Sensitive angiogenesis imaging of orthotopic bladder tumors in mice using a selective magnetic resonance imaging contrast agent containing VEGF ₁₂₁/rGel

Objectives: To investigate the efficiency of magnetic resonance imaging (MRI) contrast agents employing vascular endothelial growth factor (VEGF ₁₂₁)/rGel conjugated MnFe₂O₄ nanocrystals for imaging of neovasculature using a bladder tumor model. Materials and Methods: VEGF₁₂₁/rGel was conjugated to MnFe₂O₄ nanoparticles (MNPs). The targeting efficiency and detection capability of the VEGF₁₂₁/rGel-MNPs were investigated in both KDR-deficient (253JB-V) and KDR-overexpressing (PAE/KDR) cells using MRI. The internalization of VEGF₁₂₁/rGel-MNPs into cells was confirmed by electron microscopy. Their phosphorylation ability and cytotoxicity were compared with unconjugated VEGF₁₂₁/rGel. The orthotopic tumor mice were established by implanting low KDR-expressing 253JB-V cells into the bladder dome. After tail-vein injection of VEGF₁₂₁/rGel-MNPs, the MR signal enhancement of intratumoral vessels by VEGF₁₂₁/rGel-MNPs was observed and inhibition test using VEGF₁₂₁ was also conducted. Ex vivo MR imaging of tumor tissue, and a fluorescence immunostaining study was also performed. Results: The water-soluble VEGF₁₂₁/rGel-MNPs (44.5 ± 1.2 nm) were stably suspended in the biologic media and exhibited a high relaxivity coefficient (423 mM^-1s^-1). They demonstrated sufficient targeting capability against KDR-overexpressing PAE/KDR cells, as confirmed by dosedependent MR images and VEGF₁₂₁ inhibition tests The phosphorylation activity of KDR and cytotoxicity of VEGF₁₂₁/rGel-MNPs were evaluated. VEGF₁₂₁/rGel-MNPs successfully targeted the tumor and provided accurate anatomic details through (i) acquisition of clear neoangiogenic vascular distributions and (ii) obvious enhancement of the MR signal in T2^*-weighted images. Immunostaining and blocking studies demonstrated the specific targeting ability of VEGF₁₂₁/rGel- MNPs toward intratumoral angiogenesis. Conclusions: Synthesized VEGF ₁₂₁/rGel-MNPs as targeted MR imaging contrast agents can be specifically delivered to tumors and bind to KDR-expressing angiogenic tumor vessels.