Transferrin conjugates of triazacyclononane-based bifunctional NE3TA chelates for PET imaging: Synthesis, Cu-64 radiolabeling, and in vitro and in vivo evaluation

Three different polyaminocarboxylate-based bifunctional NE3TA (7-[2-[carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) chelating agents were synthesized for potential use in copper 64-PET imaging applications. The bifunctional chelates were comparatively evaluated using transferrin (Tf) as a model targeting vector that binds to the transferrin receptor overexpressed in many different cancer cells. The transferrin conjugates of the NE3TA-based bifunctional chelates were evaluated for radiolabeling with ⁶⁴Cu. In vitro stability and cellular uptake of ⁶⁴Cu-radiolabeled conjugates were evaluated in human serum and prostate (PC-3) cancer cells, respectively. Among the three NE3TA-Tf conjugates tested, N-NE3TA-Tf was identified as the best conjugate for radiolabeling with ⁶⁴Cu. N-NE3TA-Tf rapidly bound to ⁶⁴Cu (> 98% radiolabeling efficiency, 1 min, RT), and ⁶⁴Cu-N-NE3TA-Tf remained stable in human serum for 2 days and demonstrated high uptake in PC-3 cancer cells. ⁶⁴Cu-N-NE3TA-Tf was shown to have rapid blood clearance and increasing tumor uptake in PC-3 tumor bearing mice over a 24 h period. This bifunctional chelate presents highly efficient chelation chemistry with ⁶⁴Cu under mild condition that can be applied for radiolabeling of various tumor-specific biomolecules with ⁶⁴Cu for potential use in PET imaging applications.