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

ABSTRACT 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 [ 125 I]I‐TAT was also prepared as a control. In vitro studies carried out in CHO cells under hypoxic conditions revealed three fold uptake of [ 125 I]I‐TAT‐2‐NIM compared to [ 125 I]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, [ 125 I]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 5 I]I‐TAT‐2‐NIM showed higher accumulation in hypoxic cells compared to [ 125 I]I‐TAT in vitro, the negative influence of hypoxia on the cellular uptake of TAT‐peptides possibly indicates their limitation for hypoxia targeting applications.