S. Yoshikazu, R. Suzuki, Y. Oda, D. Omata, K. Maruyama
Laboratory of Drug and Gene Delivery Faculty of Pharma-sciences – Tokyo, Japan

Objective: The combination thrombolysis therapy with recombinant tissue plasminogen activator (rt-PA), microbubbles and ultrasound, was reported higher recanalization ratio in ischemic cerebrovascular disease. Ultrasound and microbubbles vibrate the fibrin net and accelerate thrombolysis. Recently, we developed novel liposomal submicron bubbles (Bubble Liposome (BLs)) containing ultrasound imaging gas, perfluoropropane. In this study, we developed BLs with Arg-Gly-Asp (RGD) sequence-containing peptides, which bind to the activated platelet glycoprotein IIb/IIIa complexes. The aim of this study was to examine the enhancing effects in ultrasonic thrombus imaging using these targeted BLs in vitro and in vivo.

Material and Methods: Liposomes composed of 1,2-distearoylphosphatidylcholine (DSPC) and N-[(3-Maleimide-1-oxopropyl)aminopropyl polyethyleneglycol-carbamyl] distearoylphosphatidyl-ethanolamine (DSPE-PEG-Maleimide) were manufactured, and RGD sequence contained peptide (CGGcyclic(RGDfK)) was attached liposomes by a covalent coupling reaction between cysteine and DSPE-PEG-Maleimide. RGD conjugated liposomes were sonicated with perfluorocarbon gas to form RGD-BLs. In vitro, rat activated platelets binding ability of RGD conjugated liposomes or RGD-BLs was evaluated with flowcytometry, or confocal laser scanning microscope. In vivo, thrombus accumulation ability of RGD-BLs was detected by ultrasonic diagnostic equipment (VEVO2100) on the ferric chloride induction thrombus in rat left CCA.

Results: In vitro, RGD conjugated liposomes or RGD-BLs were observed more effective binding to platelets by flowcytometry or confocal laser scanning microscope. In vivo, RGD-BLs treated group was detected higher echo signal on CCA thrombus compared with non-targeted BLs treated group.

Discussion: RGD-BLs represents a novel echo contrast agent, which can markedly enhance ultrasounic thrombus imaging in vitro and in vivo, and may be useful for noninvasively diagnosing acute thrombotic vessel occlusion and thrombolysis acceleration.

Key words: bubble liposome, RGD-peptide, thrombus imaging, ultrasound.