Targeted Ultrasound Contrast Imaging of Tumor Vasculature With Positively Charged Microbubbles.
Targeted Ultrasound Contrast Imaging of Tumor Vasculature With Positively Charged Microbubbles.
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DOI：
10.1097/rli.0000000000000699
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发表时间：
2020-11
影响因子：
6.7
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中科院分区：
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针对肿瘤血管生物标记物的微泡造影剂正在积极研究肿瘤血管的分子超声成像。然而，一种针对肿瘤血管的通用方法是可取的，这种方法不依赖于特定的生物标记物，或者在没有特定生物标志物的情况下。我们报告了使用静电相互作用来实现微泡与肿瘤血管的粘连，并由此通过超声成像来描绘肿瘤。以十氟丁烷气体为原料，采用水胶束混合法制备了微泡。分别以二羟甲基丙烷和硬脂酸聚乙二醇酯作为微泡造壳剂，阳离子脂类双硬脂酰三甲基胺丙烷(DSTAP)引入正静电。微泡在正常重力下进行浮选，以去除较大的颗粒。将小鼠结肠腺癌细胞(MC38，J.Schlom，NIH)接种于C57BL/6小鼠后腿。超声造影在异氟醚麻醉下进行，使用临床成像系统，低功率模式，组织信号抑制(CPS，7 MHz，1 Hz，MI 0.2)。超声探头定位用于监测肿瘤和对侧小腿肌肉；静脉推注2.107个微泡后，监测微泡对比信号30min或更长时间。从超声录像中提取各个时间点的帧，并用ImageJ进行分析。气泡直径平均为1.6~2微米，99.9%为5微米，以防止毛细血管内血流受阻。阳离子DSTAP微泡注射后30min以上，肿瘤内可见增强信号。随着泡壳中带正电的脂类比例的增加，肿瘤内附着的造影剂信号也增加，但正常肌肉中DSTAP-微泡的积聚也增加。对于正电荷最高的微泡，DSTAP：DSPC摩尔比为1：4，静脉注射微泡后10min，肿瘤与正常肌肉的对比信号差为1.5(p<；0.005)。30min时，肿瘤/肌肉对比信号比改善，达到2.1。DSTAP：DSTPC 1：13组肿瘤/肌肉信号比在10min时超过3.6，30min时达到5.4。DSTAP：DSPC比例为1：22的微泡最适合肿瘤靶向：10min时，肿瘤/肌肉信号比为>；7(p<；0.005)；30min时为>；16(p<；0.01)，足以显示肿瘤。阳离子微泡很容易制备。静脉给药后，它们选择性地聚集在肿瘤血管中。这些微泡提供的目标与对照的对比度可以超过一个数量级。粘附性微泡在延长的时间点、30min和更长的时间勾画出肿瘤的边界。这可以允许延长声学造影检查时间。总体而言，带正电的微泡可能成为癌症成像的通用超声造影剂。
Molecular ultrasound imaging of tumor vasculature is being actively investigated with microbubble contrast agents targeted to neovasculature biomarkers. Yet a universal method of targeting tumor vasculature independent of specific biomarkers, or in their absence, would be desirable. We report the use of electrostatic interaction to achieve adherence of microbubbles to tumor vasculature and resulting tumor delineation by ultrasound imaging. Microbubbles were prepared from decafluorobutane gas by amalgamation of aqueous micellar medium. DSPC and PEG-stearate were used as microbubble shell-forming lipids; cationic lipid distearoyl trimethylammoniumpropane (DSTAP) was included to introduce positive electrostatic charge. Microbubbles were subjected to flotation in normal gravity, to remove larger particles. Murine colon adenocarcinoma tumor (MC38, J. Schlom, NIH) was inoculated in the hind leg of C57BL/6 mice. Contrast ultrasound imaging was performed under isoflurane anesthesia, using a clinical imaging system in low power mode, with tissue signal suppression (CPS, 7 MHz, 1 Hz, MI 0.2). The ultrasound probe was positioned to monitor the tumor and contralateral leg muscle; microbubble contrast signal was monitored for 30 min or more, following intravenous bolus administration of 2.107 microbubbles. Individual time point frames were extracted from ultrasound video recording and analyzed with ImageJ. Mean bubble diameter was ~1.6–2 um; 99.9% were <5 um, to prevent blocking blood flow in capillaries. For cationic DSTAP-carrying microbubbles, contrast signal was observed in the tumor beyond 30 min after injection. As the fraction of positively charged lipid in the bubble shell was increased, adherent contrast signal in the tumor also increased, but accumulation of DSTAP-microbubbles in the normal muscle increased as well. For bubbles with the highest positive charge tested, DSTAP:DSPC molar ratio 1:4, at 10 min after intravenous administration of microbubbles the contrast signal difference between the tumor and normal muscle was 1.5 (p<0.005). At 30 min, tumor/muscle contrast signal ratio improved, and reached 2.1. For the DSTAP:DSPC 1:13 preparation, tumor/muscle signal ratio exceeded 3.6 at 10 min and reached 5.4 at 30 min. Microbubbles with DSTAP:DSPC ratio 1:22 were optimal for tumor targeting: at 10 min, tumor/muscle signal ratio was >7 (p<0.005); at 30 min, >16 (p<0.01), sufficient for tumor delineation. Cationic microbubbles are easy to prepare. They selectively accumulate in the tumor vasculature after intravenous administration. These microbubbles provide target-to-control contrast ratio that can exceed an order of magnitude. Adherent microbubbles delineate the tumor mass at extended time points, at 30 min and beyond. This may allow for an extension of the contrast ultrasound exam time. Overall, positively charged microbubbles could become a universal ultrasound contrast agent for cancer imaging.