Linking Proteins with Anionic Nanoparticles via Protamine: Ultrasmall Protein-Coupled Probes for Magnetic Resonance Imaging of Apoptosis

Magnetic resonance imaging (MRI) of a target in vivo depends on the surface, size, and particle relaxivity of the target-specific nanoparticles for MRI. Here a new method for decorating very small iron oxide particles (VSOPs) with target-specific ligands is described. The method is based on the electrostatic attraction of the strongly positively charged peptide protamine to the anionic citrate shell of the electrostatically stabilized VSOPs. The protamine coat allows linkage chemistry and chimera technology to functionalize VSOPs or other negative charged surfaces with biologics. Annexin A5 (anxA5)-VSOP utilizing thiol chemistry was generated to couple biologically active anxA5 to VSOPs for in vivo MRI of apoptosis. Annexin A5-VSOP comprises five anxA5 molecules per iron oxide nanoparticle with a high R2 particle relaxivity of 180 000 mM(-1) s(-1) yet small hydrodynamic diameter of only 14.7+/-2.9 nm beneficial for in vivo MRI of extravascular targets.