Microbubble Agents Research Articles

Randomized Feasibility Study Evaluating Multiple FDA-approved Microbubbles for CEUS Lymphography.

Prior studies on contrast-enhanced ultrasound (CEUS) lymphography for preoperative mapping before lymphaticovenous anastomosis surgery in patients with extremity lymphedema have been limited to using only Lumason and Sonazoid as microbubble contrast agents. The purpose of this study was to determine the feasibility of using two other Food & Drug Administration-approved microbubble agents, Optison and Definity, for imaging lymphatic vessels in the upper extremities. Nine female adults with unilateral upper extremity lymphedema anticipating lymphaticovenous anastomosis surgery underwent CEUS lymphography of the unaffected upper extremity randomized to either Lumason, Definity, or Optison. Lymphatic vessels were visualized in all but one case when undilated Definity was used. In the eight upper extremities where lymphatic vessels were visualized, an average of eight intradermal injections of microbubbles were performed in the extremity. Lymphatic vessels could be identified in 57% (36 of 63) of the injections. The effective dilution for each of the microbubble agents is provided. This was the first successful demonstration of lymphatic vessel visualization using either Definity or Optison. Broadening the range of available microbubble agents for CEUS lymphography could improve accessibility to the procedure and provide potentially safer alternatives.

Non-equilibrium activation dynamics of superheated phase-change contrast agents

Phase-change contrast agents, such as superheated perfluorocarbon droplets, potentially offer exciting new opportunities in diagnostic and therapeutic applications of ultrasound. An important advantage of droplets over, e.g., microbubble agents is that they can be “activated” (vaporised) by a reduction in ambient pressure, initiated by an externally applied ultrasound pulse. Controlling the timing and location of droplet activation, however, is vitally important to avoid safety concerns. Current theoretical models for droplet vaporisation assume that a critical stable bubble embryo forms that is in thermal and mechanical equilibrium. Model predictions are, however, inconsistent with some experimental observations. The aim of this study is to develop a non-equilibrium model for droplet activation wherein initial bubble growth rate and vapour pressure are dependent upon the bubble nucleus size. We demonstrate how incorporating nonequilibrium effects into theoretical models of droplet vaporisation dynamics enables better agreement of the droplet expansion with experimental data and thus provide greater insight into control of the activation process.

Lymphatic Mapping with Contrast-enhanced Ultrasound for Lymphaticovenous Anastomosis Surgery: How We Do It.

Lymphaticovenous anastomosis (LVA) surgery is an effective surgery for the treatment of lymphedema in the extremities. Indocyanine green lymphography is the reference standard for visualizing lymphatics for LVA surgery, but it has several limitations; most notably, superficial dermal congestion can mask deeper lymphatic vessels. To overcome the limitations, we add contrast-enhanced ultrasound (CEUS) lymphography. We have previously reported that CEUS lymphography can identify lymphatic vessels for LVA surgery that indocyanine green lymphography does not. Here, we describe how we perform CEUS lymphography, including workflow, technique, and documentation. Before informed consent, the patient must be screened for possible adverse reactions to microbubbles. The procedure involves multiple intradermal injections of the microbubble agent at various sites along the extremity. After each injection, imaging for microbubble uptake by lymphatic vessels is performed using an ultrasound scanner with contrast-specific software. We use sulfur hexafluoride lipid-type A microspheres (Lumason/SonoVue; Bracco Suisse SA), but we are investigating the performance of other Food & Drug Administration-approved microbubble agents for CEUS lymphography. Having a systematic approach to marking the skin can mitigate the hindrance of marking over ultrasound coupling gel. Another benefit of CEUS lymphography is the rapid identification of neighboring veins compatible in size and location for anastomosis. We hold regular scheduled multidisciplinary meetings for coordination of care, discussion of outcomes, quality assurance, and ongoing innovation.

Sonic cracking of calcium carbonate-encapsulated microbubbles observed at moderate acoustic amplitudes

Abstract Theranostic agents are materials that act both as tracers during diagnostic imaging and as vehicles carrying and releasing therapeutics during treatment. Ultrasoundtriggered theranostic agents comprise shell-encapsulated microbubbles that pulsate during low-amplitude ultrasonic imaging and release their payload upon higher-amplitude sonication whilst simultaneously assisting in the permeation of target tissue. High-amplitude release may be undesirable due to unwanted side effects related to inertial cavitation. However, low-amplitude release from microbubbles typically requires thin encapsulating shells, which in turn may be permanently disrupted under diagnostic imaging conditions. The purpose of this study was to investigate the suitability for theranostic applications of a novel microbubble agent with thick shells composed of calcium carbonate. Hydrophobised calcium carbonate-encapsulated microbubbles of radii between 1.0 μm and 11 μm were subjected to short ultrasound pulses of 1-MHz ultrasound at acoustic amplitudes of 0.5MPa or less, which corresponds to low mechanical indices. During sonication, high-speed video footage was recorded at a frame rate of ten million frames per second. We observed pulsations but no gas release at a 0.1-MPa amplitude and intra-encapsulation fragmentation during sonication at a 0.3-MPa amplitude. At 0.5-MPa amplitude sonication, release was observed from more than 70% of the microbubbles in the field of view. This finding indicates that the microbubbles were stable scatterers during 0.1-MPa sonication, but instable vehicles during 0.5-MPa sonication. The pressures used in this study to observe release were too low to allow for unwanted inertial cavitation. In conclusion, therefore, the microbubbles studied were a promising theranostic agent whose contents could be released at moderate acoustic amplitudes.

769 In Vivo Evaluation of Spinal Cord Perfusion Using Contrast-Enhanced Transcutaneous Ultrasound

INTRODUCTION: Ultrasound (US) imaging is cheap, portable, and widely available. It has been used in multiple medical disciplines, both as a means of diagnosis and therapy. Developing US as a platform for spinal cord perfusion monitoring in patients with spinal cord injury (SCI) would provide a cheap, accessible, and real-time platform for managing spinal cord perfusion in the acute setting. METHODS: Four patients with previous cervical decompression and instrumentation were evaluated at a single center with transcutaneous US. We included adult patients with at least 2 levels of continuous cervical laminectomies greater than 3 months prior to US and no history of SCI. The first two patients underwent US imaging without contrast and the second two patients underwent contrast-enhanced US imaging with Lumason, a microbubble agent. Imaging was performed by a board-certified radiologist with US expertise using a General Electric (GE) Logiq E10 ultrasound scanner ans a high frequency linear transducer. The study was approved by the institutional ethics committee. RESULTS: In the non-contrast study (n = 2), the dura, spinal cord, and vertebral body were apparent on US. Application of Doppler was insufficient to discern intramedullary perfusion, so contrast-enhanced ultrasound was pursued (n = 2). With contrast, anatomic structures were again visible, and we were able to quantify differential spinal cord perfusion within and between cross-sectional sections of the cord. We demonstrated spinal cord hemodynamic perfusion by measuring peak signal intensity and the time to peak signal intensity after microbubble contrast injection. Then, a time-intensity curve was created and the area under the curve was calculated as a surrogate of spinal cord perfusion. CONCLUSIONS: Contrast-enhanced US is a viable platform for monitoring spinal cord perfusion in patients who have undergone prior cervical laminectomies.

MR-guided ultrasound-stimulated microbubble therapy enhances radiation-induced tumor response

High intensity focused ultrasound (HIFU) systems have been approved for therapeutic ultrasound delivery to cause tissue ablation or induced hyperthermia. Microbubble agents have also been used in combination with sonication exposures. These require temperature feedback and monitoring to prevent unstable cavitation and prevent excess tissue heating. Previous work has utilized lower power and pressure to oscillate microbubbles and transfer energy to endothelial cells in the absence of thermally induced damage that can radiosensitize tumors. This work investigated whether reduced acoustic power and pressure on a commercial available MR-integrated HIFU system could result in enhanced radiation-induced tumor response after exposure to ultrasound-stimulated microbubbles (USMB) therapy. A commercially available MR-integrated HIFU system was used with a hyperthermia system calibration provided by the manufacturer. The ultrasound transducer was calibrated to reach a peak negative pressure of − 750 kPa. Thirty male New Zealand white rabbits bearing human derived PC3 tumors were grouped to receive no treatment, 14 min of USMB, 8 Gy of radiation in a separate irradiation cabinet, or combined treatments. In vivo temperature changes were collected using MR thermometry at the tumor center and far-field muscle region. Tissues specimens were collected 24 h post radiation therapy. Tumor cell death was measured and compared to untreated controls through hematoxylin and eosin staining and immunohistochemical analysis. The desired peak negative pressure of − 750 kPa used for previous USMB occurred at approximately an input power of 5 W. Temperature changes were limited to under 4 °C in ten of twelve rabbits monitored. The median temperature in the far-field muscle region of the leg was 2.50 °C for groups receiving USMB alone or in combination with radiation. Finally, statistically significant tumor cell death was demonstrated using immunohistochemical analysis in the combined therapy group compared to untreated controls. A commercial MR-guided therapy HIFU system was able to effectively treat PC3 tumors in a rabbit model using USMB therapy in combination with radiation exposures. Future work could find the use of reduced power and pressure levels in a commercial MR-guided therapy system to mechanically stimulate microbubbles and damage endothelial cells without requiring high thermal doses to elicit an antitumor response.

Effect of pulse duty ratio on temperature rise induced by focused ultrasound combined with magnetic microbubbles

Development of acoustic/magnetic contrast agent microbubbles with various diagnostic and therapeutic functions has attracted more and more attention in medical ultrasound, biomedical engineering and clinical applications. Superparamagnetic iron oxide nanoparticles (SPIO) have unique magnetic characteristics and wonderful biocompatibility, so they can be used as MRI contrast agents to improve image contrast, spatial resolution and diagnostic accuracy. Our previous work shows that the multimodal diagnostic and therapeutic microbubble agents can be successfully constructed by embedding SPIO particles into the coating shell of conventional ultrasound contrast agent (UCA) microbubbles, which in turn changes the size distribution and shell properties of UCA microbubbles, thereby affecting their acoustic scattering, cavitation and thermal effects. However, previous studies only considered the influence factors such as acoustic pressure and microbubble concentration. The relevant investigation regarding the influence of ultrasound temporal characteristics on the dynamic response of magnetic microbubbles is still lacking. This work systematically measures the temperature enhancement effect of the SPIO-albumin microbubble solution flowing in the vascular gel phantom exposed to pulsed ultrasound with various temporal settings (e.g. duty cycle, PRF and single pulse length). Meanwhile, a two-dimensional finite element model is developed to simulate and verify the experimental observations. The results show that the increase of duty cycle of pulse signal should be the crucial factor affecting the temperature enhancement effect of flowing SPIO-albumin microbubble solution under the exposure to high-intensity focused ultrasound. The current results help us to better understand the influence of different acoustic setting parameters on the thermal effect of dual-modal magnetic UCA microbubbles, and provide useful guidance for ensuring the safety and effectiveness of the application of SPIO-albumin microbubbles in clinics.

Ultrasound-activated perfluorocarbon nanodroplets in ophthalmology

Glaucoma, a leading cause of blindness, can result from increased intraocular pressure resulting from elevated outflow resistance. In this preliminary study, we consider the potential of ultrasound-activated perfluorocarbon nanodroplets (NDs) to increase the permeability of the outflow pathway. 100nm diameter NDs with a perfluoropentane core and lipid shell were introduced into the anterior chamber (AC) of ex vivo pig and in vivo rat eyes. Imaging (18 and 28 MHz) was performed with a weakly focused (F = 2.9) beam. Activation was induced by increasing the array sub-aperture to tighten the focus to F = 0.62 centrally. Images showed NDs to be distributed widely within the AC, from which they could enter the outflow pathway by virtue of their small dimension. At 28 MHz, NDs were activated at a peak negative pressure of 5 MPa, corresponding to a mechanical index of 1.8.Two days after treatment, rat eyes showed no sign of inflammation, but a few small gas bubbles were present in the AC. In this application, NDs are advantageous as cavitation nuclei with respect to conventional microbubble contrast agents because of their higher density and smaller diameter (<1/10th that of microbubble agents), which facilitate entry into the outflow channels. Future preclinical in vivo studies will assess the effect of treatment on IOP and outflow facility.

Acoustic droplet vaporization for nonthermal ablation of brain tumors

Acoustic vaporization of perfluorobutane-based phase-shift nanoemulsions (PSNE) can be used to nucleate inertial cavitation (IC) in vivo. The acoustic pressure amplitude must exceed a threshold for vaporization of PFB-based nanoemulsions. Two focused ultrasound transducers with a center frequency of 837 kHz were oriented such that their focal volumes overlapped and the acoustic pressure amplitude was amplified. In this study, the dual transducer system was combined with circulating PSNE to nucleate IC in established brain tumors, leading to nonthermal ablation of the tumors. For comparison, microbubble ultrasound agents (UCA) were used as IC nuclei for ultrasound-mediated tumor ablation. The ablation volume was confined to focal volume when PSNE were used to nucleate IC, whereas pre-focal damage was observed when UCA were used as IC nuclei. Additionally, PSNE-nucleated IC ablated a larger percentage of the brain tumors on average than MB-nucleated IC (89.46.8% vs 11.17.7%, respectively). These results suggest that PFB-based PSNE may be used to significantly reduce the inertial cavitation threshold in the cerebrovasculature, and when combined with transcranial focused ultrasound, enable efficient focal intracranial nonthermal ablation.

Application of low-boiling point phase-change nanoemulsions for sonothrombolysis

Intravascular thrombus remains a major challenge in human health, resulting in substantial morbidity and mortality for our aging population. Current treatment approaches are focused on antithrombotic treatment or mechanical thrombectomy, both of which have challenges with efficacy and side effects. Ultrasound-enhanced sonothrombolysis, including that enhanced by microbubbles, has been studied for decades as potential alternative therapy. However, clinical efficacy of ultrasound driven thrombolysis has remained elusive. One hypothesis has been that for contracted venous clots, the dense clot matrix resists permeation of microbubble cavitation agents, and superficial cavitation alone is slow and weakly effective. We hypothesized that sub-micron phase change nanoemulsions could penetrate a clot and begin lysis from the interior. Using video microscopy during clot treatment with nanoemulsions and ultrasound, we observed cavitation mediated clot erosion initiating internally to the clot. Cavitation detection imaging shows this same trend of internal cavitation with nanoemulsions, whereas traditional microbubbles cavitate only near the clot surface. Lysis rate studies demonstrate statistically faster lysis of contracted clots with phase change nanoemulsions, compared to 5x larger microbubbles of the same formulation. We conclude that phase change nanoemulsions may play a substantial role in reinvigorating sonothrombolysis potential.

Diagnostic accuracy of hepatic vein arrival time performed with contrast-enhanced ultrasonography for HCV liver cirrhosis

Background and study aims: Contrast-enhanced ultrasonography (CEUS) has increased considerably the use of ultrasound for hemodynamical analyses and quantification. Bolus injection of microbubble agents is used to evaluate transit times. This study aimed to determine the diagnostic accuracy of arrival time (seconds) to the hepatic artery (HAAT), hepatic vein (HVAT), and portal vein (PVAT), based on CEUS used for the diagnosis of cirrhosis, and to correlate these arrival times with the liver stiffness and disease severity. Patients and methods: This study evaluated 29 HCV cirrhotic and 19 chronic hepatitis C patients. History, clinical examination, laboratory investigations, abdominal ultrasonography, point shear-wave elastography (pSWE), and CEUS were conducted. Results: The mean liver stiffness increased significantly in cirrhotic versus chronic HCV (22.7 versus 5.1; p-value < 0.001). The mean HAAT (p-value = 0.001), PVAT (p-value = 0.002), and HVAT values (p-value: 0.001) were significantly prolonged in cirrhotic compared with chronic HCV. The HVAT cut-off point of cirrhotic patients was 18 s with a sensitivity, specificity, and accuracy of 96.6%, 63.2%, and 83.3%, respectively (area under curve: 0.801). Significant positive correlation was found between liver stiffness (kPa) and HVAT (s) (r = 0.585; p-value = 0.005). No significant correlation was detected between HVAT (s) and the severity of liver disease, as assessed by the Child or MELD scores in cirrhotic patients. Conclusion: Measuring HVAT by CEUS yielded high-accuracy and correlation outcomes for cirrhosis detection. It could be a valuable noninvasive method for the diagnosis of cirrhosis.

Ultrasound-Targeted Microbubble Destruction-Mediated Inhibition of Livin Expression Accelerates Ovarian Cancer Cell Apoptosis

Objective Ultrasound-targeted microbubble destruction (UTMD) technique has recently been developed as a nonviral delivery of gene therapy. This study aimed at investigating the survival and apoptosis of ovarian cancer cell line OVCA-433 by inhibiting Livin expression through ultrasound-targeted microbubble destruction. Methods We synthesized a targeted microbubble agent for UTMD-mediated shRNA against Livin gene in human ovarian cancer OVCA-433 cells. Lipid microbubbles were conjugated with a luteinizing hormone-releasing hormone analog (LHRHa) by an avidin-biotin linkage to target the ovarian cancer OVCA-433 cells expressing LHRH receptors. The microbubbles were mixed with the recombinant plasmid harboring shRNA-Livin. shRNA-Livin was transfected into OVCA-433 cells upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm2) for 8 s. Cell survival was measured by the MTT assay, cell apoptosis by flow cytometry using annexin V/PI double staining, and cell ultrastructure by using the transmission electron microscope. The mRNA and protein expression levels of caspase-3 and caspase-8 were detected by RT-qPCR and western blotting. Results UTMD-mediated delivery of shRNA-Livin remarkably reduced the survival of OVCA-433 cells but promoted the apoptosis compared with shRNA-Livin alone, shRNA-Livin plus nontargeted microbubbles, and shRNA-Livin plus LHRHa-conjugated microbubbles containing shRNA-Livin with or without exposure to ultrasound pulses. It was also found that UTMD-mediated delivery of shRNA-Livin notably declined the mRNA and protein expression levels of caspase-3 and caspase-8 in OVCA-433 cells compared with shRNA-Livin alone, shRNA-Livin plus nontargeted microbubbles, and shRNA-Livin plus LHRHa-conjugated microbubbles containing shRNA-Livin with or without exposure to ultrasound pulses. Conclusion Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles can enhance the transfection efficiency of shRNA-Livin in ovarian cancer cells.

Contrast-Enhanced Ultrasound for Intraoperative Tumor Contour Definition During Complex Partial Nephrectomy

Contrast-enhanced ultrasound (CEUS) is an imaging modality that utilizes a non-nephrotoxic, intravascular microbubble agent to capture real-time perfusion of the target organ. CEUS has demonstrated utility for the evaluation and surveillance of indeterminate renal lesions in patients who are unable to receive conventional contrast agents, yet its potential utility in renal surgery remains poorly defined in the current literature. We present a series of 4 patients who underwent successful intraoperative CEUS during partial nephrectomy (PN) to help define renal mass contour and proximity to vascular structures in an attempt to optimize renal function after PN. All cases were endophytic with moderate-high complexity (R.E.N.A.L. scores>9). CEUS was safely performed in all cases providing excellent tumor contour definition. In one case, CEUS provided better visualization of tumor proximity within the hilum than conventional US, facilitating surgical resection and reconstruction. In another case, CEUS provided better definition of tumor contour, allowing wedge resection rather than heminephrectomy. Margins were negative in all cases. The average global renal function preserved following PN was 90%. While grey-scale US with color Doppler is usually sufficient for intraoperative tumor visualization, CEUS can be considered during PN for complex endophytic, infiltrative or hilar tumors where accurate delineation of tumor contour is critical.

Consistent opening of the blood brain barrier using focused ultrasound with constant intravenous infusion of microbubble agent

The blood brain barrier (BBB) is a major obstacle to the delivery of therapeutics to the brain. Focused ultrasound (FUS) in combination with microbubbles can non-invasively open the BBB in a targeted manner. Bolus intravenous injections of microbubbles are standard practice, but dynamic influx and clearance mechanisms prevent delivery of a uniform dose with time. When multiple targets are selected for sonication in a single treatment, uniform serum concentrations of microbubbles are important for consistent BBB opening. Herein, we show that bubble infusions were able to achieve consistent BBB opening at multiple target sites. FUS exposures were conducted with different Definity microbubble concentrations at various acoustic pressures. To quantify the effects of infusion on BBB opening, we calculated the MRI contrast enhancement rate. When infusions were performed at rates of 7.2 µl microbubbles/kg/min or below, we were able to obtain consistent BBB opening without injury at all pressures. However, when infusion rates exceeded 20 µl/kg/min, signs of injury occurred at pressures from 0.39 to 0.56 MPa. When compared to bolus injections, a bubble infusion offers a more controlled and consistent approach to multi-target BBB disruption.

Abstract P1-02-11: The difference between tumor width on a contrast-enhanced ultrasound and the extent of pathological breast cancer

Abstract Background: Contrast-enhanced ultrasound (CEUS) is a modality capable of visualizing the blood vessels through the use of the microbubble agent “Perflubutane”. It can also allow for an improved visualization of breast tumors. Currently, CEUS is being used on a daily practice for breast cancer screening and treatment planning in many medical centers. This technique was approved in Japan following evidence gleaned through Phase-2 and Phase-3 clinical studies and has advanced over the past seven years. Regarding the extent of malignant breast tumor, it has been revealed that contrast-enhanced lesions observed using CEUS might be longer than those same lesions visible on B-mode. However, what contrast-enhanced images on CEUS reflect pathologically remains unclear. To resolve this issue, more complete evidence of the detailed imagery of lesions obtained by CEUS seemed to be needed. In the present study, we investigated the maximum tumor width on B-mode/CEUS images compared with the maximum pathological extent of the cancer. (Evergreen study, UMIN: 000028995). Methods: In this retrospective multi-center study, 153 operable breast cancer patients who had undergone B-mode and CEUS prior to breast surgery between August 2012 and January 2017 were registered. The maximum tumor width was measured on B-mode and CEUS, and the tumor diameter width was determined at a postoperative histopathological examination (P) by investigators. With five blind readers as independent reviewers, the lesion width of 303 images was read (B-mode and CEUS). We analyzed the difference between the lesion width on CEUS or B-mode and the pathological width using paired t-tests and the Pearson product-moment correlation coefficient. Results: The mean tumor widths (±standard deviation) measured by investigators were 15±7 mm (B-mode), 19±8 mm (CEUS) and 17±9 mm (P). The difference calculated by P from B-mode was -3±7 mm (p&amp;lt;0.0001, r=0.6142), while that from CEUS was 1±6 mm (p=0.0163, r=0.7372). CEUS showed a significantly positive high correlation (p&amp;lt;0.0001, r=0.8548) with B-mode. The mean (±standard deviation) widths measured by blind readers were 16±7 mm (B-mode) and 18±8 mm (CEUS). The difference calculated by P from B-mode was -2±8 mm (p=0.0005, r=0.5067), while that from CEUS was 1±8 mm (p=0.1256 r=0.5354). CEUS and B-mode also showed a significantly positive high correlation (p&amp;lt;0.0001, r=0.7909). Discussion: Our data suggested that the maximum lesion width might increase, in order, from B-mode to P and CEUS. Furthermore, the difference in the lesion width determined by B-mode and P appeared to be significant, although not that between P and CEUS. Moreover, a positive high correlation was detected between B-mode and CEUS. Therefore, the width on CEUS might tend to be larger than that on B-mode, regardless of the tumor size. Additional information may be determined through an analysis of the design and extent of dissection when partial mastectomy is to be performed. These findings may also provide clues as to what can be anticipated pathologically when a lesion is enhanced with CEUS. Citation Format: Aya Noro, Hiroaki Shima, Toshiko Hirai, Midori Noma, Takashi Nakamura, Emi Sato, Terumi Kaga, Yukio Mitsuzuka, Keitaro Kamei, Yumi Imayoshi, Toshikazu Ito, Shinsaku Kanazawa, Kumiko Kato, Ryuzo Sekiguchi, Goro Kutomi, Mitsuru Mori, Tadashi Hasegawa, Ichiro Takemasa, Toshitaka Okuno. The difference between tumor width on a contrast-enhanced ultrasound and the extent of pathological breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-02-11.

Engineering the Echogenic Properties of Microfluidic Microbubbles Using Mixtures of Recombinant Protein and Amphiphilic Copolymers.

Microbubbles are used as ultrasound contrast agents in medical diagnosis and also have shown great promise in ultrasound-mediated therapy. However, short lifetime and broad size distribution of microbubbles limit their applications in therapy and imaging. Moreover, it is challenging to tailor the echogenic response of microbubbles to make them suitable for specific applications. To overcome these challenges, we use microfluidic flow-focusing to prepare monodisperse microbubbles with a mixture of a recombinant amphiphilic protein, oleosin, and a synthetic amphiphilic copolymer, Pluronic. We show that these microbubbles have superior uniformity and stability under ultrasonic stimulation compared to commercial agents. We also demonstrate that by using different Pluronics, the echogenic response of the microbubbles can be tailored. Our work shows the versatility of using the combination of microfluidics and protein/copolymer mixtures as a method of engineering microbubbles. This tunability could potentially be important and powerful in producing microbubble agents for theranostic applications.

Development of 68Ga-labelled ultrasound microbubbles for whole-body PET imaging.

Microbubble (MB) contrast agents have revolutionalised the way ultrasound (US) imaging can be used clinically and pre-clinically. Contrast-enhanced US offers improvements in soft-tissue contrast, as well as the ability to visualise disease processes at the molecular level. However, its inability to provide in vivo whole-body imaging can hamper the development of new MB formulations. Herein, we describe a fast and efficient method for achieving 68Ga-labelling of MBs after a direct comparison of two different strategies. The optimised approach produces 68Ga-labelled MBs in good yields through the bioorthogonal inverse-electron-demand Diel-Alder reaction between a trans-cyclooctene-modified phospholipid and a new tetrazine-bearing HBED-CC chelator. The ability to noninvasively study the whole-body distribution of 68Ga-labelled MBs was demonstrated in vivo using positron emission tomography (PET). This method could be broadly applicable to other phospholipid-based formulations, providing accessible solutions for in vivo tracking of MBs.

Comparing Strategies for Magnetic Functionalization of Microbubbles.

The advancement of ultrasound-mediated therapy has stimulated the development of drug-loaded microbubble agents that can be targeted to a region of interest through an applied magnetic field prior to ultrasound activation. However, the need to incorporate therapeutic molecules while optimizing the responsiveness to both magnetic and acoustic fields and maintaining adequate stability poses a considerable challenge for microbubble synthesis. The aim of this study was to evaluate three different methods for incorporating iron oxide nanoparticles (IONPs) into phospholipid-coated microbubbles using (1) hydrophobic IONPs within an oil layer below the microbubble shell, (2) phospholipid-stabilized IONPs within the shell, or (3) hydrophilic IONPs noncovalently bound to the surface of the microbubble. All microbubbles exhibited similar acoustic response at both 1 and 7 MHz. The half-life of the microbubbles was more than doubled by the addition of IONPs by using both surface and phospholipid-mediated loading methods, provided the lipid used to coat the IONPs was the same as that constituting the microbubble shell. The highest loading of IONPs per microbubble was also achieved with the surface loading method, and these microbubbles were the most responsive to an applied magnetic field, showing a 3-fold increase in the number of retained microbubbles compared to other groups. For the purpose of drug delivery, surface loading of IONPs could restrict the attachment of hydrophilic drugs to the microbubble shell, but hydrophobic drugs could still be incorporated. In contrast, although the incorporation of phospholipid IONPs produced more weakly magnetic microbubbles, it would not interfere with hydrophilic drug loading on the surface of the microbubble.

The Role of US Contrast Agents in US-Guided Biopsy of Focal Liver Lesions: A Pictorial Review.

US-guided percutaneous biopsy of focal liver lesions (FLL) is a classic interventional procedure performed by almost all radiology units. Typically, an incidental focal finding on US or a focal indeterminate lesion diagnosed on CT, MRI or PET/CT is referred for US-guided biopsy for final diagnosis. The introduction of microbubble US contrast agents has overcome some of the limitations of standard US in diagnosing FLLs by displaying the microvasculature together with the US morphology, which has increased both the sensitivity and the specificity. The combination of CEUS and intervention is facilitated by newer US equipment providing split-screen mode, which displays the CEUS mode alongside the standard US mode simultaneously on a single monitor. The puncture line is displayed in both modes as well as on the monitor. The interventional device (i. e., biopsy needle) is typically best visualized in the standard US mode, while the characteristic tissue pattern in an FLL is typically best visualized in CEUS mode. There are 3 main categories in which CEUS has an impact on US-guided biopsy of FLLs: • CEUS improves the visualization of FLLs • CEUS improves the quality of the biopsy specimen from an FLL • CEUS reduces the need for US-guided biopsy of an FLL In the two first categories, CEUS is utilized simultaneously with US-guided biopsy to ensure correct needle targeting. In the last category, US-guided biopsy of the FLL becomes superfluous as a result of the CEUS examination.

Evaluation of the role of platelet derived growth factor in papillary thyroid carcinoma by targeted ultrasound contrast agent

Objective To explore the role of targeted ultrasound contrast agent in the evaluation of angiogenesis in papillary thyroid carcinoma (PTC) in nude mice by constructing platelet derived growth factor receptor α (PDGFRα)-targeted ultrasound contrast agents, and to explore the effect of platelet derived growth factor (PDGF) on angiogenesis in PTC. Methods PDGFRα-targeted ultrasound contrast agents were constructed through biotin-avidin linkage, and blank micro-bubbles were served as control group. After the siRNA-PDGF BCPAP cell line was established, well prepared BCPAP cells or siRNA-PDGF-BCPAP cells were injected subcutaneously into the back of male BABL/C nude mice. The growth of the tumors was observed closely. All tumors of the normal group and the siRNA-PDGF group were examined by PDGFRα-targeted ultrasound contrast agents or blank micro-bubbles, and the dynamic images were analyzed quantitatively by QontraXt software.All mice were sacrificed after ultrasonography. Microvessels density (MVD) in the tumors was counted by CD31 staining and the expressions of vascular endothelial growth factor(VEGF) and PDGF were detected by Western Blot. Results PTCs in nude mice were enhanced well by PDGFRα-targeted ultrasound contrast agent or blank micro-bubbles. The peak of tumors in siRNA-PDGF group was significantly lower than that in the normal group [(43.085±13.244)% vs (57.428±10.952)%, P=0.004]; MVD of the siRNA-PDGF group was significantly less than that of the normal group [(23.200±6.017)strips/view vs (35.000±8.456)strips/view, P=0.012]. The protein expression of PDGF and VEGF in siRNA-PDGF group were significantly lower than those in the normal group, respectively (0.142±0.058 vs 0.269±0.102, P=0.002; 0.096±0.036 vs 0.158±0.072, P=0.016). Conclusions PDGF plays an important role in angiogenesis of PTC, it maybe a new ultrasound molecular imaging method for monitoring tumor angiogenesis. Key words: Ultrasonography; Thyroid neoplasms; Receptor, platelet derived growth factor alpha; Contrast media, Target; Angiogenesis