Factors affecting the endothelial retention of targeted microbubbles: Influence of microbubble shell design and cell surface projection of the endothelial target molecule

Background: In biologic systems, the arrest of circulating cells is mediated by adhesion molecules projecting their active binding domain above the cell surface to enhance bond formation and tether strength. Similarly, molecular spacers are used for ligands on particle-based molecular imaging agents. The aim of this study was to evaluate the influence of tether length for targeting ligands on ultrasound molecular imaging agents. Methods: Microbubbles bearing biotin at the end of variable-length polyethylene glycol spacer arms (MB ₂₀₀₀ and MB ₃₄₀₀) were prepared. To assess in vivo attachment efficiency to endothelial counterligands that vary in their distance from the endothelial cell surface, contrast-enhanced ultrasound (CEU) molecular imaging of tumor necrosis factor-α-induced P-selectin (long distance) or intercellular adhesion molecule-2 (short distance) was performed with each agent in murine hind limbs. To assess the influence of the glycocalyx on microbubble attachment, CEU molecular imaging of intercellular adhesion molecule-2 was performed after degradation of the glycocalyx. Results: CEU molecular imaging targeted to P-selectin showed signal enhancement above control agent for MB ₂₀₀₀ and MB ₃₄₀₀, the degree of which was significantly higher for MB ₃₄₀₀ compared with MB ₂₀₀₀. CEU molecular imaging targeted to intercellular adhesion molecule-2 showed low overall signal for all agents and signal enhancement above control for MB ₃₄₀₀ only. Glycocalyx degradation increased signal for MB ₃₄₀₀ and MB ₂₀₀₀. Conclusions: Microbubble targeting to endothelial ligands is influenced by (1) the tether length of the ligand, (2) the degree to which the endothelial target is projected from the cell surface, and (3) the status of the glycocalyx. These considerations are important for designing targeted imaging probes and understanding potential obstacles to molecular imaging.