Liposomal Cu-64 labeling method using bifunctional chelators: Poly(ethylene glycol) spacer and chelator effects

Two bifunctional Cu-64 chelators (BFCs), (6-(6-(3-(2-pyridyldithio) propionamido)hexanamido)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8, 11-tetraacetic acid (TETA-PDP) and 4-(2-(2-pyridyldithioethyl)ethanamido)-11- carboxymethyl-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane (CB-TE2A-PDEA), were synthesized and conjugated to long-circulating liposomes (LCLs) via attachment to a maleimide lipid. An in vitro stability assay of ⁶⁴Cu-TETA, ⁶⁴Cu-TETA-PEG2k, and ⁶⁴Cu-CB-TE2A-PEG2k liposomes showed that more than 86% of the radioactivity remains associated with the liposomal fraction after 48 h of incubation with mouse serum. The in vivo time activity curves (TAC) for the three liposomal formulations showed that -50% of the radioactivity cleared from the blood pool in 16-18 h. As expected, the in vivo biodistribution and TAC data obtained at 48 h demonstrate that the clearance of radioactivity from the liver slows with the incorporation of a poly(ethylene glycol)-2k (PEG2k) brush. Our data suggest that ⁶⁴Cu-TETA and ⁶⁴Cu-CB-TE2A are similarly stable in the blood pool and accumulation of radioactivity in the liver and spleen is not related to the stability of Cu-64 chelator complex; however, clearance of Cu-64 from the liver and spleen are faster when injected as ⁶⁴Cu-TETA-chelated liposomes rather than ⁶⁴Cu-CB-TE2A-chelated liposomes.