Subharmonic generation from ultrasonic contrast agents

Ultrasonic contrast agents are used to enhance backscatter from blood and thus aid in delineating blood from surrounding tissue. However, behaviour of contrast agents in an acoustic field is nonlinear and leads to harmonic components in the backscattered signal. Various research groups have investigated second-harmonic emissions. In this work, the subharmonic emission from contrast agents is investigated with a view towards potential use in imaging. It is shown that the microbubbles with various surface properties, such as contrast agents, generate significant subharmonics under various insonating conditions. Theoretical results as well as experimental results using Optison indicate the generation of strong subharmonics with burst insonation at twice the resonant frequency of the microbubble. It is suggested that subharmonic imaging may provide a better modality than second-harmonic imaging to delineate blood from tissue and will be of significant importance for imaging deep vessels, such as in echocardiography and vascular diseases, due to the high signal-to-clutter ratio of the subharmonic imaging.     

Superparamagnetic nanoparticle-inclusion microbubbles for ultrasound contrast agents

We have developed a new type of ultrasound (US) contrast agent, consisting of a gas core, a layer of superparamagnetic iron oxide Fe(3)O(4) nanoparticles (SPIO) and an oil in water outermost layer. The newly developed US contrast agent microbubbles have a mean diameter of 760 nm with a polydisperity index (PI) of 0.699. Our in vitro and in vivo experiments have shown that they have the following advantages compared to gas-encapsulated microbbubbles without SPIO inclusion: (1) they provide better contrast for US images; (2) the SPIO-inclusion microbubbles generate a higher backscattering signal; the mean grey scale is 97.9, which is 38.6 higher than that of microbubbles without SPIO; and (3) since SPIO can also serve as a contrast agent of magnetic resonance images (MRI) in vitro, they can be potentially used as contrast agents for double-modality (MRI and US) clinical studies.     

A new ultrasonic transducer for improved contrast nonlinear imaging

Second harmonic imaging has provided significant improvement in contrast detection over fundamental imaging. This improvement is a result of a higher contrast-to-tissue ratio (CTR) achievable at the second harmonic frequency. Nevertheless, the differentiation between contrast and tissue at the second harmonic frequency is still in many situations cumbersome and contrast detection remains nowadays as one of the main challenges, especially in the capillaries. The reduced CTR is mainly caused by the generation of second harmonic energy from nonlinear propagation effects in tissue, which hence obscures the echoes from contrast bubbles. In a previous study, we demonstrated theoretically that the CTR increases with the harmonic number. Therefore the purpose of our study was to increase the CTR by selectively looking to the higher harmonic frequencies. In order to be able to receive these high frequency components (third up to the fifth harmonic), a new ultrasonic phased array transducer has been constructed. The main advantage of the new design is its wide frequency bandwidth. The new array transducer contains two different types of elements arranged in an interleaved pattern (odd and even elements). This design enables separate transmission and reception modes. The odd elements operate at 2.8 MHz and 80% bandwidth, whereas the even elements have a centre frequency of 900 kHz with a bandwidth of 50%. The probe is connected to a Vivid 5 system (GE-Vingmed) and proper software is developed for driving. The total bandwidth of such a transducer is estimated to be more than 150% which enables higher harmonic imaging at an adequate sensitivity and signal to noise ratio compared to standard medical array transducers. We describe in this paper the design and fabrication of the array transducer. Moreover its acoustic properties are measured and its performances for nonlinear contrast imaging are evaluated in vitro and in vivo. The preliminary results demonstrate the advantages of such a transducer design for improved contrast detection.     

Ultrasonic characterization of ultrasound contrast agents

The main constituent of an ultrasound contrast agent (UCA) is gas-filled microbubbles. An average UCA contains billions per ml. These microbubbles are excellent ultrasound scatterers due to their high compressibility. In an ultrasound field they act as resonant systems, resulting in harmonic energy in the backscattered ultrasound signal, such as energy at the subharmonic, ultraharmonic and higher harmonic frequencies. This harmonic energy is exploited for contrast enhanced imaging to discriminate the contrast agent from surrounding tissue. The amount of harmonic energy that the contrast agent bubbles generate depends on the bubble characteristics in combination with the ultrasound field applied. This paper summarizes different strategies to characterize the UCAs. These strategies can be divided into acoustic and optical methods, which focus on the linear or nonlinear responses of the contrast agent bubbles. In addition, the characteristics of individual bubbles can be determined or the bubbles can be examined when they are part of a population. Recently, especially optical methods have proven their value to study individual bubbles. This paper concludes by showing some examples of optically observed typical behavior of contrast bubbles in ultrasound fields.     

Biodegradable iodinated polydisulfides as contrast agents for CT angiography

Current clinical CT contrast agents are mainly small molecular iodinated compounds, which often suffer from short blood pool retention for more comprehensive cardiovascular CT imaging and may cause contrast-induced nephropathy. In this work, we prepared polydisulfides containing a traditional iodinated CT contrast agent in order to optimize the pharmacokinetics of the agent and improve its safety. Initially acting as a macromolecular agent and achieving sharp blood vessel delineation, the polydisulfides can be reduced by endogenous thiols via disulfide–thiol exchange reaction to oligomers that can be readily excreted via renal filtration. Short polyethylene glycol (PEG) chain was also introduced to the polymers to further modify the in vivo properties of the agents. Strong and prolonged vascular enhancement has been generated with two new agents in mice (5–10 times higher blood pool enhancement than iodixanol). The polydisulfide agents gradually degraded and excreted via renal filtration. The gradual excretion process could prevent contrast-induced nephropathy. These results suggest that the biodegradable macromolecular CT contrast agents are promising safe and effective blood contrast agents for CT angiography and image-guided interventions.     

Safety and bio-effects of ultrasound contrast agents

The use of gas-filled microbubbles as ultrasound contrast agents raises potential safety concerns for diagnostic ultrasound imaging. A number of biological effects have been seen in experimental systems, including the induction of physiological response to cardiac exposures (premature ventricular contractions) and damage at a microvascular level (microvascular rupture and petechial haemorrhage). The literature indicates that a mechanical index (MI) of 0.4 represents the threshold above which microvascular bio-effects are seen in in vivo studies. Above this value, the extent of biological effects appears to increase rapidly with both increasing in situ peak negative acoustic pressure amplitude and with contrast agent concentration. While there is no proven evidence of harm resulting from clinical use of these agents, caution is recommended when contrast-enhanced imaging is undertaken.     

Ultrasound degradation of novel polymer contrast agents

This report describes an investigation into factors affecting the degradation of novel poly(lactic-co-glycolic acid) (PLGA) contrast agents. Contrast agents fabricated by two different methods and varying in acoustic properties were compared. The effect of ultrasound frequency (5 and 10 MHz) on degradation of the microcapsules was also studied. High-performance liquid chromatography was used to quantify the production of lactic and glycolic acid to monitor agent degradation. The degradation pattern from the microcapsules was found to be closely related to capsule morphology; the more acoustically efficient capsules (maximum enhancement of 25 dB at 5 MHz with 0.004 mg/mL) degraded at a faster rate than those with lower acoustical efficiency (maximum enhancement of 25 dB at 5 MHz only achieved with 0.6 mg/mL). The capsules also degraded fastest when insonated at the frequency at which they gave highest backscatter. In addition, despite the use of a 50:50 PLGA copolymer, more glycolic than lactic acid was released at early time points, which reflects the greater hydrophilicity of the glycolic acid residues, and greater degradation rate of glycolic acid repeat units. The results from this study provided unique insight into the degradation behavior of hollow PLGA microcapsules, and their potential in ultrasound diagnosis and therapy.     

Iron oxide MR contrast agents for molecular and cellular imaging

Molecular and cellular MR imaging is a rapidly growing field that aims to visualize targeted macromolecules or cells in living organisms. In order to provide a different signal intensity of the target, gadolinium-based MR contrast agents can be employed although they suffer from an inherent high threshold of detectability. Superparamagnetic iron oxide (SPIO) particles can be detected at micromolar concentrations of iron, and offer sufficient sensitivity for T2(*)-weighted imaging. Over the past two decades, biocompatible particles have been linked to specific ligands for molecular imaging. However, due to their relatively large size and clearance by the reticuloendothelial system (RES), widespread biomedical molecular applications have yet to be implemented and few studies have been reproduced between different laboratories. SPIO-based cellular imaging, on the other hand, has now become an established technique to label and detect the cells of interest. Imaging of macrophage activity was the initial and still is the most significant application, in particular for tumor staging of the liver and lymph nodes, with several products either approved or in clinical trials. The ability to now also label non-phagocytic cells in culture using derivatized particles, followed by transplantation or transfusion in living organisms, has led to an active research interest to monitor the cellular biodistribution in vivo including cell migration and trafficking. While most of these studies to date have been mere of the 'proof-of-principle' type, further exploitation of this technique will be aimed at obtaining a deeper insight into the dynamics of in vivo cell biology, including lymphocyte trafficking, and at monitoring therapies that are based on the use of stem cells and progenitors.     

造影剂在声场中的表现形式与声学造影新技术

心肌声学造影是近年来发展的一项新技术,本文就造影剂微泡在声场中的表现形式、机械指数的调节和微泡造影剂特异性显像新技术的发展等作一介绍,并说明针对不同研究目的进行机械指数调节的方法。     

Preparation of targeted microbubbles: ultrasound contrast agents for molecular imaging

Targeted ultrasound contrast agents can be prepared by attaching targeting ligands to the lipid, protein or polymer shell coating of gas-filled microbubbles. These materials are stable on storage, fully biocompatible and can be administered parenterally. Detection of microbubble contrast agents by ultrasound is very efficient (single particles with picogram mass can be visualized). Covalent or noncovalent binding techniques can be used to attach targeting ligands. Ligand-carrying microbubbles adhere to the respective molecular targets in vitro and in vivo. Several biomechanical methods are available to improve targeting efficacy, such as the use of a flexible tether spacer arm between the ligand and the bubble, and the use of folds on the microbubble shell, that project out, enhancing the contact area and increasing the length of the lever arm.     

mRNA-Lipoplex loaded microbubble contrast agents for ultrasound-assisted transfection of dendritic cells

In cancer immunotherapy the immune system should be triggered to specifically recognize and eliminate tumor cells in the patient's body. This could be achieved by loading dendritic cells (DCs) with tumor-associated antigens (TAAs). This can be achieved by transfecting DCs with messenger RNA encoding a tumor-associated antigen. Here we demonstrate transient transfection of dendritic cells by means of mRNA-lipoplexes bound to microbubbles. Microbubble-attached lipoplexes were introduced into the cells by applying ultrasound. Our data demonstrate that ultrasound-mediated delivery of mRNA-complexes led to efficient transfection of DCs. When mRNA encoding luciferase was used, maximal levels of the enzyme activity were detected 8 h after ultrasound application. Upon longer incubation protein expression gradually declined. This treatment did not affect viability of the cells. Intracellular localisation of mRNA-lipoplexes in DCs was determined by flow cytometry using fluorescently labeled lipoplexes. Over 50% of DCs contained fluorescently labeled mRNA-complexes. In the absence of additional maturation signals, transfection of immature DCs with mRNA-lipoplex loaded microbubbles and ultrasound application induced only a minor shift in the expression level of maturation markers (CD40 and CD86). However, in the presence of the activation stimulus (LPS), cells were able to further mature as shown by a significant up-regulation of CD40 expression. Thus, our results demonstrate that mRNA-lipoplex loaded microbubbles can serve as an applicable and safe tool for efficient mRNA-transfection of cultured DCs.     

钆螯合物磁共振成像对比剂的研究进展

在综合文献的基础上,对新型细胞外、肝特异性、网状内皮系统特异性、血池和其它组织特异性钆螯合物磁共振对比剂的研究进展作了较详细的介绍。提示新型组织特异性钆螯合物磁共振对比剂更安全更有效,也是当前钆螯合物磁共振对比剂研究的发展方向。     

Sensitive MRI detection of internalized T1 contrast agents using magnetization transfer contrast

This work addresses the possibility of using Magnetization Transfer Contrast (MTC) for an improved MRI detection of T₁ relaxation agents. The need to improve the detection threshold of MRI agents is particularly stringent when the contrast agents failed to accumulate to the proper extent in targeting procedures. The herein reported approach is based on the T₁ dependence of MT contrast. It has been assessed that MT contrast can allow the detection of a Gd‐containing agent at a lower detection threshold than the one accessible by acquiring T_1W images. Measurements have been carried out either in TS/A cells or in vivo in a syngeneic murine breast cancer model. The reported data showed that in cellular experiments the MTC method displays a better sensitivity with respect to the common T_1W experiments. In particular, the reached detection threshold allowed the visualization of samples containing only 2% of Gd‐labeled cells diluted in unlabeled cells. In vivo experiments displayed a more diversified scheme. In particular, the tumor region showed two distinct behaviors accordingly with the localization of the imaging probe. The probe located in the tumor core could be detected to the same extent either by T_1w or MTC contrast. Conversely, the agent located in the tumor rim was detected with a larger sensitivity by the MTC method herein described. Copyright © 2015 John Wiley & Sons, Ltd.     

Detectability of contrast agents for confocal reflectance imaging of skin and microcirculation

Confocal reflectance microscopy of skin and other tissues in vivo is currently limited to imaging at the cellular, nuclear and general architectural levels due to the lack of microstructure-specific contrast. Morphologic and functional imaging at specific organelle and microstructure levels may require the use of exogenous contrast agents in small (nontoxic) concentrations, from which weakly backscattered light must be detected in real time. We report an analysis based on Mie theory to predict detectability, in terms of signal-to-background and signal-to-noise ratios, of reflectance contrast agents within skin and microcirculation. The analysis was experimentally verified by detectability of (a) intravenously injected polystyrene microspheres that enhance the contrast of dermal microcirculation in Sprague-Dawley rats, and (b) acetic acid-induced compaction of chromatin that enhances nuclear morphology in normal and cancerous human skin. Such analyses and experiments provide a quantitative basis for developing the opto-biochemical properties and use of contrast agents and for designing confocal instrumentation to enable real-time detectability in vivo.     

Selective imaging of adherent targeted ultrasound contrast agents

The goal of ultrasonic molecular imaging is the detection of targeted contrast agents bound to receptors on endothelial cells. We propose imaging methods that can distinguish adherent microbubbles from tissue and from freely circulating microbubbles, each of which would otherwise obscure signal from molecularly targeted adherent agents. The methods are based on a harmonic signal model of the returned echoes over a train of pulses. The first method utilizes an 'image-push-image' pulse sequence where adhesion of contrast agents is rapidly promoted by acoustic radiation force and the presence of adherent agents is detected by the signal change due to targeted microbubble adhesion. The second method rejects tissue echoes using a spectral high-pass filter. Free agent signal is suppressed by a pulse-to-pulse low-pass filter in both methods. An overlay of the adherent and/or flowing contrast agents on B-mode images can be readily created for anatomical reference. Contrast-to-tissue ratios from adherent microbubbles exceeding 30 dB and 20 dB were achieved for the two methods proposed, respectively. The performance of these algorithms is compared, emphasizing the significance and potential applications in ultrasonic molecular imaging.     

The interaction of MRI contrast agents with phospholipids

The molecular interactions of three clinically used MRI contrast agents with lipid vesicles, consisting of egg phosphatidylcholine (EPC), have been studied using high-field NMR techniques. At a molar ratio of one contrast agent molecule to five phospholipid molecules, a significant increase in the proton resonance line width occurred for certain lipid head group moieties. A large decrease in the T1 relaxation times for the head group moieties was also observed. These two effects occurred regardless of the ionic status and the chelate structure of the three contrast agents. The structure of the contrast agents did, however, affect the magnitude of the two NMR parameter changes. These NMR effects also differed in magnitude amongst the various head group entities. The NMR effects were greatest for the head group moieties at or near the vesicle-water interface. The results are discussed in terms of the structure of the phospholipid-water interface. Since the use of contrast agents has become routine in clinical MRI, our results are of importance in terms of the interaction of the agents with physiological surfaces, many of which contain phospholipids. The understanding of such interactions should be of value not only for improved diagnostics, but also in the development of new contrast agents.     

Hydrogel based tissue mimicking phantom for in-vitro ultrasound contrast agents studies

Ultrasound medical imaging (UMI) is the most widely used image analysis technique, and often requires advanced in-vitro set up to perform morphological and functional investigations. These studies are based on contrast properties both related to tissue structure and injectable contrast agents (CA). In this work, we present a three-dimensional structure composed of two different hydrogels reassembly the microvascular network of a human tissue. This phantom was particularly suitable for the echocontrastographic measurements in human microvascular system. This phantom has been characterized to present the acoustic properties of an animal liver, that is, acoustic impedance (Z) and attenuation coefficient (AC), in UMI signal analysis in particular; the two different hydrogels have been selected to simulate the target organ and the acoustic properties of the vascular system. The two hydrogels were prepared starting from cellulose derivatives to simulating the target organ parenchyma and using a PEG-diacrylate to reproduce the vascular system. Moreover, harmonic analysis was performed on the hydrogel mimicking the liver parenchyma hydrogel to evaluate the ultrasound (US) distortion during echographic measurement. The phantom was employed in the characterization of an experimental US CA. Perfect agreement was found when comparing the hydrogel acoustical properties materials with the corresponding living reference tissues (i.e., vascular and parenchimal tissue).