Nanoparticle delivery of a peptide targeting EGFR signaling

Abstract

EGFR serves as an important therapeutic target because of its over-expression in many cancers. In this study, we investigated a peptide-based therapy aimed at blocking intracellular protein-protein interactions during EGFR signaling and evaluated a targetable lipid carrier system that can deliver peptides to intracellular targets in human cancer cells. EEEEpYFELV (EV), a nonapeptide mimicking the Y845 site of EGFR which is responsible for STAT5b phosphorylation, was designed to block EGFR downstream signaling. EV was loaded onto LPH nanoparticles that are comprised of a membrane/core structure including a surface-grafted polyethylene glycol (PEG) used to evade the reticuloendothelial system (RES) and anisamide (AA) for targeting the sigma receptor over-expressed in H460 human lung cancer cells. EV formulated with PEGylated and targeted LPH (LPH-PEG-AA) was taken up by the tumor cells and trafficked to the cytoplasm with high efficiency. Using this approach, EV acted as a dominant negative inhibitor of STAT5b phosphorylation, arrested cell proliferation, and induced massive apoptosis. Intravenous administration of EV loaded in LPH-PEG-AA led to efficient EV peptide delivery to the tumor in a xenograft mouse model, and multiple injections inhibited tumor growth in a dose-dependent manner. Our findings offer proof-of-concept for an intracellular peptide-mediated cancer therapy that is delivered by carefully designed nanoparticles.

Figure 1

The inhibition effect of STAT5b phosphorylation (a) by the Y845 of EGFR and EV peptide and (b) by EV peptide with different concentrations in H460 cell lysate. And, schematic of tumor targetable nanoparticles (c).

Figure 2

Uptake of Alexa488-labeled EV peptide in different formulations by H460 cells after 4 h incubation was measured by ELISA (a). Intracellular accumulation of fluorescence labeled EV peptide (green) formulated with LPH-PEG-AA observed by confocal microscopy. Nuclei were stained with DAPI (blue) (630×) (b).

Figure 3

Cell viability at different incubation times after treatment of EV or EE peptide formulated in LPH-PEG or LPH-PEG-AA. Viability was measured by the MTT assay (P value: ** < 0.01, * < 0.05) (a). And, cell growth profile was observed by cell stain counting (b). Western blot analysis for p-STAT5b in the intact H460 cells after treating with EV or EE peptide (4 μM) formulated in LPH-PEG or LPH-PEG-AA confirmed the inhibition effect of STAT5b phosphorylation (upper panel). And, dose-response of STAT5b phosphorylation in the intact H460 cells after treating with EV peptide formulated in LPH-PEG-AA in different concentrations was observed by western blot analysis (lower panel). STAT5b serves as a loading control (c). Pull down assay for EV peptide and STAT5b was performed. Biotinylated STAT5b antibody was incubated with extracts of cells treated with fluorescence labeled EV peptide in different concentrations (from 1.5 to 17 μM). Fluorescence labeled free EE, EV and EE formulated with LPH-PEG-AA were used as the control (d). Mean ± SEM (n=3~5).

Figure 4

Change of cell morphology after treating with EV peptide formulated with LPH-PEG-AA for 48 h (top panel) and DAPI staining for the observation of fragmented DNA at 24 h after EV peptide treatment (bottom panel) (a). Flow cytometry for detection apoptotic events induced by EV peptide formulated with LPH-PEG or LPH-PEG-AA performed. PBS, EE peptide formulated with LPH-PEG or LPH-PEG-AA as the control treated for apoptosis study. Apoptosis was detected by annexin V (x-axis) and propidium iodide (PI, y-axis) (b). Cell cycle arrest of PBS treated H460 cells and those treated with EE peptide formulated with LPH-PEG-AA, EV peptide formulated with LPH-PEG or LPH-PEG-AA was observed by FACS analysis. Percentage of cells in sub G₀/G₁, G₀/G₁, S and G₂/M were determined after staining with propidium iodide (PI) (c).

Figure 5

Distribution of fluorescence labeled EV peptide formulated in LPH in major organs (heart, lung, spleen, kidney, liver and tumor) was imaged using a Xenogen IVIS imaging system The LPH was prepared by coating DOTAP liposome on the complexes of EV peptide, heparin and protamine. LPH-PEG or LPH-PEG-AA was prepared by the inserting of DSPE-PEG or DSPE-PEG-AA on LPH nanoparticles. Free peptide was injected as the control. (a). Tissue distribution of EV peptide in mouse organs after intravenously administration of Alexa-488 labeled peptide formulated with LPH, LPH-PEG or LPH-PEG-AA was quantified (b). Tumor growth retardation effect of EV (0.36 mg/kg) or EE (0.36 mg/kg) peptide formulated with LPH-PEG or LPH-PEG-AA in H460 tumor model (b) and dosage effect of EV peptide formulated LPH-PEG-AA in H460 tumor model (d) was evaluated after intravenous injection every other d. Doses were 0.36, 0.48, 0.72 and 0.96 mg/kg of EV peptide formulated with LPH-PEG-AA.. (P value: * < 0.05, ** < 0.001), Mean ± SEM (n=5).

Figure 6

Localization of AIF and caspase-3 by immunohistochemistry in the tumor sections of mouse treated with EV or EE peptide formulated with LPH-PEG or LPH-PEG-AA. Sections from mice treated with PBS (a), EE peptide with LPH-PEG (b), EE peptide with LPH-PEG-AA (c), EV peptide with LPH-PEG (d), EV peptide with LPH-PEG-AA (e) were stained with AIF or caspase-3 antibody (brown staining). In figure f of lower panel, tumor tissues of mice intravenously injected with 0.36 mg/kg of Fluorescence-conjugated EV peptide (green) formulated in LPH-PEG-AA were sectioned and microscopically examined for fluorescence. 40×