Development of antibody-carrying microbubbles based on clinically available ultrasound contrast agent for targeted molecular imaging: a preliminary chemical study.
OBJECTIVE
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.


METHODS
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.
Department 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
Kentaro
K
Otani
Kenichi
K
Yamahara
D017304:Annexin A5 / Q000378:metabolism; D000906:Antibodies; D019921:Biotinylation / Q000187:drug effects; D002122:Calcium Chloride / Q000494:pharmacology; D003287:Contrast Media; D005290:Ferric Compounds / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D007501:Iron / Q000737:chemistry; D045423:Microbubbles; D057054:Molecular Imaging / Q000379:methods*; D010087:Oxides / Q000737:chemistry; D010718:Phosphatidylserines / Q000378:metabolism; D010799:Phycoerythrin / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism; D014465:Ultrasonics
D017304:Annexin A5; D000906:Antibodies; D003287:Contrast Media; D005290:Ferric Compounds; D010087:Oxides; D010718:Phosphatidylserines; C069727:Sonazoid; D010799:Phycoerythrin; D019809:Streptavidin; D007501:Iron; D016650:Fluorescein-5-isothiocyanate; D002122:Calcium Chloride