Uptake mechanisms of EGFR-targeted TiO2 nanoparticles.

Abstract

e13583 Background: We are developing TiO2 nanoconjugates (NCs) that can be used as therapeutic and diagnostic agents. Nanoscale TiO2 can be surface conjugated with various molecules and has the unique ability to induce reactive oxygen species after radiation activation. The two major questions that we wish to answer are (1) how are NCs internalized by cells and (2) is targeting of NCs using a small peptide achievable. To address these questions, we have created NCs targeted to epidermal growth factor receptor (EGFR) which is enriched in many epithelial cancers. Methods: 3 nm TiO2 nanoparticles (NPs) were synthesized by low temperature alkaline hydrolysis. EGFR targeted NCs were created by conjugating an eleven amino acid EGF peptide to NPs through a dopamine linker. Cell growth and immunocytochemistry were done as usual. X-ray Fluorescence Microscopy (XFM) was performed at the 2-ID-E beamline of the Advanced Photon Source at Argonne National Laboratories. Results: Previous reports have shown that a part of epidermal growth factor (EGF) known as the B-loop is required for binding EGFR. We selected eleven amino acids from this region and synthesized FITC-labeled, dopamine-conjugated peptides. These peptides were then used to treat two isogenic colon cancer cell lines, one with high EGFR expression (SW480) and one with low EGFR expression (SW620). Immunofluorescent imaging of treated cells showed that FITC-labeled peptides colocalized with EGFR labeled with fluorescent antibodies on the cell surface and within the cytoplasm. The synthetic EGF peptides were then conjugated to NPs via dopamine to create EGFR targeted NCs. These NCs also colocalized with EGFR in treated cells by immunofluorescence imaging. This was confirmed using XFM which allows for direct detection of titanium as well as gold. These images showed that EGFR labeled with gold conjugated antibodies colocalized with titanium at the cell surface. Conclusions: TiO2 NCs are a promising vehicle for the delivery of therapeutic and diagnostic agents to certain cell types. Conjugating biomolecules that promote specific uptake and retention in tumor cells will allow for controlled drug delivery. In fact, we have shown that EGFR targeted NCs can successfully colocalize with EGFR on the surface of colon cancer cells. No significant financial relationships to disclose.