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Updated: Jun 12, 2026
Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release
Published on: June 12, 2021
Development of antibody-carrying microbubbles based on clinically available ultrasound contrast agent for targeted
Kentaro Otani1, Kenichi Yamahara
1Department of Regenerative Medicine and Tissue Engineering, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. otani@ri.ncvc.go.jp
Purpose:
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.
Procedures:
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.
Results:
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.
Conclusions:
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.
