Ji-Bin Liu

Ultrasound contrast agents: A review

During the past 25 years, many attempts have been made to establish effective ultrasound contrast agents for both cardiac and noncardiac applications. The ideal ultrasound contrast agent would be: (a) nontoxic; (b) injectable intravenously; (c) capable of passing through the pulmonary, cardiac and capillary circulations; and (d) stable for recirculation. A variety of potential ultrasound contrast agents have been or are now under development. Present and future ultrasound contrast agents should provide for increased diagnostic capabilities in a variety of normal and abnormal vessels and organs throughout the body. These agents will enhance tumor vascularity, delineate areas of ischemia, as well as improve visualization of vascular stenosis. Future developments with modification of ultrasound equipment should increase the capabilities of these agents to improve imaging as well as Doppler sensitivity.

Gold‐Nanoshelled Microcapsules: A Theranostic Agent for Ultrasound Contrast Imaging and Photothermal Therapy

The term theranostics, which is derived from “diagnostics” and “therapy”, refers to a treatment strategy that combines a diagnostic test and a specific therapy based on the test results. This integration of diagnostic imaging capability with therapy is critical in addressing the challenges of cancer heterogeneity and adaptation. Therefore, theranostic agents have received a great deal of recent research interest in cancer diagnosis and treatment. Among all the diagnostic imaging techniques, ultrasound imaging has a unique advantage because of its features of real-time, low-cost, high safety, and ease of incorporation into portable devices. With the use of ultrasound contrast agents (UCAs), the resolution and sensitivity of clinical ultrasound imaging have been greatly improved. Microcapsules composed of poly(lactic acid) (PLA), which has outstanding biocompatibility and biodegradability, show good ultrasound contrast-enhancing capabilities and other advantages: they have good mechanical strength and are thus stable, they can load either hydrophilic or hydrophobic species or both, and they are surface-charged and have functional groups on the surface so that they could be easily modified to introduce further practical features. Gold nanostructures exhibit good biocompatibility as well as excellent optical and electronic properties, thus allowing use in biological and medical applications. Gold nanoshells have a spherical dielectric core particle surrounded by a thin nanoscale gold shell. By controlling the thickness of the gold shell and the diameter of the core, the plasmon resonance and the resulting optical absorption of gold nanoshells can be tuned to the near-infrared (NIR) region, where the absorption of human tissues is minimal and penetration is optimal. On the other hand, the strong optical absorption of nanoshells can rapidly increase the local temperature under NIR irradiation. Therefore, the gold nanoshells can be used as photoabsorbers for remote NIR photothermal ablation therapy. Lasers and photoabsorbers such as gold nanostructures are used to carry out cancer treatment in photothermal therapy. However, the location and size of cancers must be identified before therapy, the treatment procedure needs to be monitored in real time during therapy, and the effectiveness has to be assessed after therapy. Contrast-enhanced ultrasound imaging could be the technique of choice to address these tasks. Therefore, the development of goldnanoshell-based UCAs could operate as a multifunctional theranostic agent for imaging-guided photothermal therapy. We have developed a novel multifunctional theranostic agent based on gold-nanoshelled microcapsules (GNS-MCs) by electrostatic adsorption of gold nanoparticles as seeds onto the polymeric microcapsule surfaces, followed by the formation of gold nanoshells by using a surface seeding method (Figure 1). The polymeric microcapsules were generated from PLA and polyvinyl alcohol (PVA) materials by employing the water-in-oil-in-water (W/O/W) double-emulsion method, and were negatively charged with a zeta potential of about 25 mV. Upon exposure to positively charged poly(allyl-

Subharmonic Imaging with Microbubble Contrast Agents: Initial Results

The subharmonic emission from insonified contrast microbubbles was used to create a new imaging modality called Subharmonic Imaging. The subharmonic response of contrast microbubbles to ultrasound pulses was first investigated for determining adequate acoustic transmit parameters. Subharmonic A-lines and gray scale images were then obtained using a laboratory pulse-echo system in vitro and a modified ultrasound scanner in vivo. Excellent suppression of all backscattered signals other than from contrast microbubbles was achieved for subharmonic A-lines in vitro while further optimization is required for in vivo gray scale subharmonic images.

Clinical applications of ultrasound contrast agents

Within the last decade safe and practical ultrasound contrast agents have been introduced. Most of these are based on gas-filled microbubbles, which markedly enhance Doppler signals and, in some cases, also gray-scale images. The clinical improvements expected from ultrasound contrast is reviewed. Tissue-specific contrast agents constitute another area of potential clinical significance. One particular agent is taken up by the reticulo-endothelial system and produces so-called acoustic emission signals when imaged. An introduction to the unique clinical applications of acoustic emission is given. Harmonic imaging is a new contrast-specific imaging modality, which utilizes the nonlinear properties of some agents in an attempt to alleviate current limitations of ultrasound contrast studies. Examples of harmonic images are presented.

Galactose-based intravenous sonographic contrast agent : experimental studies

A galactose‐based sonographic contrast agent, which produces stable microbubbles capable of traversing the cardiopulmonary circulation, was used to enhance Doppler signals in blood vessels of varying size after intravenous injection. A series of experiments using dogs, rabbits, and woodchucks was conducted to establish the ability of the agent to enhance the reflectivity of normal tissue, tumor tissue, and blood. Although no enhancement was perceptible in tissue on the sonogram, significant enhancement of color and spectral Doppler signals was demonstrated in a variety of vessels. These included the aorta, vena cava, and portal vein as well as such small vessels as those of the retina of the eye, renal cortex, liver parenchyma, and gallbladder wall. Both spectral and color Doppler enhancement was shown in naturally occurring woodchuck hepatomas. Peak Doppler signal enhancement after bolus injection was approximately 10 dB with a dose of 0.01 ml/kg. Recirculation of the agent provided enhancement after intravenous bolus injection for more than 3 min. With a steady intravenous infusion of 0.2 ml/min/kg, Doppler signal enhancement of about 14 dB was maintained continuously for more than 5 min. The results of these animal experiments, in particular in small vessels and with recirculation after intravenous injection, suggest excellent potential for future clinical applications.

On the feasibility of real-time, in vivo harmonic imaging with proteinaceous microspheres

Harmonic imaging is a new contrast‐specific imaging modality, which utilizes the nonlinear properties of microbubble‐based sonographic contrast agents by transmitting at the fundamental frequency but receiving at the second harmonic frequency. The feasibility of improving the detection of slow, small‐volume blood flow using real‐time harmonic imaging has been investigated in vivo. Proteinaceous microspheres (FS069) were administrated to four dogs, two woodchucks (with multiple hepatomas), and one rabbit. Three different scanners were used to obtain real‐time images of kidneys and liver (including vessels) in harmonic and conventional gray scale and color flow modes. The duration of contrast enhancement lasted significantly longer in harmonic than in conventional modes (on average 87 s; P = 0.008). Harmonic images were less susceptible to artifacts, such as acoustic shadowing, and a clear increase in the (flow) signal‐to‐noise ratio was observed. These preliminary in vivo results demonstrate the feasibility of performing real‐time, contrast‐enhanced harmonic imaging, but further studies are required to establish clinical efficacy.

Real-time ultrasound elastography: its potential role in assessment of breast lesions.

We evaluated whether real-time ultrasound elastography (USE) performed in addition to conventional ultrasound (US) can improve the differentiation of benign from malignant breast lesions. Both conventional US and real-time USE were performed in 112 consecutive patients with 139 breast lesions using a Hitachi EUB-8500 US system. Each lesion was assigned an elasticity score according to the degree and distribution of strain induced manually by mild compression. The USE scores (1 to 5) were compared with the BI-RADS assessment categories (1 to 5) obtained with conventional US. Sensitivity, specificity and overall accuracy of each method were determined with surgical pathology as the gold standard. There were 70 benign and 69 malignant lesions. The mean elasticity score was significantly higher for malignant lesions than for benign lesions (4.33 +/- 0.11 vs. 2.10 +/- 0.13, p < 0.01). When a cutoff point of 4 was used, the sensitivity, specificity and accuracy were 85.5, 88.6 and 87% for USE and 94.2, 87.1 and 90.6% for conventional US, respectively. Of the 64 lesions assessed as BI-RADS 2 or 3(i.e., benign) based on conventional US, two were scored as 4 and 5 (i.e., malignant) using USE and were subsequently proven to be malignant. Of the 75 lesions with BI-RADS 4 or 5 category from conventional US, one was scored as a category 1 (benign) with USE and found to be benign by pathology. Our study results suggest that the addition of USE imaging to conventional US could be helpful in the detection and characterization of breast masses.

Development and optimization of a doxorubicin loaded poly(lactic acid) contrast agent for ultrasound directed drug delivery

An echogenic, intravenous drug delivery platform is proposed in which an encapsulated chemotherapeutic can travel to a desired location and drug delivery can be triggered using external, focused ultrasound at the area of interest. Three methods of loading poly(lactic acid) (PLA) shelled ultrasound contrast agents (UCA) with doxorubicin are presented. Effects on encapsulation efficiency, in vitro enhancement, stability, particle size, morphology and release during UCA rupture are compared by loading method and drug concentration. An agent containing doxorubicin within the shell was selected as an ideal candidate for future hepatocellular carcinoma studies. The agent achieved a maximal drug load of 6.2 mg Dox/g PLA with an encapsulation efficiency of 20.5%, showed a smooth surface morphology and tight size distribution (poly dispersity index=0.309) with a peak size of 1865 nm. Acoustically, the agent provided 19 dB of enhancement in vitro at a dosage of 10 microg/ml, with a half life of over 15 min. In vivo, the agent provided ultrasound enhancement of 13.4+/-1.6 dB within the ascending aorta of New Zealand rabbits at a dose of 0.15 ml/kg. While the drug-incorporated agent is thought to be well suited for future drug delivery experiments, this study has shown that agent properties can be tailored for specific applications based on choice of drug loading method.

The fabrication of novel nanobubble ultrasound contrast agent for potential tumor imaging

Novel biocompatible nanobubbles were fabricated by ultrasonication of a mixture of Span 60 and polyoxyethylene 40 stearate (PEG40S) followed by differential centrifugation to isolate the relevant subpopulation from the parent suspensions. Particle sizing analysis and optical microscopy inspection indicated that the freshly generated micro/nanobubble suspension was polydisperse and the size distribution was bimodal with large amounts of nanobubbles. To develop a nano-sized contrast agent that is small enough to leak through tumor pores, a fractionation to extract smaller bubbles by variation in the time of centrifugation at 20g (relative centrifuge field, RCF) was suggested. The results showed that the population of nanobubbles with a precisely controlled mean diameter could be sorted from the initial polydisperse suspensions to meet the specified requirements. The isolated bubbles were stable over two weeks under the protection of perfluoropropane gas. The acoustic behavior of the nano-sized contrast agent was evaluated using power Doppler imaging in a normal rabbit model. An excellent power Doppler enhancement was found in vivo renal imaging after intravenous injection of the obtained nanobubbles. Given the broad spectrum of potential clinical applications, the nano-sized contrast agent may provide a versatile adjunct for ultrasonic imaging enhancement and/or treatment of tumors.

Utility of contrast-enhanced ultrasound for evaluation of thyroid nodules.

BACKGROUND No conventional imaging method reliably distinguishes between benign and malignant thyroid nodules. Our objectives were to characterize the enhancement patterns of thyroid nodules on gray-scale contrast-enhanced ultrasound (US) and to evaluate whether these patterns were useful in the differential diagnosis of thyroid nodules. METHODS Ninety-five patients, scheduled for surgery for thyroid nodules detected by gray-scale sonography, were enrolled in this prospective study. In all, there were 104 nodules (47 papillary carcinomas, 3 medullary carcinomas, 1 metastatic carcinoma, 44 hyperplasia nodule, 7 follicular adenomas, 1 suture granulomas, and 1 Hashimotos disease). After intraveneous (i.v.) injection of a 1.2 mL bolus of SonoVue, lesions were scanned with real-time gray-scale pulse inversion harmonic imaging US for at least 3 minutes at low mechanical index (MI) (0.05 to 0.08). The enhancement patterns were classified into one of four patterns by two experienced readers. RESULTS After administration of SonoVue, four enhancement patterns (homogeneous, heterogeneous, ring-enhancing, and no enhancement) were observed. Four benign and 3 malignant nodules had homogeneous enhancement pattern, 4 benign and 45 malignant nodules had heterogeneous enhancement, 44 benign and 3 malignant nodules had ring enhancement, and 1 benign nodule had no enhancement. There was a significant difference between benign and malignant nodules (p < 0.001). The benign thyroid nodules showed four enhancement patterns: ring enhancement 44/53 (83.0%), homogeneous enhancement 4/53 (7.5%), heterogeneous enhancement 4/53 (7.5%), and no enhancement 1/44 (1.9%). The malignant thyroid nodules showed three enhancement patterns: heterogeneous enhancement 45/51 (88.2%), ring enhancement 3/51 (5.9%), and homogeneous enhancement 3/51 (5.9%). Ring enhancement correlated highly with a benign diagnosis (sensitivity 83.0%, specificity 94.1%, positive predictive value 93.6%, negative predictive value 84.2%, and accuracy 88.5%). Heterogeneous enhancement correlated highly with a malignant diagnosis (sensitivity 88.2%, specificity, 92.5% positive predictive value 91.8%, negative predictive value 89.1%, and accuracy 90.4%). In both mixed and solid nodules, ring enhancement was highly predictive of a benign finding, whereas heterogeneous enhancement was highly predictive of a malignant finding. CONCLUSIONS Contrast-enhanced US enhancement patterns were different in benign and malignant lesions. Ring enhancement was predictive of benign lesions, whereas heterogeneous enhancement was helpful for detecting malignant lesions.