The Cytotoxic Effect of ⁶⁴Cu/NOTA-Terpyridine Platinum Conjugate, as a Novel Chemoradiotherapy Agent ^†

Colorectal cancer is one of the most prevalent cancers worldwide that displays both intrinsic and acquired resistance to platinum-based chemotherapeutic agents (Pt-CAs). To overcome such resistance, new classes of Pt-CAs have been proposed, including terpyridine (TP) compounds that target the G-quadruplex tertiary structure of DNA. Additionally, recent studies indicate a maximum chemoradiation benefit when radiation is administered with Pt-CAs at their highest concentrations in cancer cell DNA. Accordingly, we synthesized a novel chemo-radiotheranostic agent by conjugating a TP moiety with ⁶⁴Cu (⁶⁴Cu-NOTA-TP). The in vitro cytotoxic effects, cellular uptake, internalization and efflux of ⁶⁴Cu-NOTA-TP were measured for colorectal cancer (HCT116) and normal fibroblast (GM05757) cells. Radiolabeling NOTA-TP with ⁶⁴Cu resulted in 17,530-, 40,083- and 66,000-fold enhancements in its cytotoxicity against HCT116 cells (EC₅₀ = 0.017 ± 0.004, 0.012 ± 0.006 and 0.005 ± 0.0002 µM) as compared to ^coldCu-NOTA-terpyridine (EC₅₀ = 298 ± 2, 481 ± 25 and 330 ± 51 µM) at 24, 48 and 72 h post-administration, respectively. More importantly, the cytotoxicity of the ⁶⁴Cu-conjugate toward the HCT116 cells was about 3.8-fold higher than that of GM05757 cells at 24 and 72 h. This result was consistent with a 2–3-fold higher internalization of ⁶⁴Cu-conjugate in HCT116 cells relative to GM05757 cells at similar times. The internalized activity of the ⁶⁴Cu-conjugate steadily increased from 0.04 ± 0.02% to 18.7 ± 2.8% over a 24 h incubation time. Moreover, the efflux kinetics of the ⁶⁴Cu-conjugate showed that more than 40% of internalized activity was retained by cancer cells over a 24 h period. In conclusion, this work presents a novel chemo-radiotherapeutic agent with considerable potential for targeted cancer treatment combined with radioisotope imaging.