Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Extracellular matrix wikipedia , lookup
Cell growth wikipedia , lookup
Tissue engineering wikipedia , lookup
Signal transduction wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Cellular differentiation wikipedia , lookup
Cell culture wikipedia , lookup
Cell encapsulation wikipedia , lookup
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.