Design of a Bifunctional pHLIP-RGD Scaffold for Site-Specific [Tc] Tc Labeling: Radiochemical Evaluation and Proof-of-Concept Tumor Targeting.

[PURPOSE] To develop a novel molecular scaffold designed for dual targeting via site-specific [Tc] Tc labeling, we report its radiochemical evaluation and preliminary targeting efficacy in acidic tumor models.

[PROCEDURES] The pHLIP-RGD scaffold was synthesized by conjugating pHLIP variant 7 (var7) with cyclo(RGDfK) peptide. Pharmacokinetic assessment of [Tc] Tc-pHLIP-RGD was performed in MDA-MB-231 xenograft-bearing mice through quantitative biodistribution studies and small-animal single photon emission computed tomography (SPECT) imaging.

[RESULTS] [Tc] Tc-pHLIP-RGD demonstrated high stability in mouse serum for at least 4 h and exhibited strong binding affinity and specificity both in vitro and in vivo. Biodistribution studies revealed rapid tumor accumulation and prolonged retention, with uptake values of 7.01 ± 1.28, 4.23 ± 0.44, 8.04 ± 0.63, and 9.60 ± 1.26%ID/g at 0.5, 1.0, 2.0, and 4.0 h post-injection, respectively. Off-target accumulation was primarily observed in the liver. In blocking studies, the administration of non-radioactive pHLIP-RGD partially reduced tumor uptake of [Tc] Tc-pHLIP-RGD, with tumor distribution values at 0.5, 1.0, 2.0, and 4.0 h of 4.66 ± 0.49, 3.25 ± 0.36, 3.04 ± 1.15, and 3.75 ± 0.57%ID/g, respectively. SPECT imaging findings were consistent with biodistribution data, showing clear visualization of tumors at all time points. Tumor visibility was significantly reduced in the blocking study, with a corresponding increase in liver uptake.

[CONCLUSIONS] The heterodimeric radiotracer [Tc] Tc-pHLIP-RGD exhibited high radiochemical yield, good stability, and favorable tumor uptake and retention characteristics. These proof-of-concept results suggest the potential of the dual-targeting design strategy for developing diagnostic imaging agents for triple-negative breast cancer (TNBC).