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Uptake Mechanisms of EGFR-targeted Nanoparticles *
Ye Yuan1, Tatjana Paunesku1, Hans Arora1, Jesse Ward2, Sumita Raha1, Stefan Vogt2, Gayle E.
Woloschak1
1
Northwestern University, 303 E. Chicago Ave., Chicago IL, USA
Argonne National Laboratories, 9700 S. Cass Ave., Argonne IL, USA
2
Abstract:
We are developing TiO2 nanoconjugates (NCs) that can be used as therapeutic and diagnostic
agents. Nanoscale TiO2 can be surface conjugated with various molecules [1] and has the
unique ability to induce reactive oxygen species after radiation activation [2]. 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 cancers of
epithelial origin. Since EGFR is rapidly endocytosed upon ligand binding [3], we predict that
targeting NCs to EGFR will increase internalization by certain cancer cells.
Previous reports have shown that a part of Epidermal Growth Factor (EGF) known as the B-loop
domain is required for binding EGFR [4]. Therefore, we have selected eleven amino acids from
this region and synthesized FITC-labeled, dopamine-conjugated peptides. These peptides were
then used to treat HeLa cells as well as 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. In addition, we used nano-gold labeled antibodies to target the cytoplasmic domain of
EGFR. Finally, using X-ray Fluorescence Microscopy (XFM) we looked for overlap of gold and
titanium. This was possible because XFM allows for direct detection of titanium as well as gold.
These images showed that gold nanoparticles conjugated with anti-EGFR antibodies
colocalized with titanium from TiO2-EGF peptide nanoparticles at the cell surface.
Nanoparticles are a promising vehicle for the delivery of therapeutic and diagnostic agents to
different cell types. Conjugating biomolecules that promote specific uptake and retention in
tumor cells will allow for controlled drug delivery. As a first step, we have shown by light
microscopy and XFM that EGFR targeted NCs can indeed bind to cell surface EGFR.
References:
[1]
[2]
[3]
[4]
T. Paunesku, T. Rajh, G. Wiederrecht, J. Maser, S. Vogt, N. Stojicevic, M. Protic, B. Lai,
J. Oryhon, M. Thurnauer, and G. Woloschak, "Biology of TiO2-oligonucleotide
nanocomposites," Nature Materials, vol. 2, 2003, pp. 343-346.
Y. Kubota, T. Shuin, C. Kawasaki, M. Hosaka, H. Kitamura, R. Cai, H. Sakai, K.
Hashimoto, and A. Fujishima, "Photokilling of T-24 human bladder cancer cells with
titanium dioxide," Br J Cancer, vol. 70, 1994, pp. 1107-1111.
P. Burke, K. Schooler, and H.S. Wiley, "Regulation of epidermal growth factor receptor
signaling by endocytosis and intracellular trafficking.," Molecular biology of the cell, vol.
12, 2001, pp. 1897-910.
A. Komoriya, M. Hortsch, C. Meyers, M. Smith, H. Kanety, and J. Schlessinger,
"Biologically active synthetic fragments of epidermal growth factor: localization of a major
receptor-binding region," Proc Natl Acad Sci U S A, vol. 81, 1984, pp. 1351-1355.