Targeted delivery of CPG-oligodeoxynucleotide to breast cancer cells by poly-amidoamine dendrimer-modified magnetic nanoparticles

Download
2016
Taghavi Pourianazar, Negar
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.

Suggestions

CpG oligodeoxynucleotide- loaded PAMAM dendrimer-coated magnetic nanoparticles promote apoptosis in breast cancer cells
Pourianazar, Negar Taghavi; Gündüz, Ufuk (2016-03-01)
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...
The Preparation and characterization of dendrimer coated magnetic nanoparticles for targeted cancer therapy
Khodadust, Rouhollah; Gündüz, Ufuk; Gündüz, Güngör; Department of Biotechnology (2013)
Nanotechnology is a promising alternative to overcome the limitations of classical chemotherapy. This technology has enabled the development of particles with nano sizes that can be fabricated from a multitude of materials in a variety of compositions. These nanoparticles include; quantum dots (QDs), polymeric nanoparticles, gold nanoparticles, magnetic nanoparticles and dendrimeric nanoparticles. In first section of this study, superparamagnetic iron oxide nanoparticles were synthesized by coprecipitation ...
Investigation of in vitro cytotoxic effects of heparin coated iron oxide nanoparticles combined with tpp-dca on human hepatocellular carcinoma cell line HEPG2
Saraç, Başak Ezgi; Güray, Nülüfer Tülün; Volkan, Mürvet; Department of Biology (2018)
Nanotechnology in medicine involves the applications of nanoparticles and one of the rising field is cancer nanotechnology, which has been increasingly used in cancer diagnostics, imaging, and therapeutic drug delivery. The advantage of the use of the nanoparticles is that, they can be designed to be specific for tumor tissue. This allows increased drug delivery efficiency and reduced off-target toxicities. Iron oxide nanoparticles used in this study are smaller than 100 nm but still it gives an enhanced su...
In vitro microfluidic models of tumor microenvironment to screen transport of drugs and nanoparticles
Özçelikkale, Altuğ; Linnes, Michael; Han, Bumsoo (2017-09-01)
Advances in nanotechnology have enabled numerous types of nanoparticles (NPs) to improve drug delivery to tumors. While many NP systems have been proposed, their clinical translation has been less than anticipated primarily due to failure of current preclinical evaluation techniques to adequately model the complex interactions between the NP and physiological barriers of tumor microenvironment. This review focuses on microfluidic tumor models for characterization of delivery efficacy and toxicity of cancer ...
Expression analysis of TOP2A, MSH2 and MLH1 genes in MCF7 cells at different levels of etoposide resistance
Kaplan, Esra; Gündüz, Ufuk (2012-02-01)
Purpose: Development of resistance against anti-cancer drugs is one of the major obstacles of chemotherapy in the treatment of cancer. Etoposide is a topoisomerase II alpha (TOP2A) inhibitor, which is used in the treatment of breast cancer. Alterations in the expression of drug targets or DNA repair genes are among the important resistance mechanisms against TOP2A inhibitors. In this study, expression changes in TOP2A gene and two important mismatch repair (MMR) genes MSH2 and MLH1 were examined in order to...
Citation Formats
N. Taghavi Pourianazar, “Targeted delivery of CPG-oligodeoxynucleotide to breast cancer cells by poly-amidoamine dendrimer-modified magnetic nanoparticles,” Ph.D. - Doctoral Program, Middle East Technical University, 2016.