Enhancement of ultrasonic thrombus imaging using novel liposomal bubbles targeting activated platelet glycoprotein IIb/IIIa complex—in vitro and in vivo study

Abstract 

Background

We developed perfluorocarbon gas-containing bubble liposomes (BL) with Arg-Gly-Asp (RGD) sequence-containing peptides, which bind to activated platelet glycoprotein IIb/IIIa complexes. The aim of this study was to examine the enhancing effects in ultrasonic thrombus imaging using these targeted BL in vitro and in vivo.

Methods

Liposomes composed of phosphatidylcholine and cholesterol were manufactured, and RGD peptide was attached by a covalent coupling reaction. Sonication was used to conjugate liposomes and perfluorocarbon gas, which formed targeted BL. In vitro, targeted BL were mixed with whole blood, which was allowed to coagulate while being shaken and rotated. In vivo, we administered targeted BL to 10 rabbits with acute thrombotic occlusions in the ilio-femoral artery. Thrombi were imaged using a 7.5–9MHz linear transducer and a conventional ultrasound machine, and by scanning electron microscopy. Ultrasound images were digitized, and mean pixel gray-scale level (black=0, white=255) was measured.

Results

In vitro, mean pixel gray-scale level of the thrombi in targeted BL group was significantly higher than in control and non-targeted BL groups (93±26 vs. 58±16, 48±9, p=0.002, n=10). Scanning electron microscopy revealed large amounts of targeted BL attached to the thrombi. In vivo, mean pixel gray-scale level of the thrombi with targeted BL was significantly higher (33.2±6.4 vs. 24.8±8.5, p=0.0051, n=10) than that before targeted BL administration.

Conclusions

Perfluorocarbon gas-containing BL with RGD peptide represent a novel echo contrast agent, which can markedly enhance ultrasonic thrombus imaging in vitro and in vivo, and may be useful for noninvasively diagnosing acute thrombotic vessel occlusion.

Keywords: Echocardiography, Thrombus, Imaging, Ultrasound, Liposome