In Vitro and In Vivo Behavioral Evaluation of Condensed Lipid-Coated Perfluorocarbon Nanodroplets.

OBJECTIVE: Phase-shift contrast agents consist of a liquid perfluorocarbon core that can be vaporized by ultrasound to generate echogenic contrast with excellent spatiotemporal control. The purpose of the present work was to evaluate the in vitro and in vivo behavior of condensed lipid-shelled nanodroplets (NDs) using different analytical procedures. METHODS: Perfluorobutane NDs were prepared by condensation of precursor fluorescently labeled lipid-shelled microbubbles (MBs) and were characterized in terms of size distribution, gas core content and in vitro stability in blood, as well as for their acoustic vaporization behavior using a custom-made setup. In particular, the in vivo behavior of the NDs was thoroughly investigated after intravenous bolus injection in rats. To this end, we report, for the first time, the efficient use of three complementary detection procedures to assess the in vivo persistence of NDs: (i) ultrasound contrast imaging of vaporized NDs, (ii) gas chromatography-mass spectrometry to determine the perfluorobutane core content and (iii) fluorescence intensity measurement in the collected blood samples. RESULTS: The Coulter Counter Multisizer results confirmed the size distribution shift post-condensation. Furthermore, similar PFB concentrations from MB and ND suspensions were obtained, indicating an exceptionally low rate of MB breakage and spontaneous nanodroplet vaporization. As expected, these nanoscale droplets have longer circulation times compared with clinically approved MBs, and only slight variations in half-life were observed between the three monitoring procedures. Finally, echogenic signal observed in focal areas of the liver and spleen after vaporization was confirmed by accumulation of fluorescent nanodroplets in these organs. CONCLUSION: These results further contribute to our understanding of both the in vitro and in vivo behavior of sono-responsive nanodroplets, which is key to enabling efficient clinical translation.

Ultrasound molecular imaging of transient acute myocardial ischemia with a clinically translatable P- and E-selectin targeted contrast agent: correlation with the expression of selectins.

OBJECTIVE: The diagnosis of acute coronary syndrome remains challenging especially in patients without clear symptoms or electrocardiographic and/or biomarker features. A hallmark of ischemia/reperfusion is activation of endothelial cells leading to altered expression of molecular markers, including selectins. In this context, we aimed to validate the value of ultrasound molecular imaging for detecting transient myocardial ischemia by using a clinically translatable dual P- and E-selectin-targeted ultrasound contrast agent (UCA) and microbubble (MB(selectin)). MATERIAL AND METHODS: Transient (20 minutes) myocardial ischemia of rat heart was produced by ligation of the left anterior descending coronary artery ligation followed by 2-, 5-, or 24-hour reperfusion. Imaging of the transient ischemic event was achieved by the use of MB(selectin). Performance of this clinically translatable targeted UCA was compared with that of antibody-targeted streptavidin MBs. Finally, immunohistochemistry staining of rat myocardial ischemic tissue was performed to assess expression of selectins accessible to targeted UCA. RESULTS: In rats subjected to myocardial ischemia (20 minutes) followed by reperfusion (2 hours), injection of MB(selectin) produced high late phase (ie, 10-minute postinjection) ultrasound molecular imaging enhancement in the myocardium, which colocalized with the ischemic area. Late phase enhancement persisted 5 and 24 hours after reperfusion. Similarly, the use of MBP and MBE, comprising antibodies specific for P- and E-selectin, respectively, showed high late-phase enhancement within the ischemic area compared with remote myocardial tissue. Two and 5 hours after ischemia has resolved, a persistent expression of these 2 selectins was detected. After 24 hours of reperfusion, only MBE produced late phase enhancement within the ischemic myocardium. Immunohistochemical findings revealed that both P- and E-selectin were expressed and accessible on the surface of the activated endothelium 2 and 5 hours after the acute ischemic event, whereas only E-selectin remained accessible after 24 hours. CONCLUSIONS: Ultrasound molecular imaging of transient myocardial ischemia using dual selectin-targeted UCA is able to monitor the time course of expression of selectins after resolution of the ischemic event, paving the way for a large clinical diagnostic window.

Use of ultrasound contrast agent microbubbles in preclinical research: recommendations for small animal imaging.

Ultrasound contrast imaging techniques represent a real opportunity to improve efficiency in the preclinical drug discovery and development process. Ultrasound contrast agents (UCAs) combined with specific ultrasound contrast detection modes provide real-time, high spatial resolution of both organ and lesion blood perfusion, the so-called dynamic contrast-enhanced ultrasound imaging. With the advent of targeted UCA, ultrasound molecular imaging is gaining momentum in molecular imaging, particularly because of the simultaneous real-time anatomical and functional/molecular imaging capabilities. In preclinical research, contrast-enhanced ultrasound imaging, with either nontargeted or targeted UCA, is a fast-growing imaging modality that has not yet been standardized compared with other imaging modalities. Contrast-enhanced ultrasound imaging is an operator-dependent imaging modality, requiring adherence to rigorous step-by-step protocols. In this article, which is intended for advanced, hands-on researchers, we report key factors that can lead to variability in preclinical results and recommend some preventive methods to limit or cancel their effect on the final results. Standardized procedures are a prerequisite for acceptance of new contrast-enhanced ultrasound imaging methods to eliminate factors that could distort results, improve the reproducibility between different centers and studies, and, therefore, allow translation to clinical application.

Ultrasound molecular imaging contrast agent binding to both E- and P-selectin in different species.

OBJECTIVES: Ultrasound molecular imaging is increasingly used in preclinical studies to measure the expression of vascular markers during inflammation process. In this context, a new ultrasound contrast agent functionalized with a recombinant P-selectin glycoprotein ligand-1 analogue (rPSGL-Ig) was developed (MBrPSGL-Ig). This agent was assayed in vitro and in vivo to evaluate its binding performance and potential to image expression of inflammatory markers E- and P-selectin. Performance of this newly developed agent was compared with that of antibody (MBAb) or sialyl Lewis X (MBsLe) containing microbubbles and with control microbubbles (MBC). MATERIALS AND METHODS: The targeted ultrasound contrast agents were prepared by coupling biotin-conjugated ligands onto streptavidin-functionalized microbubbles. First, in vitro experiments were performed to measure the adhesion efficiency of these microbubble constructs under static or flow conditions (114 sec), on cell monolayer (human umbilical vein endothelial cells and bEnd.5), or coatings of E- or P-selectin of various animal species, respectively. Second, molecular imaging studies were performed in a rat inflammatory model 24 hours after intramuscular injection of lipopolysaccharide in the hind limb. Finally, immunohistochemistry staining of rat inflamed muscle tissue was performed to assess expression of E- and P-selectin. RESULTS: Microbubbles functionalized with rPSGL-Ig (MBrPSGL-Ig) displayed firm in vitro binding on the coating of both recombinant E- or P-selectin, with an efficiency similar to microbubbles comprising antibody specific for E-selectin (MBE) or P-selectin (MBP). In contrast, lower binding capacity was measured with MBsLe. At the surface of inflamed endothelial cells, MBrPSGL-Ig were able to interact specifically with E- and P-selectin. Binding specificity was demonstrated by performing blocking experiments with target-specific antibodies, resulting in an 80% to 95% decrease in binding. Ten minutes after microbubble injection, echo signal measured with MBrPSGL-Ig in the inflamed muscles was 20-fold higher compared with MBC. Moreover, the in vivo adhesion of MBrPSGL-Ig was 2- and 7-fold higher compared with P-selectin or E-selectin-specific microbubbles, respectively. Immunohistochemistry revealed a temporal coexpression of E- and P-selectin in the vascular bed of inflamed rat muscle 24 hours after lipopolysaccharide injection. CONCLUSION: The molecular imaging study demonstrates that MBrPSGL-Ig provide imaging signal higher than those measured with antibody or sialyl Lewis X containing microbubbles. These results suggest that MBrPSGL-Ig is a powerful agent to image the expression of both E- and P-selectin in the context of an inflammatory process.

Ultrasound molecular imaging of VEGFR2 in a rat prostate tumor model using BR55.

OBJECTIVES: To evaluate BR55, a new VEGFR2-specific ultrasound contrast agent, for imaging prostate tumors in an orthotopic model in the rat. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.