Date

2016

Author

Taghavi Pourianazar, Negar

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One major application of nanotechnology in cancer treatment involves designing nanoparticles to deliver drugs, oligonucleotides, and genes to cancer cells. Nanoparticles should be engineered so that they could target and destroy tumor cells with minimal damage to healthy tissues. This research aims to develop an appropriate and efficient nanocarrier, having the ability of interacting with and delivering CpG-oligodeoxynucleotides (CpG-ODNs) to tumor cells. CpG-ODNs activate Toll-like receptor 9 (TLR9), which can generate a signal cascade for cell death. In our study, we utilized three-layer magnetic nanoparticles composed of a Fe3O4 magnetic core, an aminosilane (APTS) interlayer and a cationic poly(amidoamine) (PAMAM) dendrimer. The detailed characterization of synthesized nanoparticles was performed by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), scanning electron microscope (SEM), dynamic light scattering (DLS), zeta-potential, and vibrating sample magnetometer (VSM) analyses. TEM and SEM images indicated that synthesized dendrimer-coated magnetic nanoparticles (DcMNPs) have mono-disperse size distribution with an average particle diameter of 40±10 nm. DcMNPs were found to be superparamagnetic through VSM analysis. The synthesis, aminosilane modification, and dendrimer coating of iron oxide nanoparticles were validated by FTIR and XPS analyses. Cellular internalization of nanoparticles was studied by using fluorescence microscopy for FITC-bound nanoparticles. Results demonstrated that the synthesized DcMNPs, with their functional groups, symmetry perfection, size distribution, magnetic properties, and nontoxic characteristics could be suitable nanocarriers for targeted cancer therapy upon loading with various anticancer agents. Successful transfer of the CpG-ODN to the tumor site is dependent on the development of an efficient delivery vector to overcome various hurdles, such as rapid degradation by serum nucleases and poor diffusion across the cell membrane. In the second part of this study, CpG-ODN was efficiently bound onto the surface of newly synthesized DcMNPs which can be targeted to the tumor site under magnetic field. The validation of CpG-ODN binding to DcMNPs was performed using agarose gel electrophoresis, UV-spectrophotometer, zeta-potential, and XPS analyses. Then, internalization of CpG-bound nanoparticles was evaluated in MDA-MB231, SKBR3, MCF7, and Doxorubicin-resistant MCF7 (MCF7/Dox) cell lines with Prussian blue staining method. Cytotoxicity of conjugates was assessed in fore-mentioned cell lines based on cell viability by XTT and flow cytometric analyses. Our results indicated that the synthesized DcMNPs having high positive charges on their surface could attach efficiently to CpG-ODN molecules via electrostatic means and induce cell death in breast tumor cells and could be considered a suitable targeted delivery system for CpG-ODN in biomedical applications. The magnetic core of these nanoparticles represents a promising option for selective drug targeting as they can be concentrated and held in position by means of an external magnetic field. In the next part, we investigated the expression profiles of apoptosis-related genes, such as Bax, Noxa, Puma, Bcl-2, Survivin, and C-Flip before and after treatment with CpG-loaded nanoparticles in breast cancer cells. The results indicated that after treatment of the cells, the expression profile of apoptosis-related genes showed a change compared to untreated cells which indicates triggering of the apoptotic pathways in the cells. Studies show that the TLR9 activation with CpG-ODN may induce a Th1-like cytokine response in various cells, therefore, the release of IL-6, IL-10, and TNF-α was examined after treating the cells with different concentrations of free CpG, unloaded nanoparticles, and CpG-loaded nanoparticles and compared with the amount of release by the untreated cells. The results showed an increase in the released amount of IL-6 after treatment with CpG-loaded nanoparticles in breast cancer cells.

Subject Keywords

Breast, Poly(amidoamine)., Cancer, Drug delivery systems., Dendrimers in medicine.

URI

http://etd.lib.metu.edu.tr/upload/12619835/index.pdf
https://hdl.handle.net/11511/25554

Collections

Graduate School of Natural and Applied Sciences, Thesis