Ultrasound Signal Enhancement Research Articles

Dual-Mode Fluorescence/Ultrasound Imaging with Biocompatible Metal-Doped Graphene Quantum Dots.

Sonography offers many advantages over standard methods of diagnostic imaging due to its non-invasiveness, substantial tissue penetration depth, and low cost. The benefits of ultrasound imaging call for the development of ultrasound-trackable drug delivery vehicles that can address a variety of therapeutic targets. One disadvantage of the technique is the lack of high-precision imaging, which can be circumvented by complementing ultrasound contrast agents with visible and, especially, near-infrared (NIR) fluorophores. In this work, we, for the first time, develop a variety of lightly metal-doped (iron oxide, silver, thulium, neodymium, cerium oxide, cerium chloride, and molybdenum disulfide) nitrogen-containing graphene quantum dots (NGQDs) that demonstrate high-contrast properties in the ultrasound brightness mode and exhibit visible and/or near-infrared fluorescence imaging capabilities. NGQDs synthesized from glucosamine precursors with only a few percent metal doping do not introduce additional toxicity in vitro, yielding over 80% cell viability up to 2 mg/mL doses. Their small (<50 nm) sizes warrant effective cell internalization, while oxygen-containing surface functional groups decorating their surfaces render NGQDs water soluble and allow for the attachment of therapeutics and targeting agents. Utilizing visible and/or NIR fluorescence, we demonstrate that metal-doped NGQDs experience maximum accumulation within the HEK-293 cells 6-12 h after treatment. The successful 10-fold ultrasound signal enhancement is observed at 0.5-1.6 mg/mL for most metal-doped NGQDs in the vascular phantom, agarose gel, and animal tissue. A combination of non-invasive ultrasound imaging with capabilities of high-precision fluorescence tracking makes these metal-doped NGQDs a viable agent for a variety of theragnostic applications.

Intracellular vesicle entrapment of nanobubble ultrasound contrast agents targeted to PSMA promotes prolonged enhancement and stability in vivo and in vitro.

Gas-core nanoscale bubbles (or nanobubbles) have gained significant recent attention as promising contrast agents for cancer molecular imaging using medical ultrasound. Previous work has shown that active targeting of nanobubbles to tumor biomarkers such as the prostate-specific membrane antigen (PSMA) significantly prolongs ultrasound signal enhancement, which is a critical feature for successful tumor diagnosis. However, the specific mechanism behind this effect is not well understood, and has not been previously studied in detail. Thus, in the current work, we investigated the process of PMSA- targeted nanobubble transport in tumors across different scales from in vivo whole tumor imaging using high-frequency dynamic contrast-enhanced ultrasound to intracellular confocal imaging and, molecularly using headspace gas chromatography/mass spectrometry. Data demonstrated that, indeed, molecular targeting of nanobubbles to the PSMA biomarker prolongs their tumor uptake and retention across the entire tumor volume, but with variability due to the expected tumor heterogeneity. Importantly, in vitro, the active targeting of NBs results in internalization via receptor-mediated endocytosis into the target cells, and the co-localization with intracellular vesicles (late-stage endosomes/lysosomes) significantly prolongs perfluorocarbon gas retention within the cells. This has not been directly observed previously. These results support the potential for nanobubbles to enable highly specific, background-free diagnostic imaging of the target cells/tissues using ultrasound.

Acidic pH-Activated Gas-Generating Nanoparticles with Pullulan Decorating for Hepatoma-Targeted Ultrasound Imaging.

Contrast-enhanced ultrasound (US) is a widely used imaging modality for hepatocellular carcinoma diagnosis. Mostly, US imaging is confined to the intravascular process because of the limitation of the microbubble contrast agent currently utilized. Targeted contrast agents that incline to accumulate in tumor tissue or tumor cells and enhance the US signal may advance the sensitivity of ultrasonography and exploit the dimension of US imaging of tumor at the molecular level. In this study, we developed CaCO3/pul-PCB (CPP) hybrid nanoparticles with hepatoma-targeting pullulan decorating on the surface through a mineralization route using the pullulan- graft-poly(carboxybetaine methacrylate) (pul-PCB) copolymer as a modifier. This particle was stable in blood physiological pH and generated echogenic CO2 bubbles under tumoral acidic conditions, which enabled the US signal enhancement. Upon intravenous injection, CPP hybrid nanoparticles accumulated efficiently in tumor tissue and exhibited sixfold contrast enhancement in 35 min at the tumor site in the hepatoma-bearing mice model. By contrast, there was barely any signal change in normal liver tissue. Therefore, the presented CPP hybrid nanoparticle is a promising contrast agent for effective US imaging of hepatoma.

Dual photoacoustic monitoring in laser ablation synthesis of silver nanoparticles to find in situ their fluence threshold formation

Abstract In this study, a dual pulsed photoacoustic technique was applied to monitor, in real time, the laser ablation synthesis in solution (LASiS) of silver nanoparticles. It was found that, at low optical fluence values, the ultrasound signal associated to the laser ablation process exhibited constant amplitude, independently of the ablation time. Further analysis confirmed that, under these conditions, the ablation process provided only suspended micro-sized Ag fragments. However, fluence values above 400 mJ cm−2 promoted the formation of nanoparticles, accompanied by amplitude changes in the photoacoustic signal. Determination of this threshold condition establishes a fingerprint in the photoacoustic signal that can be used to identify, in situ and in real time, the beginning of nanoparticle synthesis. Supplementary, the nanoparticle formation was also photoacoustically monitored during the ablation process introducing an additional laser pulse train of low fluence value propagating perpendicularly to the pulse train used for ablation. These laser pulses did not interfere with the nanoparticle LASiS process, but renders an ultrasound signal enhancement due to the surface plasmon resonance of metal nanoparticles. The characterization of the LASiS process through the photoacoustic technique can provide information about nanoparticle concentration and other synthesis features of interest.

Gas-generating nanoparticles for molecular ultrasound imaging

Diagnostic ultrasound imaging often relies on contrast agents. Most common contrast agents are microbubbles that are confined to vascular compartments and molecular targets because micrometer size particles cannot escape through endothelial barriers. Therefore, a desired ultrasound contrast agent should consist of nanometer scale particles that are capable of escaping from vasculature, penetrating into tissue, and then generating sufficient contrast once they reach the target site. We developed a novel contrast agent—plasmonic nanoparticles covered by azide compounds (AzNPs), capable of on-demand laser-induced gas generation via photolysis of azide functional groups. The 50 nm diameter AzNPs were small enough to penetrate the endothelial barrier. Upon laser activation, the AzNPs generated nitrogen gas which served as ultrasound contrast agent. The feasibility of the AzNPs to produce ultrasound contrast was tested within polyethylene tube under laminar flow. Upon laser irradiation, significant ultrasound signal enhancement was observed due to the N2 gas bubbles generated by the photolysis of AzNPs. These and other studies suggest that the AzNPs may enable the ultrasound diagnosis of various diseases that conventional microbubbles cannot detect. Furthermore, these optically absorbing particles can also be used for imaging and therapeutic applications of light and sound such as ultrasound-guided photoacoustic imaging.

Renal Retention of Lipid Microbubbles: A Potential Mechanism for Flank Discomfort During Ultrasound Contrast Administration

The etiology of flank pain sometimes experienced during the administration of ultrasound contrast agents is unknown. The aim of this study was to investigate whether microbubble ultrasound contrast agents are retained within the renal microcirculation, which could lead to either flow disturbance or local release of vasoactive and pain mediators downstream from complement activation. Retention of lipid-shelled microbubbles in the renal microcirculation of mice was assessed by confocal fluorescent microscopy and contrast-enhanced ultrasound imaging with dose-escalating intravenous injection. Studies were performed with size-segregated microbubbles to investigate physical entrapment, after glycocalyx degradation and in wild-type and C3-deficient mice to investigate complement-mediated retention. Urinary bradykinin was measured before and after microbubble administrations. Renal contrast-enhanced ultrasound in human subjects (n= 13) was performed 7 to 10 min after the completion of lipid microbubble administration. In both mice and humans, microbubble retention was detected in the renal cortex by persistent contrast-enhanced ultrasound signal enhancement. Microbubble retention in mice was linearly related to dose and occurred almost exclusively in cortical glomerular microvessels. Microbubble retention did not affect microsphere-derived renal blood flow. Microbubble retention was not influenced by glycocalyx degradation orby microbubble size, thereby excluding lodging, but was reduced by 90% (P < .01) in C3-deficient mice. Urinary bradykinin increased by 65% 5 min after microbubble injection. Lipid-shelled microbubbles are retained in the renal cortex because of complement-mediated interactions with glomerular microvascular endothelium. Microbubble retention does not adversely affect renal perfusion but does generate complement-related intermediates that are known to mediate nociception and could be responsible for flank pain.

Effect of Microbubble Ligation to Cells on Ultrasound Signal Enhancement

Molecular imaging with contrast-enhanced ultrasound (CEU) relies on the detection of microbubbles retained in regions of disease. The aim of this study was to determine whether microbubble attachment to cells influences their acoustic signal generation and stability. Biotinylated microbubbles were attached to streptavidin-coated plates to derive density versus intensity relations during low- and high-power imaging. To assess damping from microbubble attachment to solid or cell surfaces, in vitro imaging was performed for microbubbles charge-coupled to methacrylate spheres and for vascular cell adhesion molecule-1-targeted microbubbles attached to endothelial cells. Signal enhancement on plates increased according to acoustic power and microbubble site density up to 300 mm. Microbubble signal was reduced by attachment to solid spheres during high- and low-power imaging but was minimally reduced by attachment to endothelial cells and only at low power. Attachment of targeted microbubbles to rigid surfaces results in damping and a reduction of their acoustic signal, which is not seen when microbubbles are attached to cells. A reliable concentration versus intensity relationship can be expected from microbubble attachment to 2-dimensional surfaces until a very high site density is reached.

Prolonging the Ultrasound Signal Enhancement From Thrombi Using Targeted Microbubbles Based on Sulfur-Hexafluoride–Filled Gas

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. 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. 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. 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.

Characteristics of SonoVuetrade mark.

The advances made by ultrasonography in the last decade, in parallel with the development of ultrasound contrast agents, have opened a wide range of potential breakthroughs in the field of ultrasound imaging. SonoVue(trade mark) is a new echocontrast agent made of microbubbles stabilized by phospholipids and containing sulphur hexafluoride (SF6), an innocuous gas. The suspension of the microbubbles is stable over the time following reconstitution. The bubble concentration of SonoVue(trade mark) is between 100 and 500 million per ml. The mean bubble diameter is 2.5 µm and more than 90% of the bubbles are smaller than 8 µm. Following intravenous injection, the bubble suspension is submitted to pressure increases. SF(6), a high molecular weight gas with low solubility in water, was selected since laboratory tests showed that it confers to the bubbles a good resistance to pressure changes as those that occur in the left ventricle, in the pulmonary capillaries, or in the coronary circulation. The high bubble concentration, combined with a favorable size distribution profile, provides SonoVue(trade mark) with a strong echogenicity. SonoVue(trade mark) shows a peak in the backscatter coefficient at about 3 MHz. With regard to the gas contained in the bubbles, its pharmacokinetics have been assessed during a study in human volunteers. Following intravenous administration of 0.3 ml/kg of SonoVue(trade mark) (i.e., approximately ten times the imaging dose), the blood level curve showed a distribution half-life of about 1 minute and an elimination half-life of about 6 minutes. More than 80% of the administered gas is exhaled via the lungs after 11 minutes. Extensive studies in animals and humans have confirmed the outstanding safety profile of this second generation contrast agent and its capability in providing a clinically useful ultrasound signal enhancement for the evaluation of cardiac function and extracardiac vessel abnormalities. Thanks to the long persistence of SF(6) microbubbles, SonoVue(trade mark) is also potentially useful in the assessment of myocardial perfusion, as well as microcirculatory disorders.