Microbubble Accumulation Research Articles

Kalman Filter-Based Microbubble Tracking for Robust Super-Resolution Ultrasound Microvessel Imaging.

Contrast microbubble (MB)-based super-resolution ultrasound microvessel imaging (SR-UMI) overcomes the compromise in conventional ultrasound imaging between spatial resolution and penetration depth and has been successfully applied to a wide range of clinical applications. However, clinical translation of SR-UMI remains challenging due to the limited number of MBs detected within a given accumulation time. Here, we propose a Kalman filter-based method for robust MB tracking and improved blood flow speed measurement with reduced numbers of MBs. An acceleration constraint and a direction constraint for MB movement were developed to control the quality of the estimated MB trajectory. An adaptive interpolation approach was developed to inpaint the missing microvessel signal based on the estimated local blood flow speed, facilitating more robust depiction of microvasculature with a limited amount of MBs. The proposed method was validated on an ex ovo chorioallantoic membrane and an in vivo rabbit kidney. Results demonstrated improved imaging performance on both microvessel density maps and blood flow speed maps. With the proposed method, the percentage of microvessel filling in a selected blood vessel at a given accumulation period was increased from 28.17% to 74.45%. A similar SR-UMI performance was achieved with MB numbers reduced by 85.96%, compared to that with the original MB number. The results indicate that the proposed method substantially improves the robustness of SR-UMI under a clinically relevant imaging scenario where SR-UMI is challenged by a limited MB accumulation time, reduced number of MBs, lowered imaging frame rate, and degraded signal-to-noise ratio.

Combining Acoustic Trapping With Plane Wave Imaging for Localized Microbubble Accumulation in Large Vessels.

The capability of accumulating microbubbles using ultrasound could be beneficial for enhancing targeted drug delivery. When microbubbles are used to deliver a therapeutic payload, there is a need to track them, for a localized release of the payload. In this paper, a method for localizing microbubble accumulation with fast image guidance is presented. A linear array transducer performed trapping of microbubble populations interleaved with plane wave imaging, through the use of a composite pulse sequence. The acoustic trap in the pressure field was created parallel with the direction of flow in a model of a vessel section. The acoustic trapping force resultant from the large gradients in the acoustic field was engendered to directly oppose the flowing microbubbles. This was demonstrated numerically with field simulations, and experimentally using an Ultrasound Array Research Platform II. SonoVue microbubbles at clinically relevant concentrations were pumped through a tissue-mimicking flow phantom and exposed to either the acoustic trap or a control ultrasonic field composed of a single-peak acoustic radiation force beam. Under the flow condition at a shear rate of 433 s-1, the use of the acoustic trap led to lower speed estimations ( ) in the center of the acoustic field, and an enhancement of 71% ± 28%( ) in microbubble image brightness.

A Combined Magnetic‐Acoustic Device for Simultaneous, Coaligned Application of Magnetic and Ultrasonic Fields

AbstractAcoustically‐responsive microbubbles have been widely researched as agents for both diagnostic and therapeutic applications of ultrasound. There has also been considerable interest in magnetically‐functionalised microbubbles as multi‐modality imaging agents and carriers for targeted drug delivery. In this paper, we present a design for an integrated device capable of generating co‐aligned magnetic and acoustic fields in order to accumulate microbubbles at a specific location and to activate them acoustically. For this proof‐of‐concept study, the device was designed to concentrate microbubbles at a distance of 10 mm from the probe's surface, commensurate with relevant tissue depths in preclinical small animal models. Previous studies have indicated that both microbubble concentration and duration of cavitation activity are positively correlated with therapeutic effect. The utility of the device was assessed in vitro tests in a tissue‐mimicking phantom containing a single vessel (1.2 mm diameter). At a peak fluid velocity of 4.2 mm s−1 microbubble accumulation was observed under B‐mode ultrasound imaging and the corresponding cavitation activity was sustained for a period more than 4 times longer than that achieved with an identical acoustic field but in the absence of a magnet. The feasibility of developing a larger scale device for human applications is discussed.

Dynamics of Targeted Microbubble Adhesion Under Pulsatile Compared with Steady Flow

Hemodynamic flow variations at low fluid shear stress are thought to play a critical role in local atherosclerotic plaque initiation and development and to affect plaque instability. Targeted microbubbles are being developed as intravascular agents for identifying atherosclerotic lesions using ultrasound. How variations in local hydrodynamic flow influence the adhesiveness of targeted microbubbles is not well understood. We postulated that rates of targeted microbubble binding and accumulation differ when subjected to steady flow (SF) as compared with oscillatory or pulsatile flow (PF), because PF imposes non-uniform blood rheology and periodic acceleration and deceleration of blood velocity, when compared with SF. We assessed the binding rates of targeted microbubbles in seven randomly assigned PF and seven matched SF replicate runs at low (<1 Pa) and intermediate (≥1 and <2.5 Pa) wall shear stress (WSS) by drawing 4.8 × 106 microbubbles mL−1 over streptavidin-coated substrates, immobilized within a parallel plate flow chamber at a calculated density of 81 binding sites μm-2. Selective binding and accumulation of targeted microbubbles was recorded in a single field of view using real-time video microscopy. Microbubble accumulation was modeled to obtain flow-mediated microbubble binding kinetics (amplitude, A, and rate constant, k). PF elicited higher microbubble accumulation rates, in comparison to SF. The rates of microbubble accumulation differed significantly between PF and SF (p < 0.05) at intermediate WSS but not at low WSS (p > 0.05). The rate of microbubble accumulation decreased as WSS increased.

The use of MMP2 antibody-conjugated cationic microbubble to target the ischemic myocardium, enhance Timp3 gene transfection and improve cardiac function

The objective of this study was to synthesize a cationic microbubble (CMB) conjugated with an antibody against matrix metalloproteinase 2 (CMBMMP2) to increase microbubble accumulation and gene transfection in the infarcted myocardium and to restore ventricular function following an ischemic insult. We previously reported that our CMBs enhanced the efficiency of gene transfection following ultrasound-targeted microbubble destruction (UTMD) in rodent hearts. Therefore, we conjugated a thiolated MMP2 antibody to the PEG chains on the CMB surface, which was verified by fluorescent microscopy. Rats underwent ischemia/reperfusion injury 3 days prior to UTMD delivery of the control or Timp3 plasmid. The CMBMMP2 improved microbubble accumulation in the infarct region, with 57% more contrast intensity compared to the non-conjugated CMB. UTMD-mediated CMBMMP2 delivery of the Timp3 gene significantly increased TIMP3 protein levels in the infarct scar and border zone at 3 days post-UTMD compared to delivery by the non-conjugated CMB. Both MMP2 and MMP9 activity were reduced in the CMBMMP2Timp3 group, which resulted in smaller and thicker infarcts and improved cardiac function. UTMD therapy with this CMBMMP2 provides an efficient platform for the targeted delivery of factors intended to preserve ventricular structure and improve cardiac function after ischemic injury.

Molecular Ultrasound Imaging Using a Targeted Contrast Agent for Assessing Early Tumor Response to Antiangiogenic Therapy

Contrast-enhanced ultrasound (US) and targeted microbubbles have been shown to be advantageous for angiogenesis evaluation and disease staging in cancer. This study explored molecular US imaging of a multitargeted microbubble for assessing the early tumor response to antiangiogenic therapy. Target receptor expression of 2LMP breast cancer cells was quantified by flow cytometric analysis and characterization established with antibodies against mouse α(V)β3- integrin, P-selectin, and vascular endothelial growth factor receptor 2. Tumor-bearing mice (n = 15 per group) underwent contrast-enhanced US imaging of multitargeted microbubbles. Microbubble accumulation was calculated by destruction-replenishment techniques and time-intensity curve analysis. On day 0, mice underwent baseline imaging. Next, therapy group mice were injected with a 0.2-mg dose of bevacizumab, and controls received matched saline injections. Imaging was repeated on days 1 and 3. After imaging was completed on day 3, the mice were euthanized and tumors excised. Histologic analysis of microvessel density and intratumoral necrosis was completed on tumor sections. On day 3 after bevacizumab dosing, a 71.8% change in tumor vasculature was shown between the therapy and control groups (P = .01). The therapy group had a 15.4% decrease in tumor vascularity, whereas the control group had a 56.4% increase. Molecular US imaging of angiogenic markers can detect the early tumor response to drug therapy.

Intravascular ultrasound catheter to enhance microbubble-based drug delivery via acoustic radiation force

Previous research has demonstrated that acoustic radiation force enhances intravascular microbubble adhesion to blood vessels in the presence of flow for moleculartargeted ultrasound imaging and drug delivery. A prototype acoustic radiation force intravascular ultrasound (ARFIVUS) catheter was designed and fabricated to displace a microbubble contrast agent in flow representative of conditions encountered in the human carotid artery. The prototype ARFIVUS transducer was designed to match the resonance frequency of 1.4- to 2.6-μm-diameter microbubbles modeled by an experimentally verified 1-D microbubble acoustic radiation force translation model. The transducer element was an elongated Navy Type I (hard) lead zirconate titanate (PZT) ceramic designed to operate at 3 MHz. Fabricated devices operated with center frequencies of 3.3 and 3.6 MHz with -6-dB fractional bandwidths of 55% and 50%, respectively. Microbubble translation velocities as high as 0.86 m/s were measured using a high-speed streak camera when insonating with the ARFIVUS transducer. Finally, the prototype was used to displace microbubbles in a flow phantom while imaging with a commercial 45-MHz imaging IVUS transducer. A sustained increase of 31 dB in average video intensity was measured following insonation with the ARFIVUS, indicating microbubble accumulation resulting from the application of acoustic radiation force.

Heterogeneity of Microbubble Accumulation: A Novel Approach to Discriminate Between Well-Differentiated Hepatocellular Carcinomas and Regenerative Nodules

This prospective study aimed to elucidate the possibility of differentiating well-differentiated hepatocellular carcinoma (wHCC) from regenerative nodule (RN) on the basis of the heterogeneity of microbubble accumulation. Intensity analysis was conducted on early-phase and late-phase (60 s and 900 s post-injection; perflubutane microbubble) harmonic sonograms in 33 focal hepatic lesions (≤15 mm; 30 patients with chronic liver disease) that were histologically proven as wHCC or RN. Heterogeneity of enhancement, an average of standard deviation of late-phase enhancement in three different sections in the lesions with late-phase iso-enhancement, was examined with respect to the histologic findings. Heterogeneity of enhancement was higher in wHCC (28.7 ± 3.8) than RN (19.8 ± 2.1, p = 0.0213) in the 29 late-phase iso-enhancement lesions. The best cut-off value of the heterogeneity for the diagnosis of wHCC was 25.58, and the sensitivity and specificity were 77.8% and 100%, respectively. A novel parameter, heterogeneity of microbubble accumulation, facilitates differentiation between wHCC and RN showing a late-phase, iso-enhancement appearance.

A Triple‐Targeted Ultrasound Contrast Agent Provides Improved Localization to Tumor Vasculature

Actively targeting ultrasound contrast agents to tumor vasculature improves contrast-enhanced sonography of tumor angiogenesis. This report summarizes an evaluation of multitargeted microbubbles, comparing single-, dual-, and triple-targeted motifs. Microbubbles were avidin-biotin linked to antibodies against mouse α(V)β(3)-integrin, P-selectin, and vascular endothelial growth factor receptor 2. These receptors are constitutively overexpressed in tumor vasculature. Binding comparisons between targeted microbubble groups were evaluated on mouse SVR angiosarcoma endothelial cells. Levels of the targeted receptors were characterized with flow cytometry. Targeted microbubble groups were administered to human MDA-MB-231 breast cancer tumor-bearing mice (n = 3) followed by contrast-enhanced sonography in a microbubble-sensitive harmonic imaging mode implemented on an ultrasound scanner equipped with a linear array transducer (5 MHz transmit and 10 MHz receive) to evaluate differences in microbubble accumulation in the tumor vasculature. In vitro analysis showed a 50% increase (P < .001) in triple-targeted microbubble binding over dual-targeted microbubble groups in mouse SVR cells. Mice bearing MDA-MB-231 tumors showed a 40% increase in tumor image intensity after dosing with triple-targeted microbubbles compared with single- and dual-targeted microbubbles (P = .006). Histologic staining confirmed the presence of α(V)β(3)-integrin, P-selectin, and vascular endothelial growth factor receptor 2 in the tumors. Microbubble accumulation in the tumor vasculature was improved using a triple-targeted microbubble approach.

Ultrasound Molecular Imaging of VEGFR2 in a Rat Prostate Tumor Model Using BR55

To evaluate BR55, a new VEGFR2-specific ultrasound contrast agent, for imaging prostate tumors in an orthotopic model in the rat. Rat prostate adenocarcinoma were established by injection of G Dunning R-3327 tumor cells in one lobe of the prostate of Copenhagen rats. Imaging experiments were performed with BR55, SonoVue, and streptavidin-functionalized microbubbles coupled with an anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody using a clinical ultrasound scanner. Contrast enhancement in the tumor and healthy prostate was followed over time by intermittent imaging at low acoustic power. Signal quantification and statistical analysis were performed in the tumor and healthy tissue to compare the behavior of the 3 contrast agents. Immunohistochemistry was performed on the prostate and tumor specimen to determine the expression of VEGFR2. Comparable contrast enhancement was observed in tumors at peak intensity for BR55 and SonoVue. Then, once unbound microbubbles had cleared from the circulation, a strong enhancement of the tumor was obtained with BR55, whereas no significant microbubble accumulation was detected in the healthy prostate tissue. SonoVue microbubbles were rapidly eliminated, and no significant binding was observed in the tumor. The tumor to prostate ratio calculated after signal quantification was about 20 for the 3 doses of BR55 tested. The enhancement obtained with BR55 in the tumor was not significantly different from the one observed with antibody-coupled streptavidin microbubbles. Intense staining for VEGFR2 was detected in the tumor vessels by immunohistochemistry. This study showed that BR55 binding to prostate tumors resulted in a strong enhancement of the lesions as early as a few minutes after contrast injection, whereas minimal nonspecific accumulation occurred in the healthy part of the gland. BR55, like SonoVue, provide information on tissue perfusion during the early vascular phase, but BR55 binding to the tumoral endothelium allows to gain additional information by highlighting the sites of active angiogenesis. The late phase enhancement of the tumor should be particularly valuable for prostate cancer detection and for biopsy guidance.

Changes in tumor vascularity precede microbubble contrast accumulation deficit in the process of dedifferentiation of hepatocellular carcinoma

Purpose To elucidate the changes in tumor vascularity and microbubble accumulation on contrast-enhanced sonograms, in relation to the dedifferentiation of hepatocellular carcinoma (HCC). Materials and methods This prospective study enrolled 10 patients with histologically proven HCC (14.4–39.0 mm, 26.1 ± 7.4) showing nodule-in-nodule appearance upon contrast-enhanced computed tomography. Contrast-enhanced ultrasound was performed by harmonic imaging under a low mechanical index (0.22–0.25) during the vascular phase (agent injection to 1 min) and late phase (15 min) following the injection of Sonazoid™ (0.0075 ml/kg). Contrast enhancement in the inner and outer nodules was assessed in comparison with that in adjacent liver parenchyma as hyper-, iso-, or hypo-enhanced. Results Vascular-phase enhancement of all 10 inner nodules was hyper-enhanced, and that of outer nodules was hyper-enhanced in 3, iso-enhanced in 2, and hypo-enhanced in 5. Late-phase enhancement of inner nodules was hypo-enhanced in 8 and iso-enhanced in 2. Furthermore, late-phase enhancement of outer nodules was iso-enhanced in the 7 lesions that showed iso- or hypo-enhancement in the vascular phase, and hypo-enhanced in the 3 with hyper-enhancement in the vascular phase. Late-phase hypo-enhancement was significantly more frequent in the nodules showing early-phase hyper-enhancement (11/13) than in the nodules showing early-phase iso- or hypo-enhancement (0/7) in both the inner and outer nodules. Conclusion Dedifferentiation of HCC may be accompanied by changes in tumor vascularity prior to a reduction in microbubble accumulation. Observation of the vascular phase may be more useful than late-phase imaging for the early recognition of HCC dedifferentiation when using contrast-enhanced ultrasound with Sonazoid.

Decrease in accumulation of ultrasound contrast microbubbles in non‐alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is one of the representative liver diseases in developed countries. Diagnosis of NASH is dependent on histological findings from liver biopsy. The usefulness of contrast ultrasound with Levovist for diagnosis of NASH is described. 2.5 g of ultrasound contrast agent Levovist was injected intravenously. The liver was scanned at 5, 10, 15, 20, 30, 40 and 50 min and changes in microbubble accumulation were evaluated. The signal intensity from regions of interest (ROI) on the contrast images was measured and estimated using time intensity curves (TICs). Twenty-one patients with NASH, 33 with non-alcoholic fatty liver disease (NAFLD) and 10 healthy volunteers (HV) were studied. The signal intensity was measured quantitatively at 5 and 20 min after injection. There was a statistically significant decrease in NASH, when compared with NAFLD and HV groups. These changes in signal intensity were not correlated to the degree of fibrosis and steatosis in histological study. The sensitivity, specificity and overall accuracy obtained from the receiver operating characteristic (ROC) curve were 100% when the cut-off value was set at 43.6 of signal intensity at 20 min. The Levovist contrast study is a useful screening examination which picks up NASH among fatty liver patients.

Diagnosis of NASH using delayed parenchymal imaging of contrast ultrasound

Non alcoholic steatohepatitis (NASH) is one of the representative liver diseases in the developed countries. Diagnosis of NASH is dependent on histological findings from liver biopsy. Usefulness of contrast ultrasound with Levovist for diagnosis of NASH is described. Clinical study: Ultrasound contrast agent, Levovist of 2.5g was injected intravenously. The liver was scanned at 5, 10, 15, 20, 30, 40, and 50min after Levovist injection in different planes using a contrast specific ultrasound mode. Changes in microbubble accumulation in the liver were evaluated. The signal intensity from regions of interest (ROI) on the contrast images was measured and accumulation and decrescence of microbubbles were estimated using the time intensity curves (TICs). The image data and TICs were evaluated by blind reviewers. Fifteen patients with NASH, 8 with alcoholic steatohepatitis (ASH), 45 with non alcoholic fatty liver (NAFL), 10 with chronic hepatitis C (CHC) and 10 healthy volunteers were studied. Animal study: Methionine-choline-deficient diet (MCDD) fed rats were used for NASH model. Correlation between microbubble accumulation and morphological and functional changes of sinusoidal endothelium and macrophage was evaluated. The maximum intensity of contrast ultrasound was decreased and time course decrescence was more rapid in NASH than the other groups. These changes were correlated to the degree of centrilobular and pericellular fibrosis but not to steatosis in histological study. Disturbance of microbubble accumulation was correlated with sinusoidal function rather than morphological changes such as fibrosis and parenchymatitis in the animal studies. The Levovist contrast study enables differential diagnosis between NASH and other diseases that provoke steatosis and fibrosis.

Acoustic radiation force enhances targeted delivery of ultrasound contrast microbubbles: in vitro verification

Recent research has shown that targeted ultrasound contrast microbubbles achieve specific adhesion to regions of intravascular pathology, but not in areas of high flow. It has been suggested that acoustic radiation can be used to force free-stream microbubbles toward the target, but this has not been verified for actual targeted contrast agents. We present evidence that acoustic radiation indeed increases the specific targeted accumulation of microbubbles. Lipid microbubbles bearing an antibody as a targeting ligand were infused through a microcapillary flow chamber coated with P-selectin as the target protein. A 2.0 MHz ultrasonic pulse was applied perpendicular to the flow direction. Microbubble accumulation was observed on the flow chamber surface opposite the transducer. An acoustic pressure of 122 kPa enhanced microbubble adhesion up to 60-fold in a microbubble concentration range of 0.25 x 10(6) to 75 x 106) ml(-1). Acoustic pressure mediated the greatest adhesion enhancement at concentrations within the clinical dosing range. Acoustic pressure enhanced targeting nearly 80-fold at a wall shear rate of 1244 s(-1), suggesting that this mechanism is appropriate for achieving targeted microbubble delivery in high-flow vessels. Microbubble adhesion increased with the square of acoustic pressure between 25 and 122 kPa, and decreased substantially at higher pressures.