Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan.
Mol Imaging Biol. 2011 Apr;13(2):250-6. doi: 10.1007/s11307-010-0344-7.
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 μL) 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 μL, 3.3 vs. 50 μL, 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.
本研究旨在探讨使用临床可用的含磷脂酰丝氨酸（PS）的全氟丁烷填充微泡制备携带抗体的靶向微泡用于分子超声成像的可行性。
首先，我们使用膜联蛋白 V 通过流式细胞术（荧光激活细胞分选（FACS））检测全氟丁烷填充微泡表面 PS 是否可以被检测到。微泡与荧光素异硫氰酸酯（FITC）标记的膜联蛋白 V（多达 50μL）在冰上孵育 15min 后，使用 FACSCalibur 进行评估。其次，我们研究了 PS 标记的藻红蛋白（PE）标记的链霉亲和素是否可以通过生物素化膜联蛋白 V 的中介作用附着到含 PS 的全氟丁烷填充微泡上。与生物素化膜联蛋白 V 结合的微泡与 PE-链霉亲和素在冰上孵育 30min，然后进行 FACS 分析。最后，我们研究了 PS 标记的全氟丁烷填充微泡是否可以通过生物素化膜联蛋白 V 和亲和素-生物素结合来完成生物素化 IgG 的附着。带亲和素-生物素复合物的微泡与 Alexa488 标记的生物素化 IgG 在冰上孵育 30min。
FITC 标记的膜联蛋白 V 结合后可检测到 FITC 阳性微泡。此外，Sonazoid 微泡的平均荧光强度呈剂量依赖性增加（0μL，3.3 与 50μL，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（平均荧光强度，327.1 与 14.8）。通过生物素化膜联蛋白 V 和链霉亲和素的中介作用，实现了 Alexa488 信号的显著放大。
我们的结果支持使用临床可用的含 PS 的全氟丁烷填充微泡制备携带抗体的靶向微泡的可行性。尽管还需要进一步研究，但该技术可能适用于体内分子超声成像。
