Novel single-chain antibody-targeted microbubbles for molecular ultrasound imaging of thrombosis and thrombolysis.

Molecular imaging is a fast emerging technology allowing non-invasive detection of vascular pathologies. However, imaging modalities offering high resolution currently do not allow real-time imaging. We hypothesized that contrast enhanced ultrasound with microbubbles selectively targeted to activated platelets would offer high-resolution, real-time molecular imaging of evolving and dissolving arterial thrombi. Lipid-shell based gas-filled microbubbles (MB) were conjugated to either a single-chain antibody (scFv) specific for activated GPIIb/IIIa via binding to a Ligand-Induced Binding Site (LIBS-MB), or a non-specific scFv (control-MB). LIBS-MB, but not control-MB, strongly adhered to both immobilized activated platelets and micro-thrombi under flow. Thrombi, induced in carotid arteries of C57Bl6-mice in vivo by ferric chloride injury, were then assessed with ultrasound before and 20 minutes after MB injection, using grayscale area intensity measurement. Grayscale units converted to decibels demonstrated a significant increase after LIBS-MB but not after control-MB injection (p<0.001). Furthermore after thrombolysis with urokinase, LIBS-MB ultrasound-imaging allows monitoring of the reduction of thrombus size (p<0.001). A lower systematic dose of targeted fibrinolytic agent is required to achieve the same fibrinolytic potency. We demonstrate that GPIIb/IIIa-targeted microbubbles specifically bind to activated platelets in vitro and allow real-time molecular imaging of acute arterial thrombosis as well as monitoring of pharmacological thrombolysis in vivo. This non-invasive and cost effective imaging modality provides a unique approach to rapidly detect (micro)thrombi with high resolution, allowing for early diagnosis and therapy, as well as enabling the fast identification of success or failure of thrombolytic therapy.