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.