Design and Evaluation of a ⁶⁸ Ga-Labeled Dimeric Cyclic Peptide as a Large-Volume Linker Strategy for Balancing Affinity and Retention in CXCR4-Targeted PET Imaging

CXCR4 is overexpressed in various malignancies and represents an attractive target for PET imaging. However, currently available peptide-based tracers often exhibit rapid clearance and a narrow imaging window, which limit their clinical implication. Here, we report the design and preclinical characterization of a ⁶⁸Ga-labeled dimeric cyclic peptide, implementing a large-volume linker strategy aimed at achieving an optimized balance between receptor affinity and tumor retention for CXCR4-targeted PET imaging. The tracer was synthesized, radiolabeled with the ⁶⁸Ga³⁺ ion, and evaluated for radiochemical purity. <i>In vitro</i> stability, binding affinity, CXCR4-specific cellular uptake, pharmacokinetics, biodistribution, and PET/CT imaging were also assessed. The tracer showed high radiochemical purity and excellent stability. Although its binding affinity was moderate (IC₅₀ = 161.5 nM), the tracer exhibited clear CXCR4-specific uptake and sustained tumor retention, with PET-derived tumor uptake of 3.4-3.8%ID/g between 30 and 240 min and tumor-to-muscle ratios increasing from ∼10 to ∼62 over the same period. However, notable hepatic uptake was observed, which may be attributed to the peptide size and moderate hydrophobicity. Further structural optimization such as PEGylation or scaffold minimization may reduce hepatic uptake and enhance the clinical applicability.