Magnetic Resonance Imaging of Inducible E-Selectin Expression in Human Endothelial Cell Culture

Abstract

Covalent conjugates of the cross-linked iron oxide nanoparticles (CLIO) and high-affinity (K_d^app = 8.5 nM) anti-human E-selectin (CD62E) F(ab‘)₂ fragments were prepared and tested in vitro to establish feasibility of endothelial proinflammatory marker magnetic resonance (MR) imaging. The conjugates were obtained by using thiol−disulfide exchange reaction between 3-(2-pyridyl)propionyl-CLIO and S-acetylthioacetate-modified F(ab‘)₂ fragments. The purified CLIO−F(ab‘)₂ conjugates (average hydrodynamic diameter 40.6 nm) were used in experiments with the live human endothelial umbilical vein cells (HUVEC). Cells treated with IL-1β expressed E-selectin and showed a 100−200 times higher binding of CLIO particles (83−104 ng iron/million cells) than control cells. The binding resulted in a high superparamagnetism of HUVEC with the transverse water proton relaxation time (T2) decrease to 30−40 ms in cell precipitates. Cells did not bind/internalize CLIO−F(ab‘)₂ conjugates prepared using a control fragment or nonconjugated iron oxide particles before or after treatment with IL-1β. MR imaging of cells showed a highly specific T2-weighted signal darkening associated with cells treated with IL-1β and incubated with anti-E selectin. Demonstration of MR imaging of E-selectin expression justifies further development of MR-targeted agents for monitoring tumor vascular endothelial proliferation, angiogenesis, and atherosclerosis.