16.8 sec^?1 for MION‐free and MION‐entrapped polymeric microbubbles at 5% volume fraction, respectively. ΔR₂* and apparent transverse relaxivities (r₂*) for MION‐entrapped polymeric microbubbles and MION‐entrapped solid microspheres (without gas core) were also compared, showing the synergistic effect of the gas core with MIONs. This is the first experimental demonstration of microbubble susceptibility enhancement for MRI application. This study indicates that gas‐filled polymeric microbubble susceptibility effect can be substantially increased by incorporating iron oxide nanoparticles into microbubble shells. With such an approach, microbubbles can potentially be visualized with higher sensitivity and lower concentrations by MRI. Magn Reson Med, 2010. © 2009 Wiley‐Liss, Inc.     

Polyclonal human immunoglobulin G labeled with polymeric iron oxide: antibody MR imaging

Human polyclonal immunoglobulin (Ig) G was attached to a monocrystalline iron oxide nanocompound (MION), a small superparamagnetic probe developed for receptor and antibody magnetic resonance (MR) imaging. The resulting complex, MION-IgG, had a slightly negative surface charge, a molecular weight of 150-180 kDa, and 0.36 microgram of IgG attached per milligram of iron. After intravenous administration of MION-IgG to normal rats, most of the compound localized in liver, spleen, and bone marrow. In an animal model of myositis, MION-IgG caused reduced signal intensity (most apparent on T2-weighted spin-echo and gradient-echo images) at the site of inflammation. No change in signal intensity existed after an injection of unlabeled MION. Site-specific localization of MION-IgG was corroborated with scintigraphic imaging with indium-111 IgG and MION-In-111-IgG and was confirmed histologically with iron staining. These results indicate that antibody MR imaging is feasible in vivo. Target-specific and antibody MR imaging could be easily extended to other applications, including detection of cancer, infarction, and degenerative diseases.     

Pulse inversion imaging of liver blood flow: improved method for characterizing focal masses with microbubble contrast

RATIONALE AND OBJECTIVES: To create a microbubble contrast image of vessels that lie below the resolution of an ultrasound system, a technique is required that detects preferentially the agent echo, rejecting that from tissue. Harmonic imaging exploits the nonlinear behavior of microbubbles but forces a compromise between image sensitivity and axial resolution. The authors describe and evaluate a new method that overcomes this compromise and improves contrast imaging performance: pulse inversion imaging. METHODS: Sequences of pulses of alternate phase are transmitted into tissue and their echoes summed. A prototype scanner equipped with pulse inversion was used to image phantoms and 16 patients with focal liver masses. RESULTS: Pulse inversion images show contrast sensitivity and resolution superior to that of harmonic images. Vessels can be imaged at an incident power sufficiently low to avoid destroying the agent, allowing unique visualization of tumor vasculature. Distinct patterns were seen in hemangiomas, metastases, and hepatocellular carcinomas. CONCLUSIONS: Pulse inversion imaging is an improved bubble-specific imaging method that extends the potential of contrast ultrasonography.