Exploring CPP Enhanced Hypoxia Targeting: An Approach Using Radiolabeled 2‐nitroimidazole TAT Conjugate

Mediated by their protein transduction domain, cell-penetrating peptides (CPPs) have been widely explored for facilitating intracellular delivery of drugs and small molecules. In this study, a radiolabeled, 2-nitroimidazole conjugated TAT (TAT - transactivator of transcription) peptide was prepared and evaluated for targeting tumor hypoxia. The conjugate was radiolabeled with iodine-125 and comprehensive in vitro and in vivo evaluations were conducted to assess its potential to target hypoxic cells. A radiolabeled [¹²⁵I]I-TAT was also prepared as a control. In vitro studies carried out in CHO cells under hypoxic conditions revealed three fold uptake of [¹²⁵I]I-TAT-2-NIM compared to [¹²⁵I]I-TAT at 1 h post-incubation, which further increased sixfold at 4 h post-incubation, clearly demonstrating the role of nitroimidazole in targeting hypoxic cells. However, the study also revealed a negative influence of hypoxia in cellular uptake, wherein the radiolabeled TAT peptides showed a lower uptake in cells under hypoxic conditions than in normoxic conditions. In vivo, [¹²⁵I]I-TAT-2-NIM showed uptake and retention in the tumor with the tumor/muscle ratio above 2 at all time points studied. However, the tumor/blood ratio was found to decrease with time. Though [¹² ⁵I]I-TAT-2-NIM showed higher accumulation in hypoxic cells compared to [¹²⁵I]I-TAT in vitro, the negative influence of hypoxia on the cellular uptake of TAT-peptides possibly indicates their limitation for hypoxia targeting applications.