Development of Antibody-Carrying Microbubbles Based on Clinically Available Ultrasound Contrast Agent for Targeted Molecular Imaging: A Preliminary Chemical Study
Development of Antibody-Carrying Microbubbles Based on Clinically Available Ultrasound Contrast Agent for Targeted Molecular Imaging: A Preliminary Chemical Study
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DOI：
10.1007/s11307-010-0344-7
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发表时间：
2011-04-01
影响因子：
3.1
通讯作者：
Yamahara, Kenichi
中科院分区：
文献类型：
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作者：
Otani, Kentaro;Yamahara, Kenichi
该研究的目的是检查使用临床上可用的含有磷脂酰丝氨酸（PS）的全氟丁烷填充的微泡用于分子超声成像的携带抗体的靶向泡制剂的可行性。我们检测了填充全氟丁烷的微泡表面的PS是否可以通过流式细胞术检测到在冰上与异硫氰酸荧光素（FITC）标记的膜联蛋白V（至多50 μ L）缀合15分钟后，使用FACSCalibur评估微泡。其次，我们研究了藻红蛋白（PE）标记的链霉亲和素是否可以通过生物素化的膜联蛋白V的中间体连接到含有PS的全氟丁烷填充的微泡。微泡与生物素化的膜联蛋白V缀合与PE-链霉亲和素孵育30分钟在冰上，然后进行流式细胞仪分析。最后，我们研究了是否可以使用生物素化的膜联蛋白V和抗生物素蛋白-生物素结合来完成生物素化的IgG附着到含有PS的全氟丁烷填充的微泡上。将含有抗生物素蛋白-生物素复合物的微泡与Alexa 488标记的生物素化IgG在冰上孵育30 min。在与FITC-膜联蛋白V偶联后可检测到FITC阳性微泡。此外，Sonazoid气泡的平均荧光强度以剂量依赖性方式增加（0 μ L，3.3 vs. 50 μ L，617.1）。存在生物素化膜联蛋白V时微泡的PE信号高于不存在生物素化膜联蛋白V时的PE信号（平均荧光强度，327.1 vs. 14.8）。Alexa 488-信号的显着放大是通过生物素化的膜联蛋白V和链霉亲和素的中介完成的。我们的研究结果支持基于临床上可用的含PS的全氟丁烷填充的微泡的抗体携带靶向气泡制剂的可行性。虽然需要进一步的研究，这种技术可能适用于在体内的分子超声成像。
The purpose of the study was to examine the feasibility of an antibody-carrying targeted-bubble preparation using clinically available phosphatidylserine (PS)-containing perfluorobutane-filled microbubbles for molecular ultrasound imaging.Firstly, we examined whether PS on the surface of perfluorobutane-filled microbubbles could be detected by means of flow cytometry (fluorescence activated cell sorting (FACS)) using annexin V. After conjugation with fluorescein isothiocyanate (FITC)-labeled annexin V (up to 50 mu L) for 15 min on ice, microbubbles were assessed using a FACSCalibur. Secondly, we examined whether phycoerythrin (PE)-labeled streptavidin could be attached onto PS-containing perfluorobutane-filled microbubbles through the intermediacy of biotinylated annexin V. Microbubbles conjugated with biotinylated annexin V were incubated with PE-streptavidin for 30 min on ice, then FACS analysis was performed. Finally, we examined whether attachment of biotinylated IgG onto PS-containing perfluorobutane-filled microbubbles could be accomplished using biotinylated annexin V and avidin-biotin binding. Microbubbles with avidin-biotin complexes were incubated with Alexa488-labeled biotinylated IgG for 30 min on ice.FITC-positive microbubbles could be detected after conjugation with FITC-annexin V. Additionally, the mean fluorescence intensity of Sonazoid bubbles increased in a dose-dependent manner (0 mu L, 3.3 vs. 50 mu L, 617.1). The PE signal of microbubbles in the presence of biotinylated annexin V was higher than that in the absence of biotinylated annexin V (mean fluorescence intensity, 327.1 vs. 14.8). Significant amplification of the Alexa488-signal was accomplished through the intermediation of biotinylated annexin V and streptavidin.Our results support the feasibility of an antibody-carrying targeted-bubble preparation based on clinically available PS-containing perfluorobutane-filled microbubbles. Although further study is needed, this technique could be applicable for in vivo molecular ultrasound imaging.