Neuroblastoma is the most common pediatric malignancy diagnosed in the first year of life. More than one-half of these children have disseminated disease at the time of diagnosis. Magnetic resonance imaging (MRI) is crucial in the diagnostic imaging evaluation of neuroblastoma, however, no neuroblastoma-specific MRI contrast agents currently exist. We propose to create neuroblastoma-specific MRI contrast agents by conjugating low-molecular weight peptide ligands (<2000 MW) that bind to the surface of neuroblastoma cells with monocrystalline iron oxide nanoparticles (MIONs). In addition to their ease of synthesis, low-molecular weight ligands have several attractive pharmacological and biochemical properties, including rapid biodistribution, excellent tissue penetration, and lack of immunogenicity. MIONs are ultrasmall (approximately 20 nm hydrodynamic radius) dextran-coated iron oxide superparamagnetic colloids that alter T2-weighted MRI contrast enhancement. Recent reports have suggested that MIONs can be conjugated to peptides and proteins to generate cell-specific contrast enhancement. To generate neuroblastoma-specific MRI imaging reagents, we will employ the following systematic approach. The conjugation of peptides to MIONs will be optimized using a previously described neuropeptide Y1 (NPY-1)-Iike molecule that binds with high affinity to neuroblastoma cells. Complete in vitro characterization of the MION/NPY-1 conjugate will be performed including binding affinity, kinetics, sub-cellular localization, and MRI contrast enhancement of living cells. In addition, NPY/MION conjugates will be evaluated in vivo using human neuroblastoma tumor xenografts established in nu/nu mice. Since NPY-1 has relative, but not absolute specificity for neuroblastoma cells, we will, in parallel, search for novel peptide ligands that bind to the surface of these cells. Using differential peptide phage display screening of two cell lines, one an aggressive neuroblastoma and the other its chemically-differentiated benign counterpart, we will attempt to discover peptides specific for the malignant phenotype. Conjugation of these novel peptides to MIONs could result in contrast agents with higher specificity than is possible with NPY-1 derivatives. As imaging technologies such as MRI have continued to improve, the integration of basic genetic and molecular biologic discoveries into development of novel tissue-specific contrast agents has lagged behind. This study presents a generalizable approach to producing tumor cell-specific MRI contrast reagents, and should permit improved diagnosis, and hence treatment, of neuroblastoma in children.
