Characterization of thein vitro adherence behavior of ultrasound responsive double-shelled microspheres targeted to cellular adhesion molecules

Abstract

We have developed novel adhesion molecule-targeted double-shelled microspheres which encapsulate nitrogen. We report in vitro targeting studies utilizing these microspheres conjugated to target-specific antibodies directed towards ICAM-1 and VCAM-1. In static adherence experiments, the adherence patterns of microspheres conjugated to three different monoclonal antibodies (two targeted to ICAM-1 and one to VCAM-1) to their target surfaces were very different. Maximum microsphere adherence at the lowest target and/or ligand densities was observed with the VCAM-1 system. Differences in target-specific adherence were also observed between anti-ICAM-1 and anti-VCAM-1 microsphere conjugates in flow adherence studies. Equilibrium binding studies of the target proteins in solution to the microsphere-bound ligands showed that the affinity constants of two microsphere-bound monoclonal antibodies for their target proteins are similar. Thus, ligand-target affinity is not the only determinant of microsphere adherence to the target surface in our systems. Shear stress was found to have an effect on the mean diameter of adhered microspheres; a decrease in the mean diameter with increasing shear was observed. The magnitude of this effect was dependent on both microsphere-bound ligand and target surface densities, with a more pronounced change at lower densities. Adhered microspheres were readily detectable using ultrasound at the lowest tested surface density of 40 mm(-2).

Targeted ultrasound contrast agents for ultrasound molecular imaging and therapy

“…Combinations of different shell materials have also been reported; Ottoboni et al [17] used microcapsules with a cross-linked albumin outer layer and a poly-(DL-lactide) inner layer. The advantage of the two different layers is the possibility to tweak the acoustic performance via the inner polymer layer in terms of their stiffness and thickness, and to change the biological activity via the protein outer layer.…”

“…Specific inflammation markers that have been used for tUCAs are intracellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, and P-selectin [17,19,35,36]. MBs targeted to VCAM-1 can be used to discriminate the severity of inflammatory burden in mice with various degrees of atherosclerosis [31].…”

Preparation of targeted microbubbles: ultrasound contrast agents for molecular imaging

“…Streptavidin is then used to attach biotinylated antibody [32]. Thus, combined (especially in the preclinical animal studies) benefits of covalent (i.e., readymade microbubbles requiring minimal washes) and noncovalent techniques (i.e., universal choice of ligand from a wide range of available biotinylated materials) can be realized.…”

“…This method applies to the water-insoluble polymers. A simpler singleemulsion variant of this method exists, with the preparation of oil-in-water emulsion containing a polymer solution in a very volatile organic solvent and a moderately volatile organic nonsolvent [4,32]) that precipitates polymer and forces it to form the shell around the nonsolvent core, as soon as the volatile component evaporates from the emulsion mixture particles. Extended evaporation process, such as lyophilization, is subsequently applied to remove the nonsolvent, which leaves the empty internal core, to be filled with gas.…”

Ultra-fast bright field and fluorescence imaging of the dynamics of micrometer-sized objects

“…The conjugation of biotinylated R-Phycoerythrin (R-PE) (Invitrogen, Life Technologies, Grand Island, NY) was carried out using biotin streptavidin interaction. 29 In brief, 0.2 ml of MB (5 e8/ml concentration) was mixed with 0.2 ml biotin R-PE (0.36 mg/ml) in phosphor buffered saline (PBS) for 1 h at room temperature. The mixture was washed by adding 1 ml PBS followed with centrifugation at 200 g for 3 min.…”