Microbubbles conjugated with anti-vascular cell adhesion molecule-1 nanobody cAbVCAM1-5

Ultrasound is a widely used imaging modality (1), and its role in noninvasive molecular imaging with ligand-carrying microbubbles (MBs) is expanding (2). MBs are comprised of spherical cavities encapsulated in a shell and filled with a gas. The shells are made of phospholipids, surfactants, denatured human serum albumin, or synthetic polymers. Ligands and antibodies can be incorporated into the MB shell surface. MBs are usually 2–8 μm in size. MBs of this size provide a strongly reflective interface and resonate to ultrasound waves. They are used as ultrasound contrast agents in imaging of inflammation, angiogenesis, intravascular thrombus, and tumors (2-4). They also can potentially be used for drug and gene delivery (5). Endothelial cells are important cells in inflammatory responses (6, 7). Bacterial lipopolysaccharide (LPS), virus, inflammation, and tissue injury increase tumor necrosis factor α (TNFα), interleukin-1 (IL-1), and other cytokine and chemokine secretion. Leukocyte emigration from blood is dependent on their ability to adhere to endothelial cell surfaces. Inflammatory mediators and cytokines induce chemokine secretion from endothelial cells and other vascular cells and increase their expression of cell surface adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), integrins, and selectins. Chemokines are chemotactic toward leukocytes and toward sites of inflammation and tissue injury. The movements of leukocytes through endothelial junctions into the extravascular space are highly orchestrated through various interactions with different adhesion molecules on endothelial cells (8). VCAM-1 is found in very low amounts or non-detectable on the cell surface of resting endothelial cells and other vascular cells, such as smooth muscle cells and fibroblasts (9-13). VCAM-1 binds to very late antigen-4 (VLA-4) integrin on the cell surface of leukocytes. IL-1 and TNFα increase expression of VCAM-1 and other cell adhesion molecules on the vascular endothelial cells, which leads to leukocyte adhesion to the activated endothelium. Furthermore, VCAM-1 expression was also induced by oxidized low-density lipoproteins under atherogenic conditions (14). Overexpression of VCAM-1 by atherosclerotic lesions plays an important role in their progression toward vulnerable plaques, which may erode and rupture. MBs targeted with monoclonal antibody against VCAM-1 are being developed as a noninvasive agent for VCAM-1 expression in vascular endothelial cells during different stages of inflammation in atherosclerosis (15). Nanobodies are the smallest intact antigen-binding fragments (15 kDa) isolated from heavy-chain camelid antibodies with efficient and specific tumor targeting (16-18). Hernot et al. (19) prepared an anti-VCAM-1 nanobody (cAbVCAM1-5) that was site-specifically biotinylated in bacteria. cAbVCAM1-5 was coupled to biotinylated lipid MBs streptavidin-biotin bridging to form MB-cAbVCAM1-5 for use with ultrasound imaging of VCAM-1 expression.