Ultrasound Contrast Agents for Inflammation and Angiogenesis

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DESCRIPTION (provided by applicant): We propose to develop molecular imaging probes for imaging angiogenesis and inflammation using contrast-enhanced ultrasound. These probes are gas-encapsulated microbubbles that can be coated with ligands specific for molecular markers of disease using robust covalent coupling chemistries. The agents proposed here will be used to image the severity and spatial extent of disease in preclinical animal models, and have strong potential for clinical translation. We propose to develop microbubbles bearing versatile covalent ligand-coupling chemistries, which will allow preclinical users to couple their own ligands. Additionally, we will develop two pre-targeted agents bearing covalently bound ligands specific for biomarkers of inflammation and angiogenesis. Although site-targeted microbubble contrast agents are currently commercially available for preclinical research, these agents utilize time consuming and potentially immunogenic biotin/avidin ligand coupling chemistry. Covalent coupling of the ligand to the agent surface eliminates the biotin/avidin system, greatly increasing the convenience and utility of the agents. The goals of this proposal are (1) to develop covalent ligand coupling strategies suitable for a variety of ligand classes, (2) to create ultrasound contrast agents bearing covalently bound ligands specific for biomarkers of inflammation and angiogenesis, and (3) to quantitatively test targeted adhesion of these ligand-bearing agents to relevant molecular targets in vitro. We will test two ligands for biomarkers of inflammation and angiogenesis, respectively: a selectin-binding oligosaccharide (sialyl Lewisx) and a VEGF receptor binding single-chain VEGF construct. The selectins are critically involved in numerous inflammatory disorders, and have been implicated in therapeutic and pathogenic angiogenesis. With the goal of producing convenient probes for imaging inflammation and angiogenesis, we propose to construct microbubbles bearing covalently bound sLex, a single-chain VEGF, or a combination of both ligands. These ligands are superior to antibodies commonly used as ligands due to the lack of immunogenicity, species-independence, and small size. Additionally, sLex possesses an excellent ability to mediate high-efficiency retention of microparticles, even under conditions of rapid blood flow. We hypothesize that dual-targeted microbubbles bearing both scVEGF and sLex may exhibit synergistic adhesion resulting in enhanced microbubble retention to angiogenic endothelium expressing E- selectin and VEGFR-2. The rapid bond kinetics of sLex can mediate initial tethering of the agent, and once localized at the endothelial surface scVEGF can mediate firm adhesion to VEGF receptors. Microbubbles bearing the sLex ligand alone will be useful for imaging a wide variety of inflammatory disorders, and VEGF- bearing microbubbles will be useful for imaging general angiogenic processes. The knowledge gained will serve to develop a universal targeted microbubble platform able to be conjugated to user-selected ligands using a robust covalent coupling system, and will provide a platform for subsequent pre-targeted formulations. Project Narrative Molecular imaging of biomarker expression can greatly accelerate the pace of drug discovery, and has tremendous potential for clinical translation. The development of microbubbles bearing a robust and versatile covalent ligand-coupling chemistry will expand the utility of these agents in preclinical research, and is necessary for subsequent clinical implementation. Additionally, microbubbles pre-targeted to biomarkers of inflammation and angiogenesis will serve as a convenient and user-friendly product for preclinical molecular imaging.