Mitochondria-tropic radioconjugates to enhance the therapeutic potential of terbium-161

Background: Strategies that focus on delivering Auger electron emitters to highly radiosensitive intracellular targets—such as the nucleus, cell membrane, or mitochondria—are gaining attention. Targeting these organelles could enhance therapeutic efficacy while minimizing off-target toxicity by allowing lower administered doses. In this context, this study explores the therapeutic potential of ¹⁶¹Tb-labeled radiocomplexes that integrate the mitochondria-targeting triphenylphosphonium (TPP) moiety with a prostate-specific membrane antigen (PSMA) targeting vector. The goal is to assess these dual-targeted radiocomplexes for their ability to deliver conversion electrons (CE) and Auger electrons (AEs) to prostate cancer (PCa) cells, specifically targeting the mitochondria to enhance therapeutic efficacy. Results: Two novel radiocomplexes, [¹⁶¹Tb]Tb-TPP-PSMA and [¹⁶¹Tb]Tb-TPP-G₃-PSMA, were synthesized with high radiochemical yield and purity. The proposed structures were validated using HPLC and ESI-MS analysis, with their ^natTb counterparts serving as reference compounds. In vitro experiments included cellular uptake, internalization, mitochondrial uptake, and DNA damage assays in PSMA-positive PCa cell lines. Clonogenic assays were performed to evaluate cell survival post-treatment. In vivo studies were conducted using SCID/Beige mice bearing PCa xenografts and involved µSPECT/CT imaging and radiometabolite analysis to evaluate biodistribution, pharmacokinetics, tumor uptake and in vivo stability of the radiocomplexes. Both [¹⁶¹Tb]Tb-TPP-PSMA and [¹⁶¹Tb]Tb-TPP-G₃-PSMA showed high radiochemical stability and were efficiently internalized by PSMA-positive cells, while showing minimal uptake in PSMA-negative cells. These dual-targeted radiocomplexes demonstrated significantly higher mitochondrial uptake compared to the non-TPP-containing [¹⁶¹Tb]Tb-PSMA-617, leading to increased DNA damage and enhanced radiocytotoxicity. In vivo, the dual-targeted complexes demonstrated PSMA-specific tumor uptake and pharmacokinetics comparable to [¹⁶¹Tb]Tb-PSMA-617, with effective clearance from non-target tissues. Conclusions: The TPP-modified ¹⁶¹Tb-radiocomplexes effectively targeted the mitochondria of PSMA-positive PCa cells, leading to increased DNA damage and reduced cell viability compared to single-targeted radiocomplexes. These findings suggest that dual-targeting strategies, which combine PSMA and mitochondrial targeting, can enhance the therapeutic potential of radiopharmaceuticals for prostate cancer treatment.