Bi-Functional Radiotheranostics of 188Re-Liposome-Fcy-hEGF for Radio- and Chemo-Therapy of EGFR-Overexpressing Cancer Cells

Abstract

Epidermal growth factor receptor (EGFR) specific therapeutics is of great importance in cancer treatment. Fcy-hEGF fusion protein, composed of yeast cytosine deaminase (Fcy) and human EGF (hEGF), is capable of binding to EGFR and enzymatically convert 5-fluorocytosine (5-FC) to 1000-fold toxic 5-fluorocuracil (5-FU), thereby inhibiting the growth of EGFR-expressing tumor cells. To develop EGFR-specific therapy, ¹⁸⁸Re-liposome-Fcy-hEGF was constructed by insertion of Fcy-hEGF fusion protein onto the surface of liposomes encapsulating of ¹⁸⁸Re. Western blotting, MALDI-TOF, column size exclusion and flow cytometry were used to confirm the conjugation and bio-activity of ¹⁸⁸Re-liposome-Fcy-hEGF. Cell lines with EGFR expression were subjected to treat with ¹⁸⁸Re-liposome-Fcy-hEGF/5-FC in the presence of 5-FC. The ¹⁸⁸Re-liposome-Fcy-hEGF/5-FC revealed a better cytotoxic effect for cancer cells than the treatment of liposome-Fcy-hEGF/5-FC or ¹⁸⁸Re-liposome-Fcy-hEGF alone. The therapeutics has radio- and chemo-toxicity simultaneously and specifically target to EGFR-expression tumor cells, thereby achieving synergistic anticancer activity.

Keywords: 5-fluorocuracil; cytosine deaminase; epidermal growth factor receptor; liposome; prodrug; rhenium-188.

Figure 1

Identification of Fcy and Fcy-hEGF purified proteins. (A) The genes encoding Fcy–hEGF- and Fcy-myc-his₆ were digested with the restriction enzymes, BamHI and EcoRI, and cloned into the yeast vector, pPCIZ-αA. The alpha-secreting signaling peptide is present to assist in the secretion of the proteins. (B) SDS-Gel results of Fcy-hEGF fusion protein (left) and Fcy purified protein (right) stained by coomassie blue. (C) MALDI-TOF analysis of Fcy-hEGF (left) and Fcy (right) purified proteins.

Figure 2

Preparation and characterization of liposome-Fcy-hEGF and -Fcy. (A) Fcy-hEGF and Fcy were reduced by Trout’s reagent to synthesis the thiol-conjugation of Fcy-hEGF-SH and Fcy-SH, and then conjugation with maleimide-PEG2000-DSPE to synthesis the pegylated Fcy-hEGF and Fcy, which were further inserted onto the liposome surface. (B) Size exclusion of liposome-Fcy-hEGF (red line) and liposome-Fcy (blue line) was mediated by sepharose^TM 4B column. (C) The size analysis of liposome (red line of top and bottom panel), liposome-Fcy-hEGF (green line of top panel) and liposome-Fcy (green line of bottom panel) by nano-ZS size analyzer. (D) Flow cytometric analysis of HNE buffer and liposome for gating the liposome particles (red cycle). (E) Flow cytometric analysis of liposome without antibody staining (gray line) was as negative control, whereas liposome (black line), liposome-Fcy-hEGF (red line) and -Fcy (green line) were stained with Fluorochrome-conjugated anti-myc antibody.

Figure 3

Cytotoxicity of liposome-Fcy-hEGF/5-FC and liposome-Fcy/5-FC. (A) MTT assay was conducted for A431 (left), MDA-MB-231 (middle) and MCF-7 (right) cells treated with different concentration of liposome-Fcy-hEGF, liposome-Fcy, Fcy-hEGF and Fcy in combination with 5-FC (1 mg/mL). (B) Percentage of viable A431, MDA-MB-231 and MCF-7 cells treated with different concentration of liposome-Fcy-hEGF (right) and liposome-Fcy (left) in combination with 5-FC (1 mg/mL). The data are means ± SEM of three independent experiments performed in triplicate.

Figure 4

Cytotoxicity of ¹⁸⁸Re-liposome-Fcy-hEGF/5-FC for A431 cells. (A) ¹⁸⁸Re was conjugated with BMEDA first, followed by encapsulation into the interior of liposome-Fcy-hEGF. (B) Percentage of viable A431 cells treated with different concentration of ¹⁸⁸Re. (C) Percentage of viable A431 cells treated with liposome-Fcy-hEGF, liposome-Fcy-hEGF/5-FC, ¹⁸⁸Re-liposome-Fcy-hEGF, and ¹⁸⁸Re-liposome-Fcy-hEGF/5-FC. The specific amount of ¹⁸⁸Re, liposome (phospholipid), Fcy-hEGF, and 5-FC were 100 μCi, ~82 nmol, ~3.33 nM, and ~10 μg in 100 μL, respectively. The data are means ± SEM of three independent experiments performed in triplicate. *** p < 0.005.