Application of copper (I) selective ligands for PET imaging of reactive oxygen species through metabolic trapping

Reactive oxygen species (ROS) are attractive targets for clinical PET imaging. In this study, we hypothesized that PET imaging of ROS would be possible by using chelating ligands (L) that form stable complexes with copper (I) but not with copper (II), based on metabolic trapping. Namely, when [⁶⁴Cu][Cu^I(L)₂]⁺ is oxidized by ROS, the oxidized complex will release [⁶⁴Cu]Cu²⁺. Then, the released [⁶⁴Cu]Cu²⁺ will be trapped inside the cell, resulting in PET signal depending on the redox potential of ROS. To examine the potential of this novel molecular design for ROS imaging, we synthesized copper (I) complexes with bicinchoninic acid (BCA) disodium salt and bathocuproinedisulfonic acid (BCS) disodium salt and evaluated their reactivity with several kinds of ROS. In addition, the cellular uptake of [⁶⁴Cu][Cu^I(BCS)₂]^3- and the stability of [⁶⁴Cu][Cu^I(BCS)₂]^3- in a biological condition were also evaluated.

[⁶⁴Cu]Cu²⁺ was reduced to [⁶⁴Cu]Cu⁺ by ascorbic acid and coordinated with BCA and BCS in the acetate buffer to synthesize [⁶⁴Cu][Cu^I(BCA)₂]^3- and [⁶⁴Cu][Cu^I(BCS)₂]^3-. The radiochemical yields were determined by thin-layer chromatography (TLC). After [⁶⁴Cu][Cu^I(BCS)₂]^3- was incubated with hydroxyl radical, lipid peroxide, superoxide, and hydrogen peroxide, the percentage of released [⁶⁴Cu]Cu²⁺ from the parent complex was evaluated by TLC. HT-1080 human fibrosarcoma cells were treated with 0.1 % Dimethyl sulfoxide (control), imidazole ketone erastin (IKE), or IKE + ferrostatin-1 (Fer-1). Then, the uptake of [⁶⁴Cu][Cu^I(BCS)₂]^3- to HT-1080 cells in each group was evaluated as %Dose/mg protein. Lastly, [⁶⁴Cu][Cu^I(BCS)₂]^3- was incubated in human plasma, and its intact ratio was determined by TLC.

The radiochemical yield of [⁶⁴Cu][Cu^I(BCS)₂]^3- (86 ± 1 %) was higher than that of [⁶⁴Cu][Cu^I(BCA)₂]^3- (44 ± 3 %). [⁶⁴Cu][Cu^I(BCA)₂]^3- was unstable and partially decomposed on TLC. After [⁶⁴Cu][Cu^I(BCS)₂]^3- was reacted with hydroxyl radical, lipid peroxide, and superoxide, 67 ± 2 %, 44 ± 13 %, and 22 ± 3 % of total radioactivity was detected as [⁶⁴Cu]Cu²⁺, respectively. On the other hand, the reaction with hydrogen peroxide did not significantly increase the ratio of [⁶⁴Cu]Cu²⁺ (4 ± 1 %). These results suggest that [⁶⁴Cu][Cu^I(BCS)₂]^3- could be used for detecting high-redox-potential ROS such as hydroxyl radical and lipid peroxide with high selectivity. The cellular uptake values of [⁶⁴Cu][Cu^I(BCS)₂]^3- in the control, IKE, and Fer-1 group were 42 ± 2, 54 ± 2, and 47 ± 5 %Dose/mg protein (n = 3), respectively, suggesting the ROS specific uptake of [⁶⁴Cu][Cu^I(BCS)₂]^3-. On the other hand, the intact ratio after the incubation of [⁶⁴Cu][Cu^I(BCS)₂]^3- in human plasma was 9 ± 5 %.

PET imaging of ROS would be possible by using a copper (I) selective ligand, based on metabolic trapping. Although improvement of the membrane permeability and the stability of copper (I) complexes is required, the present results pave the way for the development of novel ⁶⁴Cu-labeled complexes for PET imaging of ROS.