Multi-modality imaging of bone formation by PET/CT and near-infrared imaging

Objectives Aberrant matrix metalloproteinase (MMP) expression regulates disease and ossification processes including invasion, migration and degradation of extracellular matrices. In this study, we used a dual-labeled targeting agent specific for activated MMP-2/9 to non-invasively monitor the early stages of bone formation. We selected a model of heterotopic ossification (HO) which involves the delivery of bone morphogenetic protein (BMP)-2 via AdBMP2-transduced cells.

Methods A peptide sequence (KKAHWGFTLD) specific for MMP-2/9 was used as a targeting agent. The peptide was conjugated to DOTA (DOTA-MMP) and compared to a control peptide (DOTA-SCR). IRDye 800CW was conjugated to each peptide and bioactivity was assessed using a plate-based MMP-2/9 inhibition assay. The dye-conjugated peptides were radiolabeled with 64Cu and purified by C-18 columns. Radiochemical yield was determined by radio-TLC. At 2-6 days prior to imaging, human fibroblast MRC5 cells transduced with Ad5F35BMP2 or Ad5F35empty (control) were injected (i.m.) into the hind-legs of NOD.SCID mice. MicroPET/CT imaging was performed with 64Cu-DOTA-MMP or 64Cu-DOTA-SCR (without dye) at 5 and 24 hours post-injection to determine effect of MMP expression on tracer accumulation.

Results Dye-labeled DOTA-MMP showed a similar MMP-2/9 inhibition level relative to that of unlabeled DOTA-MMP while DOTA-SCR did not inhibit the active enzymes. Radiolabeling studies demonstrated that dye-labeled DOTA-MMP could be dual-labeled with 64Cu at high yields (>95%) without affecting the integrity of the dye. Micro PET/CT images showed tracer accumulation at the target site at 5 h post-injection, while DOTA-SCR did not localize at the site of HO.

Conclusions A novel targeting agent for multi-modality imaging of bone growth was developed. Further studies are required to evaluate the specificity of the dual-labeled agent in vivo and determine the optimal time point for MMP imaging post-fibroblast implantation.

Research Support Work supported by DOD (DARPA-BAA-08-50)