A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro

Abstract

The epidermal growth factor receptor (EGFR) serves an important function in the proliferation of tumors in humans and is an effective target for the treatment of cancer. In this paper, we studied the targeting characteristics of small peptides (AEYLR, EYINQ, and PDYQQD) that were derived from three major autophosphorylation sites of the EGFR C-terminus domain in vitro. These small peptides were labeled with fluorescein isothiocyanate (FITC) and used the peptide LARLLT as a positive control, which bound to putative EGFR selected from a virtual peptide library by computer-aided design, and the independent peptide RALEL as a negative control. Analyses with flow cytometry and an internalization assay using NCI-H1299 and K562 with high EGFR and no EGFR expression, respectively, indicated that FITC-AEYLR had high EGFR targeting activity. Biotin-AEYLR that was specifically bound to human EGFR proteins demonstrated a high affinity for human non-small-cell lung tumors. We found that AEYLR peptide-conjugated, nanostructured lipid carriers enhanced specific cellular uptake in vitro during a process that was apparently mediated by tumor cells with high-expression EGFR. Analysis of the MTT assay indicated that the AEYLR peptide did not significantly stimulate or inhibit the growth activity of the cells. These findings suggest that, when mediated by EGFR, AEYLR may be a potentially safe and efficient delivery ligand for targeted chemotherapy, radiotherapy, and gene therapy.

Keywords: EGFR; NLC; lung cancer; small peptide; tumor targeting.

Figure 1

Binding activities of the small peptides. The expression of EGFR on (A) NCI-H1299 and (Β) Κ562 was confirmed by flow cytometry. The gray background indicates non-treatment and the black lines indicate the results obtained for the EGFR antibody treatment. (C) NCI-H1299 cells binding with FITC-labeled peptide using flow cytometry. (D) Comparison of binding percentages of FITC-labeled peptides with NCI-H1299 and K562 cells using flow cytometry. (Ε) Κ562 cells binding with FITC-labeled peptide by flow cytometry. Note: Data shown are presented as mean ± SEM and were obtained from three independent experiments. Abbreviations: EGFR, epidermal growth factor receptor; FITC, fluorescein isothiocyanate; SEM, standard error of the mean.

Figure 2

Internalization of peptides. NCI-H1299 cells were incubated with 10 μΜ (A1) FITC-AEYLR, (B1) FITC-LARLLT, (C1) FITC-PDYQQD, (D1) FITC-NYQQN, (E1) FITC-EYINQ, (F1) FITC-RALEL, and (G1) FITC at 37°C for 3 hours; K562 cells were incubated with 10 μΒ FITC-peptide (H1) under the same conditions. Phase contrast micrographs of the same fields (A2 to H2). Notes: Images were taken using a fluorescence microscope. Magnification: ×200. Abbreviation: FITC, fluorescein isothiocyanate.

Figure 3

Immunohistochemical results of human non-small-cell lung cancer tissue. (A) EGFR expressed on human non-small-cell lung cancer tissue identified by EGFR mAb (1:100). (B) Biotin-AEYLR (10 μΒ) co-localized with human non-small-cell lung cancer tissue, but (C) the control peptide biotin-RALEL did not. (D) The same result as (C) were observed for PBS. Note: Magnification: ×200. Abbreviations: EGFR, epidermal growth factor receptor; mAb, monoclonal antibody; PBS, phosphate-buffered solution.

Figure 4

(A) Biotin-AEYLR-bound or biotin-RALEL-bound hEGFR proteins or BSA proteins. (B) Activity of AEYLR and RALEL peptides on NCI-H1299 cell growth was studied via MTT assay. Fluorescence microscopy images and flow cytometry of (C) FITC-AEYLR-NLC, (D) FITC-RALEL-NLC, and (E) FITC-NLC in the presence of NCI-H1299 cells after incubation for 3 hours at 37°C. Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; hEGFR, human epidermal growth factor receptor; NLC, nanostructured lipid carrier; OD, optical density.