99mTc-HYNIC-(tricine/EDDA)-FROP peptide for MCF-7 breast tumor targeting and imaging

Abstract

Background: Breast cancer is the most common malignancy among women in the world. Development of novel tumor-specific radiopharmaceuticals for early breast tumor diagnosis is highly desirable. In this study we developed ^99mTc-HYNIC-(tricine/EDDA)-Lys-FROP peptide with the ability of specific binding to MCF-7 breast tumor.

Methods: The FROP-1 peptide was conjugated with the bifunctional chelator hydrazinonicotinamide (HYNIC) and labeled with ^99mTc using tricine/EDDA co-ligand. The cellular specific binding of ^99mTc-HYNIC-FROP was evaluated on different cell lines as well as with blocking experiment on MCF-7 (human breast adenocarcinoma). The tumor targeting and imaging of this labeled peptide were performed on MCF-7 tumor bearing mice.

Results: Radiochemical purity for ^99mTc-HYNIC-(tricine/EDDA)-FROP was 99% which was determined with ITLC method. This radiolabeled peptide showed high stability in normal saline and serum about 98% which was monitored with HPLC method. In saturation binding experiments, the binding constant (K_d) to MCF-7 cells was determined to be 158 nM. Biodistribution results revealed that the ^99mTc-HYNIC-FROP was mainly exerted from urinary route. The maximum tumor uptake was found after 30 min post injection (p.i.); however maximum tumor/muscle ratio was seen at 15 min p.i. The tumor uptake of this labeled peptide was specific and blocked by co-injection of excess FROP. According to the planar gamma imaging result, tumor was clearly visible due to the tumor uptake of ^99mTc-HYNIC-(tricine/EDDA)-FROP in mouse after 15 min p.i.

Conclusions: The ^99mTc-HYNIC-(tricine/EDDA)-FROP is considered a promising probe with high specific binding to MCF-7 breast cancer cells.

Keywords: Breast cancer; FROP; Imaging; MCF-7; Radiopharmaceutical; Tumor targeting.

Fig. 1

The chemical structure of the HYNIC-K-FROP peptide

Fig. 2

Radio high-performance liquid chromatography (HPLC) analysis of ^99mTc- HYNIC-(tricine/EDDA)-FROP peptide at 1 h after labeling (a) and incubated in human serum at 1 h after labeling (b). The retention time of peptide was from 20 to 22 min and for mixture of ^99mTCO₄⁻ and ^99mTc-co-ligand was 3–5 min

Fig. 3

a The ability binding of ^99mTc- HYNIC-(tricine/EDDA)-FROP peptide on MCF-7 cells and comparison of this binding with binding to the SKOV-3 (ovarian cancer), A-549 (non-small cell lung cancer), T47D (breast cancer) and PC-3 (prostate cancer) cell lines and b HFFF2 (normal human fibroblast) cell line. The data are shown as the means ± SD. Data indicates significant difference (p values< 0.0001) in uptake of radiolabeled peptide between different cancer and normal cell lines. c In vitro specific binding of ^99mTc-HYNIC-(tricine/EDDA)-FROP to MCF-7 cells. Pre-saturation was done by blocking unlabeled FROP (500-fold excess) peptide after 0.5 h incubation at 37 °C

Fig. 4

Internalization of the ^99mTc-HYNIC-(tricine/EDDA)-FROP peptide by MCF-7 cells at different times after incubation at 37 °C: total binding represents uptake on the surface membrane plus that inside of the cell; internalization represents the activity inside of the cell only

Fig. 5

Saturation binding assay curves of ^99mTc-HYNIC-(tricine/EDDA)-FROP peptide upon incubation with MCF-7 cells

Fig. 6

Imaging of MCF-7 breast cancer xenograft nude mouse using ^99mTc-HYNIC-(tricine/EDDA)-FROP peptide; planar γ-camera images were acquired at 15 min after injection