| This study aims to prepare a novel nano-scale ultrasound contrast agent, which is lipid-coated and perfluorooctyl bromide (PFOB) inside, and to compare its characteristics and imaging enhancement with lipidic microbubbles of perfluoropropane (C3F8). Systematic studies were designed to evaluate which kind of ultrasound contrast agent is more suitable for site-targeted imaging enhancement. We hypothesized echocardiography with a site-targeted ultrasound contrast agent, whose specificity was imparted by use of biotinylated monoclonal antibodies, avidin and biotinylated nanoparticles, could provide a means to identify viable myocardium after ischemia-reperfusion injury. The present study includes two parts:Part 1. Comparison of characteristics and imaging enhancement between two nano-scale ultrasound contrast agents:PFOB lipidic particles and C3F8 lipidic microbubbles, a vitro experimental studyObjective Two kinds of nano-scale ultrasound contrast agents, PFOB lipidic particles and C3F8 lipidic microbubbles, were prepared. Systematic studies were designed to compare their characteristics, imaging enhancement, and resistance to acoustic pressure in vitro.Methods Lipidic particles were prepared with phospholipid and PFOB. Lipidic microbubbles were created with phospholipid and C3F8. Physico-chemical properties of the two kinds of ultrasound contrast agents were measured, including morphous, size distribution, Zeta potential, concentration and stability. PFOB lipidic particles with and without biotin, C3F8.lipidic microbubbles with and without biotin were prepared, respectively. Then, four kinds of ultrasound contrast agents were imaged with both fundamental and CPS imaging before and after avidin was added. Their signal intensity were compared with METLAB software. Both PFOB particles and C3F8 microbubbles were exposed in ultrasound field of lower(MI=0.28) and higher(MI=0.56) ultrasound pressure levels. Their signal intensity after different exposure time were compared with METLAB software. Results①Both PFOB lipidic particles and C3F8 microbubbles were sphericity under microscope. Their particle diameters were (152.30±35.99)nm and (774.59±108.59)nm, respectively (P< 0.05). Their Zeta potential were (-40.90±6.51)mV, (-14.80±3.97)mV, respectively (P< 0.05).②Concentration and size distribution of PFOB lipidic particles didn't alter during the whole observation. Concentration of C3F8 lipidic microbubbles were stable within 12 hours after they were prepared. However, its concentration began to reduce significantly after 24 hours (P＜0.05). Its particle diameters didn't alter within 2 days, but were larger after 4 days (P＜0.05).③Under microscope, dispersity of control lipidic particles/microbubbles were good before and after avidin was added. Before the addition of avidin, dispersity of biotinylated lipid particles/microbubbles were also good. While after avidin was added, both of them began to aggregate. Addition of avidin had no effect on the particle diameters of control lipidic particles/microbubbles. However, the particle diameters of biotinylated lipidic particles and microbubbles were both lager with the addition of avidin (P< 0.05).③With fundamental imaging, neither control nor biotinylated PFOB particles were visible before avidin was added. Addition of avidin produced a significant imaging enhancement in biotinylated PFOB particles (P< 0.05). While control PFOB particles were still unvisible. However, with fundamental imaging, both control and biotinylated C3F8 microbubbles manifested ultrasonic backscatter before and afer the addition of avidin, without significant difference in their signal intensity.⑤CPS imaging couldn't detect nonlinear signal from biotinylated PFOB particles niether before nor after avidin was added. However, CPS imaging could detect nonlinear signal from biotinylated C3F8 microbubbles both before and after the addition of avidin, without significant difference in their signal intensity.⑥The signal intensity of PFOB particles were stable in the ultrasound pressure field of both lower(MI=0.28) and higher(MI=0.56) levels. However, signal intensity of C3F8 microbubbles were obviously decreased afer exposure for 20 seconds at a lower acoustic pressure (P< 0.05), and their signal intensity were even lower after they were exposed for 30 seconds(P< 0.05). At a higher ultrasound pressure, signal intensity of C3F8 microbubbles were obviously decreased afer exposure for 10 seconds (P< 0.05), and their signal intensity were even lower after exposure for 20 and 30 seconds (P< 0.05). Conclusions Compared to C3F8 microbubbles, PFOB particles with better penetrativity, stability, resistance to acoustic pressure and higher signal-to-noise (target to untarget signal) ratio, are more suitable for targeted contrast ultrasound imaging.Part 2. Imaging enhancement of viable myocardium after ischemia-reperfusion by site-targeted lipidic nanoparticles of PFOB:a vivo study Objective Our purpose was to confirm specific imaging enhancement of viable myocardium with a kind of site-targeted PFOB lipidic nanoparticles. Methods The contrast agent is a biotinylated, fluorescent-labelled, lipid-coated, perfluorooctyl bromide emulsion. Physico-chemical properties of the agent were measured, including size distribution, Zeta potential, concentration and so on. Ischemia-reperfusion models were created in rats, and then exposed to biotinylated anti-monocyte chemoattactant protein-1(MCP-1) monoclonal antibody, rhodamine avidin and biotinylated, fluorescence-labelled particles, successively. Echocardiographic images were obtained before and after injection and were recorded onto CD-ROM. Frozen sections of their hearts were observed under fluorescence microscope. TTC-EB and HE dyeing were performed to observe the distribution of viable myocardium.Results①The particle diameter, zeta potential and concentration of lipid particles were (172.30±52.06)nm, (-33.10±6.50)mV and (2.28±0.46)×1011/ml, respectively.②As TTC-EB and HE dyeing show, myocardium of anterior wall and interventricular septum was necrotic, but there were still viable myocardium in anterior wall. Myocardium of posterior, inferior and lateral wall were healthy.③From the short-axis view, the myocardium under endocardium of anterior wall exhibited increased echogenicity after contrast exposure than before (P< 0.05). While the ultrasound signal of myocardium of other walls were still.④The PFOB lipid particles located in the myocardium of anterior wall under light micrograph, and gave out bright green and red fluorescence under fluorescence microscope. Conclusions The viable myocardium can be targeted and acoustically enhanced by the site-targeted PFOB lipidic nanoparticles. This new agent has potential to improve sensitivity and specificity for noninvasive identifying viable myocardium.
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