Design and Preclinical Evaluation of a ⁶⁸ Ga-Labeled Macrocyclic Peptide PET Probe Targeting Transferrin Receptor 1 in Colorectal Cancer

Transferrin Receptor 1 (TfR1/CD71) is frequently overexpressed in colorectal cancer (CRC) and is associated with poor clinical outcomes, making it a compelling target for molecular imaging. However, conventional immunohistochemical (IHC) assessment is limited by spatial heterogeneity. To address this, we developed ⁶⁸Ga-NOTA-TR01, a novel ⁶⁸Ga-labeled macrocyclic peptide probe designed for noninvasive positron emission tomography (PET) imaging of TfR1 expression, aiming to enhance precision diagnosis and guide therapy stratification in CRC. The macrocyclic peptide precursor NOTA-TR01 was synthesized and radiolabeled with ⁶⁸Ga. Radiochemical purity (RCP), molar activity, stability in phosphate-buffered saline and serum, and lipophilicity (log <i>P</i>) were systematically evaluated. <i>In vitro</i> characterization included surface plasmon resonance (SPR) to determine binding affinity, flow cytometry to assess TfR1 expression in CRC cell lines (HT29: high expression; LOVO: low expression), and cellular binding/blocking assays. <i>In vivo</i> performance was evaluated using microPET/CT imaging and biodistribution studies in HT29 and LOVO tumor-bearing mice, with specificity confirmed by blocking with excess unlabeled peptide. Tumoral TfR1 expression was further validated by IHC. ⁶⁸Ga-NOTA-TR01 was synthesized with excellent RCP (>99%), high molar activity (>20 GBq/μmol), robust stability (>98% at 24 h), and hydrophilic character (log <i>P</i> = -1.709). SPR demonstrated strong binding affinity (<i>K</i> _D = 2.23 × 10^{- 8} M). <i>In vitro</i>, binding in TfR1^high HT29 cells significantly exceeded that in TfR1^low LOVO cells (9.56 ± 0.17% vs 4.51 ± 0.22% AD/5 × 10⁵ cells at 120 min; <i>P</i> < 0.001) and could be inhibited by over 85% with cold peptide. microPET imaging revealed specific accumulation in HT29 tumors (peak tumor-to-muscle ratio = 7.88 ± 0.79 at 120 min), which was markedly suppressed (83% reduction) by coinjection of excess unlabeled peptide. Biodistribution data confirmed significantly higher uptake in HT29 tumors (1.12 ± 0.16%ID/g vs 0.90 ± 0.21%ID/g in LOVO tumors at 120 min; <i>P</i> < 0.05), consistent with IHC results. ⁶⁸Ga-NOTA-TR01 was a high-affinity, metabolically stable PET imaging agent capable of specifically visualizing TfR1 expression <i>in vivo</i>. This novel probe demonstrated promising potential for noninvasive patient stratification, treatment monitoring, and prognostic assessment in TfR1-positive CRC.