Synthesis and Evaluation of Technetium-99m-labeled pH (low) Insertion Peptide Variant 7 for Early Diagnosis of MDA-MB-231 Triple-Negative Breast Cancer by Targeting the Tumor Microenvironment

Objective To prepare technetium-99m (99mTc)-labeled pH (low) insertion peptide variant 7 (pHLIP(Var7)) and carry out imaging of tumor-bearing nude mice in vivo, to study its value in the early diagnosis of triple-negative breast cancer (TNBC).Methods The pHLIP(Var7) sequence was synthesized by solid-phase polypeptide synthesis. Four amino acids, Gly-(D)-Ala-Gly-Gly, were attached to the N-terminus of pHLIP(Var7) to form a strong chelating group containing an N4 structure. The polypeptide was directly labeled with 99mTc to determine the in vitro binding propensity of 99mTc-pHLIP(Var7) to MDA-MB-231 cells, to measure the in vivo biodistribution of 99mTc-pHLIP(Var7) in tumor-bearing nude mice, and to perform small-animal single-photon-emission computed tomography (SPECT)/computed tomography (CT) of these mice.Results The radiochemical yield and radiochemical purity of 99mTc-pHLIP(Var7) were 99.49%±0.17% and 99.63±0.44%, respectively. The radiochemical purity was still more than 96% after 24 h in serum. The binding fraction of 99mTc-pHLIP(Var7) and MDA-MB-231 cells continuously increased in an acidic environment, being significantly higher than their cell binding fraction (P < 0.01) when pH = 7.4 and the cell binding fraction (P < 0.01) of 99mTc-kVar7 at different pH values (pH = 6.0, 6.5, 7.0 and 7.4) at each time point (P < 0.01). The biodistribution assay showed that the distribution of 99mTc-pHLIP(Var7) in tumors at each time point was significantly higher than that of 99mTc-kVar7 (P < 0.01). 99mTc-pHLIP(Var7) and 99mTc-kVar7 were also significantly distributed in the liver. SPECT/CT imaging was basically consistent with the biodistribution results. The tumor was clearly imaged at each time point after injection of 99mTc-pHLIP(Var7) but could not be imaged after injection of 99mTc-kVar7. The liver was clearly imaged at each time point in both groups.Conclusion 99mTc-pHLIP(Var7) has high radiochemical yield and stability, and it concentrates highly in tumor tissues. The presence of a very strong radioactive background in the abdomen of tumor-bearing nude mice does not preclude the early diagnosis of TNBC.