Objective To develop a radiolabeled peptide molecular tracer ⁹⁹Tc^m-His-Arg-Pro-Tyr-Ile-Ala-His (⁹⁹Tc^m-T7) targeting transferrin receptor and evaluate its micro SPECT/CT imaging effect in tumor-bearing nude mice models.

Methods The peptide probe ⁹⁹Tc^m-T7 was developed by indirect labeling method with the coordination of co-ligands N-tri (hydroxymethyl) methylglycine and ethylenediamine diacetate. The expression levels of TfR on the surface of human pancreatic PANC-1 tumor cells and human breast MX-1 tumor cells were measured through flow cytometry. Cell binding and competitive blocking assays was conducted to analyze the binding affinity and specificity of ⁹⁹Tc^m-T7 in vitro. Micro SPECT/CT imaging and biodistribution after the establishment of mouse xenograft models were performed in vivo to evaluate the affinity and feasibility of noninvasive tumor imaging. Radio-autograph assay and immunohistochemical staining were conducted to validate the correlation between the uptake of ⁹⁹Tc^m-T7 and expression of TfR in tumor tissues. Independent sample t-test was used for the comparison between the two groups.

Results The radiolabeled probe ⁹⁹Tc^m-T7 was successfully synthesized with a radiolabeling yield of greater than 95%. It exhibited great stability in vitro, with radiochemical purities of (95.3±0.3)% and (90.6±0.4)% after incubation in normal saline and fetal bovine serum for 4 hours, respectively. The results of flow cytometry showed that PANC-1 tumor cells overexpressed TfR on the surface with a high tendency to bind TfR monoclonal antibody ((98.9±0.1)%), whereas MX-1 tumor cells showed low TfR expression on the membrane( (0.2±0.1)%). In vitro cell binding assay results showed that the binding rate of PANC-1 cells to ⁹⁹Tc^m-T7 reached a peak ((16.12±0.01)%) after 60 minutes of incubation, which was higher than that of MX-1 cells ((1.20±0.01)%), and the difference between them was statistically significant (t=28.67, P<0.001). The results of cell competition inhibition experiment showed that the binding rate of PANC-1 blocking group to ⁹⁹Tc^m-T7 decreased to (2.40±0.01)%, which was significantly different from that of PANC-1 experimental group(t=26.91, P<0.001). The results of micro SPECT/CT imaging in nude mice bearing tumor showed that ⁹⁹Tc^m-T7 could be quickly cleared from the blood and mainly eliminated from the kidneys. PANC-1 tumor-bearing nude mice models showed clear tumor contour 30 minutes after injection of ⁹⁹Tc^m-T7, with a tumor-to-muscle ratio of 2.80±0.22. The results of biological distribution experiments showed that the uptake of ⁹⁹Tc^m-T7 by tumors and organs (percentage injection dose rate (%ID/g) per gram of tissue) was consistent with the imaging results, and the uptake of ⁹⁹Tc^m-T7 in PANC-1 cells ((0.55±0.18)%ID/g) was higher than that in MX-1 cells ((0.16±0.11)%ID/g), and the difference was statistically significant (t=6.42, P<0.001). The radio-autograph assay showed that PANC-1 cells significantly absorbed ⁹⁹Tc^m-T7 compared with MX-1 cells 30 minutes after injection of ⁹⁹Tc^m-T7. The highest uptake in normal organs was observed in the kidney, followed by the liver. Hematoxylin-eosin and immunohistochemical staining revealed no obvious necrosis in the tumor parenchyma. The PANC-1 cells overexpressed TfR, and whereas the MX-1 cells had low TfR expression.

Conclusion A specific polypeptide molecular probe ⁹⁹Tc^m-T7 targeting TfR was successfully prepared, which has excellent imaging efficiency in tumor-bearing nude mice models, and is expected to provide a new imaging method for monitoring the expression of tumor TFR in vivo.