Enhancement of gas‐filled microbubble R2* by iron oxide nanoparticles for MRI

Gas‐filled microbubbles have the potential to become a unique intravascular MR contrast agent due to their magnetic susceptibility effect, biocompatibility, and localized manipulation via ultrasound cavitation. However, microbubble susceptibility effect is relatively weak when compared with other intravascular MR susceptibility contrast agents. In this study, enhancement of microbubble susceptibility effect by entrapping monocrystalline iron oxide nanoparticles (MIONs) into polymeric microbubbles was investigated at 7 T in vitro. Apparent T₂ enhancement (ΔR₂*) induced by microbubbles was measured to be 79.2 ± 17.5 sec^?1 and 301.2 ± 16.8 sec^?1 for MION‐free and MION‐entrapped polymeric microbubbles at 5% volume fraction, respectively. ΔR₂* and apparent transverse relaxivities (r₂*) for MION‐entrapped polymeric microbubbles and MION‐entrapped solid microspheres (without gas core) were also compared, showing the synergistic effect of the gas core with MIONs. This is the first experimental demonstration of microbubble susceptibility enhancement for MRI application. This study indicates that gas‐filled polymeric microbubble susceptibility effect can be substantially increased by incorporating iron oxide nanoparticles into microbubble shells. With such an approach, microbubbles can potentially be visualized with higher sensitivity and lower concentrations by MRI. Magn Reson Med, 2010. © 2009 Wiley‐Liss, Inc.     

Targeting an Ultrasound Contrast Agent to Folate Receptors on Ovarian Cancer Cells

Objective. The purpose of the study was to synthesize and characterize folate‐targeted microbubbles (MB_F) as an ultrasound contrast agent and to evaluate their affinity to the folate receptor (FR) in vitro. Methods. Folate‐targeted microbubbles were prepared by incorporating 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[amino(polyethylene glycol)‐2000]‐folate into the lipid membrane of microbubbles. The diameter and concentration of the MB_F were determined by a cell counter and sizer. The MB_F, control microbubbles (MB_C), and MB_F with a free folic acid block‐ade were tested for binding specificity to human ovarian carcinoma SKOV3 cells, which overexpress the FR, by microscopy and confocal imaging, respectively. Results. The basic physical characteristics of MB_F were similar to those of MB_C. In the cell binding test, the adherence efficiency of MB_F to the SKOV3 cells (mean ± SD, 16 ± 5 microbubbles per cell; P < .01) was significantly higher than that of MB_C (0.7 ± 0.4 microbubbles per cell) or MB_F with the free folic acid blockade (0.7 ± 0.6 microbubbles per cell). Conclusions. Folate‐targeted microbubbles showed high affinity to SKOV3 cells with FR overexpression. They are potentially useful for ultrasonic molecular imaging and treatment of FR‐positive tumors and warrant further investigation.     

Analysis of in vitro Transfection by Sonoporation Using Cationic and Neutral Microbubbles

The objective of the study was to examine the role of acoustic power intensity and microbubble and plasmid concentrations on transfection efficiency in HEK-293 cells using a sonoporator with a 1-MHz transducer. A green fluorescent protein (GFP) reporter plasmid was delivered in as much as 80% of treated cells, and expression of the GFP protein was observed in as much as 75% of cells, using a power intensity of 2 W/cm² with a 25% duty cycle. In addition, the relative transfection abilities of a lipid noncationic and cationic microbubble platform were investigated. As a positive control, cells were transfected using Lipofectamine reagent. Cell survival and transfection efficiency were inversely proportional to acoustic power and microbubble concentration. Our results further demonstrated that high-efficiency transfection could be achieved, but at the expense of cell loss. Moreover, direct conjugation of plasmid to the microbubble did not appear to significantly enhance transfection efficiency under the examined conditions, although this strategy may be important for targeted transfection in vivo. (E-mail: mbl2a@virginia.edu)     

Nonlinear Emission from Individual Bound Microbubbles at High Frequencies

Targeted microbubbles detected with high-frequency ultrasound can establish the molecular expression of blood vessels with submillimeter resolution. To improve microbubble-specific imaging at high frequencies, the subharmonic and second harmonic signal from individual microbubbles were measured as a function of size and pressure. Single phospholipid-shell microbubbles (1.1 to 5.0 μm in diameter) bound to gelatin, co-aligned with an optical microscope and transducer, were insonated with 30 MHz Gaussian-enveloped pulses at pressures from 20 kPa to 1 MPa with –6 dB one-way bandwidths of 11%, 20% and 45%. A subharmonic signal (15 MHz) was detected above a pressure threshold of 110 kPa—independent of bandwidth. The signal peaked for microbubbles 1.60 μm in diameter subject to 20% and 11% bandwidth pulses, and 1.80 μm for 45% bandwidth pulses, for pressures up to 400 kPa, agreeing with the notion that microbubbles insonated at twice their resonant frequency preferentially emit a subharmonic component. For pressures between 400 kPa and 1 MPa, a broader range of microbubbles emitted a subharmonic signal, and microbubbles below 1.70 μm in diameter were disrupted. The second harmonic signal measured, within the limited experimental conditions, was consistent with nonlinear propagation. Further, the results shed light on the effect of the shell on the phase of the subharmonic signal with respect to the fundamental signal. (E-mail: michael.sprague@sri.utoronto.ca)     

携Sialyl Lewis^X超声微泡靶向探查缺血心肌的炎症分子“印记”

目的探讨应用靶向超声分子成像探查心肌缺血再灌注损伤炎症“印记”的可行性。方法采用“亲和素-生物素”桥接法构建携唾液酸化路易斯（Sibyl Lewis^X）靶向超声微泡（MBsLex）和同型对照微泡（MBc）。10只心肌缺血再灌注小鼠随机先后注入MBsLex和MBc（间隔30min）,分别于注入5min后行心肌对比超声检查,测量心肌缺血区和非缺血区的声强度（Ⅵ）。结果对比超声图像显示MBsLex组缺血区心肌造影见显著增强,Ⅵ值高达23．52±1．08,而在MBc组缺血区心肌造影仅见轻度增强,Ⅵ缸为9．81±0．41,两者之间差异有统计学意义（P〈0．05）。但无论MBsLex组还是MBc组,缺血区心肌Ⅵ值均明显高于非缺血区心肌Ⅵ值（P〈0．05）。两组非缺血区心肌之间Ⅵ值未见明显差异。结论应用携sLex超声微泡行对比超声能够靶向探查心肌缺血再灌注损伤的炎症“印记”。     

Therapeutic Ultrasound for Gene Delivery

In vitro and in vivo studies using perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles to enhance gene delivery are reviewed. In vitro studies show PESDA binds to oligonucleotides and that ultrasound can be used to deposit these nucleotides. In addition, in vitro studies show that drug release from microspheres is dependent on ultrasound transmission frequency as well as pulsed or continuous application. Early in vivo studies confirm that ultrasound in combination with microbubbles can be used to facilitate gene deposition. However, the role of ultrasound targeting gene delivery remains to be determined.     

Enhanced methods for visualizing myocardial perfusion with peripheral venous injection of Levovist?: Application of triggered harmonic imaging and triggered harmonic power Doppler imaging techniques

Objectives:The purpose of this study was to determine whether triggered harmonic imaging (THI) or triggered harmonic power Doppler imaging (THPDI) could obtain the myocardial contrast enhancement using peripheral venous injection of a first generation echocardiographic contrast agent, Levovist^®. Methods:In a phantom model, we examined the influence of an acoustic power, harmonic filters, transmitted frequencies and focus positions of transducer on Levovist^®. Then fundamental, harmonic or harmonic power Doppler imaging were performed with ECG-triggered imaging in eight closed-chest dogs using bolus injection of Levovist^®. Results:In a phantom model, the highest transmission power (Mechanical index 1.6), a medium harmonic filter and a focus position (6 cm) resulted in the best enhanced contrast in both THI and THPDI. Furthermore, higher pulse repetition frequency (5500 Hz) of harmonic power Doppler made clearer enhancement. In animal models, we could not observe the apparent myocardial contrast using triggered fundamental imaging, and the intensity of each region of interest (ROI) of myocardium had not changed significantly. However, homogeneous myocardial contrast could be obtained using THI, which was conditioned on the highest transmission power, a medium harmonic filter same as the phantom model, at a lower transmitted frequency (1.8 MHz) and a focus position, which were located in the middle portion of the left ventricle. The peak intensity of each ROI increased significantly in a gray level. Furthermore, THPDI caused emphasized myocardial contrast visually. Conclusions:These results indicate that THI and THPDI produce obvious MCE using peripheral venous injection of Levovist^®.     

实时心肌超声造影评价糖尿病早期心肌血流灌注改变的实验研究

目的：探讨实时心肌超声造影（RT-MCE）技术检测糖尿病（DM）早期大鼠心肌血流灌注异常的价值。方法：24只雄性SD大鼠中随机选取12只通过腹腔注射链脲佐菌素建立DM模型（DM组）,剩余12只大鼠作为对照,腹腔注射等量枸橼酸缓冲液。模型建成8周后,在静息状态下,对两组大鼠行RT-MCE检查,检测两组大鼠乳头肌水平左室壁感兴趣区的峰值声学强度（A）以及造影剂的灌注速度（β）,并计算出心肌血流量（A×β）。RT-MCE检查结束后,处死大鼠取心肌组织,行CD31免疫组织化学染色检测心肌微血管密度（MVD）,然后对DM组A与MVD进行相关分析。结果：与对照组比较,在静息状态下DM组的A及A＆#215;β较对照组明显减低,差异具有统计学意义（P＜0.01）,β较对照组减低,但差异无统计学意义（P＞0.05）。DM组的MVD较对照组明显减低（P＜0.01）。DM组A与MVD呈线性正相关（r=0.903,P＜0.01）。结论：DM早期即可发生心肌血流灌注的异常,RT-MCE在检测DM早期心肌血流灌注异常方面具有重要的应用价值。