Investigation of the Therapeutic Effects of Palbociclib Conjugated Magnetic Nanoparticles on Different Types of Breast Cancer Cell Lines

Introduction-Drug targeting and controlled drug release systems in cancer treatment have many advantages over conventional chemotherapy in terms of limiting systemic toxicity, side effects, and overcoming drug resistance.Methods and Results-In this paper, fabricating nanoscale delivery system composed of magnetic nanoparticles (MNPs) covered with poly-amidoamine (PAMAM) dendrimers and using its advantages were fully used to help the chemotherapeutic drug, Palbociclib, effectively reach tumors, specifically and stay stable in the circulation longer. In order to determine whether conjugate selectivity can be increased for the specific drug type, we have reported different strategies for loading and conjugation of Palbociclib to different generations of magnetic PAMAM dendrimers. The best method leading to the highest amount of Palbociclib conjugation was chosen, and the characterization of the Palbociclib conjugated dendrimeric magnetic nanoparticles (PALDcMNPs) were performed. In vitro pharmacological activity of the conjugation was demonstrated by measuring the cell viability and lactate dehydrogenase (LHD) release. Obtained results indicated that PAL-DcMNPs treatment of the breast cancer cell lines, leads to an increase in cell toxicity compared to free Palbociclib. The observed effects were more evident for MCF-7 cells than for MDA-MB231 and SKBR3 cells, considering that viability decreased to 30% at 2.5 lM treatment of PAL-DcMNPs at MCF-7 cells. Finally, in Palbociclib and PAL-DcMNPs treated breast cancer cells, the expression levels of some pro-apoptotic and drug resistance related genes were performed by RT-PCR analysis.Conclusion-Our knowledge indicates that the proposed approach is novel, and it can provide new insight into the development of Palbociclib targeting delivery system for cancer treatment

CPG-oligodeoksinükleotitlerin meme kanseri hücrelerine poli-amidoamin dendrimer kaplı manyetik nanoparçacıklar ile hedeflenmesi.

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.Ph.D. - Doctoral Progra

ANTİ-HER2 BAĞLI PHB POLİMERİ İLE KAPLI MANYETİK NANOPARÇAÇIKLARIN HAZIRLANMASI VE MCF-7 MEME KANSERİ HÜCRE HATTINA UYGULANMASI

ANTİ-HER2 BAĞLI PHB POLİMERİ İLE KAPLI MANYETİK NANOPARÇAÇIKLARIN HAZIRLANMASI VE MCF-7 MEME KANSERİ HÜCRE HATTINA UYGULANMAS

Changes in apoptosis-related gene expression and cytokine release in breast cancer cells treated with CpG-loaded magnetic PAMAM nanoparticles

CpG-oligodeoxynucleotide (CpG-ODN) can function as an immune adjuvant. Previously, we showed that stimulation of breast cancer cells with CpG-ODN conjugated with PAMAM dendrimer-coated magnetic nanoparticles (DcMNPs) has induced apoptosis. The aim of the current study was to evaluate the expression levels of some apoptosis-regulating genes in several human breast cancer cells treated with CpG/DcMNPs. Treated MDA-MB231 cells showed an increase in Noxa and Bax gene expression levels, whereas the expression level of Survivin decreased. Similarly, Noxa gene was overexpressed in treated MCF7 cells. In treated SKBR3 cells, a decline in the c-Flip mRNA level was determined. Furthermore, release of cytokines, IL-6, IL-10, and TNF-alpha, was determined in cell culture supernatants. CpG/DcMNP treatment leads to an increase in the release of IL-6 in MDA-MB231 and SKBR3 cells, whereas release of IL-10 and TNF-alpha did not change significantly. It is indicated that CpG-ODN may show its cytotoxic effect by regulating the expression of apoptosis-related genes and the release of cytokine in breast cancer cells

CpG oligodeoxynucleotide- loaded PAMAM dendrimer-coated magnetic nanoparticles promote apoptosis in breast cancer cells

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. This will be a novel targeted delivery system to enhance the accumulation of CpG-ODN molecules in tumor cells. The validation of CpG-ODN binding to DcMNPs was performed using agarose gel electrophoresis, UV-spectrophotometer, XPS analyses. Cytotoxicity of conjugates was assessed in MDA-MB231 and SKBR3 cancer cells based on cell viability by XTT assay and flow cytometric analysis. Our results indicated that the synthesized DcMNPs having high positive charges on their surface could attach to CpG-ODN molecules via electrostatic means. These nanoparticles with the average sizes of 40 +/- 10 nm bind to CpG-ODN molecules efficiently and induce cell death in MDA-MB231 and SKBR3 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. (C) 2016 Elsevier Masson SAS. All rights reserved

Bioapplications of poly(amidoamine) (PAMAM) dendrimers in nanomedicine

Poly(amidoamine) (PAMAM) dendrimers are a novel class of spherical, well-designed branching polymers with interior cavities and abundant terminal groups on the surface which can form stable complexes with drugs, plasmid DNA, oligonucleotides, and antibodies. AmineaEuroterminated PAMAM dendrimers are able to solubilize different families of hydrophobic drugs, but the cationic charges on dendrimer surface may disturb the cell membrane. Therefore, surface modification by PEGylation, acetylation, glycosylation, and amino acid functionalization is a convenient strategy to neutralize the peripheral amine groups and improve dendrimer biocompatibility. Anticancer agents can be either encapsulated in or conjugated to dendrimer and be delivered to the tumor via enhanced permeability and retention (EPR) effect of the nanoparticle and/or with the help of a targeting moiety such as antibody, peptides, vitamins, and hormones. Biodegradability, non-toxicity, non-immunogenicity, and multifunctionality of PAMAM dendrimer are the key factors which facilitate steady increase of its application in drug delivery, gene transfection, tumor therapy, and diagnostics applications with precision and selectivity. This review deals with the major topics of PAMAM dendrimers including structure, synthesis, toxicity, surface modification, and also possible new applications of these spherical polymers in biomedical fields as dendrimer-based nanomedicine

DETERMINATION OF THE RELATIONSHIP BETWEEN DOXORUBICIN RESISTANCE AND WNT SIGNALING PATHWAY IN HELA AND K562 CELL LINES

WOS: 000431691800003PubMed ID: 29805346Activation of the Wnt signaling in some types of cancer and its relation with chemotherapy resistance is a very interesting issue that has been emphasized in recent years. Although, it is known that increase in the activity of beta-catenin is important in blast transformation and drug resistance, the underlying mechanisms are still unclear. In this study, changes in the expression levels of 186 genes that are thought to be important in drug resistance and Wnt signaling pathways were determined by using qPCR method in doxorubicin-sensitive and -resistant HeLa and K562 cell lines. It has been observed that the genes involved in the Wnt signaling pathways are involved in more changes in HeLa/Dox cells (36 genes) than in the K562/Dox cells (17 genes). Genes important for the development of cancer resistance have been found to be significantly different in expression levels of 18 genes in HeLa/Dox cells and 20 genes in K562/Dox cells. In both cell lines, the expression of ABCB1 gene was significantly increased to 160 and 103 fold, respectively. However, despite the resistance to same drug in HeLa and K562 cell lines, it appears that the expression levels of different oncogenes and genes involved in Wnt signaling pathways have been altered. It has been found that although resistance develops to the same drug in both cell lines, the expression levels of different genes have changed. If functional analysis of these genes is performed on patient population groups, these molecules may become candidates for novel therapeutic target molecules.TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [214S634]This work was supported by TUBITAK 3001 project (Project No: 214S634)

Determination of the relationship between doxorubicin resistance and Wnt signaling pathway in HeLa and K562 cell lines

Activation of the Wnt signaling in some types of cancer and its relation with chemotherapy resistance is a very interesting issue that has been emphasized in recent years. Although, it is known that increase in the activity of β-catenin is important in blast transformation and drug resistance, the underlying mechanisms are still unclear. In this study, changes in the expression levels of 186 genes that are thought to be important in drug resistance and Wnt signaling pathways were determined by using qPCR method in doxorubicin-sensitive and –resistant HeLa and K562 cell lines. It has been observed that the genes involved in the Wnt signaling pathways are involved in more changes in HeLa/Dox cells (36 genes) than in the K562/Dox cells (17 genes). Genes important for the development of cancer resistance have been found to be significantly different in expression levels of 18 genes in HeLa/Dox cells and 20 genes in K562/Dox cells. In both cell lines, the expression of ABCB1 gene was significantly increased to 160 and 103 fold, respectively. However, despite the resistance to same drug in HeLa and K562 cell lines, it appears that the expression levels of different oncogenes and genes involved in Wnt signaling pathways have been altered. It has been found that although resistance develops to the same drug in both cell lines, the expression levels of different genes have changed. If functional analysis of these genes is performed on patient population groups, these molecules may become candidates for novel therapeutic target molecules