Egfr-Mediated Nuclear Delivery Improves The Genotoxic Effect Of Fe3o4@Tio2 Nanoparticles

Metal oxide nanoparticles (NPs) composed of an Fe3O4 core and a TiO2 (Fe3O4@TiO2) shell can be activated by light and radiation to produce genotoxic reactive oxygen species (ROS). However, ROS are short-lived and in order to induce DNA damage these potentially therapeutic NPs must reach the cell nucleus. Certain receptors that can act as cell surface targets for NPs, such as the Epidermal Growth Factor Receptor (EGFR), are transported to the nucleus after ligand binding. Therefore, the goals of this study are to determine if Fe3O4@TiO2 NPs can be made to bind the Epidermal Growth Factor Receptor (EGFR) in order to (1) improve internalization, (2) promote nuclear translocation, and (3) increase DNA damage after activation. Epidermal Growth Factor (EGF) derived B-loop peptides and control Scrambled peptides were synthesized by Northwestern University's Institute for BioNanotechnology in Medicine. Peptides were conjugated to the surface of 6-7 nm Fe3O4@TiO2 NPs for 18 hours at 4°C. The binding of cellular proteins to B-loop and Scrambled NPs was tested using a pull down assay and the presence of EGFR, and the nuclear transport protein importin-β was determined by Western Blotting. The internalization of fluorescently labeled B-loop and Scrambled NPs was quantified by Flow Cytometry at several time points post-treatment. The subcellular distribution of NPs and EGFR was determined by confocal microscopy and high-resolution X-ray Fluorescence Microscopy (XFM). Cells treated with B-loop or Scrambled NPs for 30 minutes were exposed to UV-irradiation for 3 minutes. The amount of DNA damage induced by activation of NPs was determined via a neutral comet assay by measuring the Olive tail moment of single cells. B-loop NPs but not Scrambled NPs could bind EGFR and the nuclear transport protein importin-β from EGFR-expressing HeLa cells. Internalization of fluorescently labeled B-loop NPs was increased 3.9-fold over Scrambled NPs. Confocal microscopy imaging of fluorescent NPs as well as XFM imaging showed colocalization of B-loop NPs and EGFR initially at the cell surface and then in the nucleus 30 minutes after treatment. The amount of double-stranded DNA breaks as measured by the average comet tail length after UV-irradiation was significantly greater in B-loop NP vs Scrambled NP treated cells (13.32 vs 5.01, p < 0.0001) We have identified an eleven amino acid peptide (B-loop peptide) derived from EGF that can bind and activate EGFR. B-loop NPs can improve the internalization and nuclear localization of NPs by binding both EGFR and importin-β. This improvement in nuclear localization of B-loop NPs resulted in a significant increase in DNA damage after treatment with UV light.