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Öğe Co-Delivery of VEGF siRNA and IL-4 into Chitosan Nanoparticles in Breast Tumor Model of Rat(Nature Publishing Group, 2013) Salva, Emine; Ozbas-Turan, Suna; Kabasakal, Levent; Eren, Fatih; Alan, Saadet; Ozkan, Naziye; Akbuga, Julide[Abstract Not Available]Öğe Combination therapy with chitosan/siRNA nanoplexes targeting PDGF-D and PDGFR-? reveals anticancer effect in breast cancer(Wiley, 2023) Salva, Emine; Ozbas, Suna; Alan, Saadet; Ozkan, Naziye; Ekentok-Atici, Ceyda; Kabasakal, Levent; Akbuga, JulideBackground: Platelet derived growth factors (PDGF)-D and the expression of its receptor increase in neoplastic progression of cancer. Co-silencing of growth factor and receptor can be suggested as an important strategy for effective cancer therapy. In the present study, we hypothesized that suppression of PDGF-D signaling pathway with small interfering RNAs (siRNAs) targeting both PDGF-D and PDGF receptor (PDGFR)-beta is a promising strategy for anticancer therapy. Methods: Chitosan nanoplexes containing dual and single siRNA were prepared at different weight ratios and controlled by gel retardation assay. Characterization, cellular uptake, gene silencing and invasion studies were performed. The effect of nanoplexes on breast tumor growth, PDGF expression and apoptosis was investigated. Results: We have shown that downregulation of PDGF-D and PDGFR-beta with chitosan/siRNA nanoplex formulations reduced proliferation and invasion in breast cancer cells. In the in vivo breast tumor model, it was determined that the intratumoral administration of chitosan/siPDGF-D/siPDGFR-beta nanoplexes markedly decreased the tumor volume and PDGF-D and PDGFR-beta mRNA and protein expression levels and increased apoptosis. Conclusions: According to the results obtained, we evaluated the effect of PDGF-D and PDGFR-beta on breast tumor development and showed that RNAi-mediated inhibition of this pathway formulated with chitosan nanoplexes can be considered as a new breast cancer therapy strategy.Öğe Comparison of VEGF gene silencing efficiencies of chitosan and protamine complexes containing shRNA(Wiley, 2014) Erdem-Cakmak, Fulden; Ozbas-Turan, Suna; Salva, Emine; Akbuga, JulideVEGF is an angiogenic factor promoting the proliferation and migration of endothelial cells. Inhibition of VEGF by RNAi mechanism is one of the novel and the most important strategies in antiangiogenesis therapy. In this study, the tumor silencing efficiency of ternary complexes after addition of protamine to chitosan complexes containing VEGF targeting shRNA was investigated. Besides chitosan, protamine is an effective gene delivery material. Binary and ternary complexes consisting of chitosan, protamine, and shRNA were prepared to target VEGF, their morphology, size, and zeta potential of the complexes being measured. The average size of the complexes was between 173 and 284nm and zeta potential was between +10 and 16mV. In the ternary complexes, size decreased as the chitosan ratio increased; however, its molecular weight had no effect on the size of complexes. HeLa, HEK293, and MCF-7 cell lines were used for in vitro transfection. VEGF was assayed by ELISA. A higher silencing effect was obtained using ternary complexes. Transgene expression was increased by adding protamine to chitosan complexes. Gene inhibition values in cell lines followed the rank HEK293>HeLa>MCF-7. The addition of protamine to the chitosan/shRNA (VEGF) complexes increased the knockdown of VEGF genes in the cell lines, and no cytotoxicity was found after the complexes had been incorporated into the cells.Öğe The Development of Ternary Nanoplexes for Efficient Small Interfering RNA Delivery(Pharmaceutical Soc Japan, 2013) Salva, Emine; Turan, Suna Ozbas; Akbuga, JulideTargeted posttranscriptional gene silencing by RNA interference (RNAi) has garnered considerable interest as an attractive new class of drugs for several diseases, such as cancer. Chitosan and protamine are commonly used as a vehicle to deliver and protect small interfering RNA (siRNA), but the strong interaction still remains to be modulated for efficient siRNA uptake and silencing. Therefore, in this study, ternary nanoplexes containing chitosan and protamine were designed to substantially enhance the siRNA efficiency. Binary and ternary nanoplexes were prepared at different the ratios of moles of the amine groups of cationic polymers to those of the phosphate ones of siRNA (N/P) ratios and characterized in terms of size, zeta potential, morphology and serum stability. The silencing efficiencies and cytotoxicities of prepared nanoplexes were evaluated by enzyme-linked immunosorbent assay (ELISA) (for human vascular endothelial growth factor; hVEGF) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. The mean diameter of ternary nanoplexes ranged from 151 to 282nm, depending on the weight ratio between polymers and siRNA. The gene silencing effect after transfection with ternary nanoplexes (chitosan/siRNA/protamine 83%) was significantly higher than that with binary nanoplexes (chitosan/siRNA 71% and protamine/siRNA 74%). Ternary nanoplexes showed the highest cellular uptake ability when compared with binary nanoplexes. Ternary nanoplexes did not induce substantial cytotoxicity. Serum stability and the lack of cytotoxicity of the nanoplexes provided advantages over other gene silencing studies. These results suggest ternary nanoplexes have the potential to be an effective siRNA carrier to study the gene silencing effect.Öğe The effectiveness of chitosan-mediated silencing of PDGF-B and PDGFR-? in the mesangial proliferative glomerulonephritis therapy(Academic Press Inc Elsevier Science, 2019) Alan, Saadet; Salva, Emine; Yilmaz, Ismet; Turan, Suna Ozbas; Akbuga, JulidePlatelet-derived growth factor-B (PDGF-B) is a growth factor that plays an important role in the progression of mesangial proliferative glomerulonephritis (MsPGN). PDGF-B may contribute to mesangioproliferative changes and is overexpressed in MsPGN. Recently, small interfering RNAs (siRNAs) have been widely used for gene silencing effects in experimental models of renal diseases. Nanoparticle-based therapeutics are preferred for reasons such as increasing therapeutic efficacy and reducing toxic effects caused by high doses. The distribution of nanoparticles to the kidney is a significant advantage in siRNA delivery. The aim of this study was to investigate the efficacy of chitosan/siRNA nanoplexes in silencing of PDGF-B and PDGFR-beta genes in kidney and to decrease mesangial cell proliferation and matrix accumulation in MsPGN model induced by anti-Thy-1.1 antibody. The therapeutic effects of chitosan/siPDGF-B + siPDGFR-beta nanoplexes in glomerulonephritic rats were studied by molecular, biochemical, and histopathologic evaluations. Chitosan/siPDGF-B + siPDGFR-beta nanoplexes markedly reduced PDGF-B and PDGFR-beta mRNA and protein expressions in experimental MsPGN model. Histopathologic examination results showed that the silencing of PDGF-B and its receptor PDGFR-beta led to reduction in mesangial cell proliferation and matrix accumulation. The use of chitosan/siPDGF-B + siPDGFR-beta nanoplexes for silencing the PDGF-B pathway in MsPGN can be considered as a new effective therapeutic strategy.Öğe The effects of chitosan/miR-200c nanoplexes on different stages of cancers in breast cancer cell lines(Elsevier Science Bv, 2016) Kaban, Kubra; Salva, Emine; Akbuga, JulideDysregulation of miR-200c in breast cancer has been associated with migration, epithelial mesenchymal transition (EMT), angiogenesis and metastasis of the tumor cells. Therefore, the modulation of miR-200c offers a promising therapeutic approach in breast cancer. However, the major obstacles in the usage of miRNAs in therapy are their low stability, rapid clearance, and poor cellular uptake. The development of efficient and safe delivery systems is important in effective therapy with miRNA. The purpose of this study was to investigate the therapeutic role of chitosan/miR-200c nanoplexes in angiogenesis, EMT, invasion, and apoptosis in breast cancer cell lines. We found that miR-200c levels were downregulated in various breast cancer cell lines by qRT-PCR After transfection with chitosan/miRNA nanoplexes in the appropriate size (294 nm) and zeta potential (12.3 mV), levels of miR-200c increased and reached the endogenous miR-200c levels in the MCF-7, MDA-MB-231, and MDA-MB-435 cells. While the chitosan/miR-200c nanoplexes decreased angiogenesis, invasion, EMT, and metastasis in the cells, the apoptosis levels increased by 3.1, 1.3, and 3 fold in the MCF-7, MDA-MB-231, MDA-MB-435 cell lines, respectively. Consequently, chitosan is a suitable carrier for miR-200c and formed stable nanoplexes with miR-200c. The effect of the chitosan/miRNA nanoplexes on tumor angiogenesis, EMT, invasion, metastasis, and apoptosis, changed depending on the cell-types. Therefore, during the treatment with the chitosan based miR-200c nanoplexes in breast cancers, the type of tumor cells must be considered. (C) 2016 Elsevier B.V. All rights reserved.Öğe The Effects to GM-CSF Expression and Fibroblast Proliferation of pGM-CSF Containing Chitosan/PVP Hydrogels(Marmara Univ, Fac Pharmacy, 2017) Salva, Emine; Akbuga, JulideThe aim of this study was to develop pGM-CSF containing chitosan/PVP hydrogel system for gene delivery. The hydrogels in semi-interpenetrating polymer network (semi-IPN) structure were synthesized by glutaraldehyde crosslinking between 2% chitosan and 2%, % 4, % 6 PVP. The viscosity, swelling properties and surface morphology of hydrogels were examined. These hydrogels showed highly porous structure. The porosity was increased with enhanced PVP concentration. Viscosity decreased with increasing PVP concentration of the hydrogels. The swelling percentage increased with increasing PVP concentration. In order to examine the effect of cell attachment and proliferation of hydrogels, NIH-3T3 fibroblast cell was used. The cells were able to attach on the top surface of hydrogels. However, the attachment of cells were slow at days 1 and 3. At days 7 and 14, cells looked healthy and normal and proliferated around and top of the hydrogels. With the ELISA study, the highest GM-CSF expression was obtained with pGM-CSF containing 2% chitosan+4% PVP hydrogels. MTT assay showed that chitosan/PVP hydrogels did not induce significant cytotoxic effect. As a result, these hydrogels could be served candidate for local gene delivery in tissue regeneration.Öğe The enhancement of gene silencing efficiency with chitosan-coated liposome formulations of siRNAs targeting HIF-1? and VEGF(Elsevier Science Bv, 2015) Salva, Emine; Turan, Suna Ozbas; Eren, Fatih; Akbuga, JulideRNA interference (RNAi) holds considerable promise as a novel therapeutic strategy in the silencing of disease-causing genes. The development of effective delivery systems is important for the use of small interfering RNA (siRNA) as therapy. In the present study, we investigated the effect on breast cancer cell lines and the co-delivery of liposomes containing siHIF1-alpha and siVEGF. In order to achieve the co-delivery of siHIF1-alpha and siVEGF and to obtain lower cytotoxicity, higher transfection and silencing efficiency, in this study, we used chitosan-coated liposomal formulation as the siRNA delivery system. The obtained particle size and zeta potential values show that the chitosan coating process is an effective parameter for particle size and the zeta potential of liposomes. The liposome formulations loaded with siHIF1-alpha and siVEGF showed good stability and protected siRNA from serum degradation after 24-h of incubation. The expression level of VEGF mRNA was markedly suppressed in MCF-7 and MDA-MB435 cells transfected with chitosan-coated liposomes containing siHIF1-alpha and VEGF siRNA, respectively (95% and 94%). In vitro co-delivery of siVEGF and siHIF1-alpha using chitosan-coated liposome significantly inhibited VEGF (89%) and the HIF1-alpha (62%) protein expression when compared to other liposome formulations in the MDA-MB435 cell. The co-delivery of siVEGF and siHIF1-alpha was greatly enhanced in the vitro gene silencing efficiency. In addition, chitosan-coated liposomes showed 96% cell viability. Considering the role of VEGF and HIF1-alpha in breast cancer, siRNA-based therapies with chitosan coated liposomes may have some promises in cancer therapy. (C) 2014 Elsevier B.V. All rights reserved.Öğe The enhancement of gene silencing efficiency with chitosan-coated liposome formulations of siRNAs targeting HIF-1? and VEGF (vol 478, pg 147, 2015)(Elsevier Science Bv, 2017) Salva, Emine; Turan, Suna Ozbas; Eren, Fatih; Akbuga, Julide[Abstract Not Available]Öğe Generation of stable cell line by using chitosan as gene delivery system(Springer, 2016) Salva, Emine; Turan, Suna Ozbas; Ekentok, Ceyda; Akbuga, JulideEstablishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.Öğe In Vitro Dose Studies on Chitosan Nanoplexes for microRNA Delivery in Breast Cancer Cells(Mary Ann Liebert, Inc, 2017) Kaban, Kubra; Salva, Emine; Akbuga, JulideChanges in microRNA (miRNA) expression levels that play important roles in regulation lead to many pathological events such as cancer. The miR-200 family is an important target in cancer therapy. The aim of this study is to equilibrate endogenous levels between cancer and noncancerous cells to prevent serious side effects of miR-200c- and miR-141-like metastatic colonization. For the first time, the characterization of miR-200c and miR-141 cluster containing chitosan nanoplexes was shown, and the optimization of miRNA expression levels by conducting dose studies in breast cancer cell lines was made. The mean diameter of chitosan/miR-141 and chitosan/miR-200c nanoplexes ranged from 296 to 355 nm and from 294 to 380 nm depending on the N/P ratio, respectively. The surface charge of nanoplexes was positive with zeta potential of +12 to +26 mV. While naked miRNA was degraded after 0 min in a 10% serum-containing medium, chitosan/miRNA nanoplexes were protected for 72 h. During the in vitro cellular uptake study, nanoplexes were observed to be accumulating in the cytoplasm or nucleus. After using different doses for miR-200c, the determined doses are 750, 100, and 750 ng in the MCF-7, MDA-MB-231, and MDA-MB-435 cell lines, respectively. Doses were determined as 100 ng for MDA-MB-231 and 150 ng for MDA-MB-435 to reach endogenous miR-141 levels of MCF-10A. Our results suggest that chitosan nanoplexes for miR-200c and miR-141 are an efficient delivery system in terms of formulation and transfection. As a conclusion, dose studies are important to provide effective treatment with miRNAs.Öğe In Vitro PDGF-B Gene Silencing Studies and In Vivo Delivery of siRNA to the Rat Kidney Using Chitosan/siRNA Nanoplexes(Marmara Univ, Fac Medicine, 2016) Salva, Emine; Ozbas Turan, Suna; Alan, Saadet; Akbuga, JulideThe targeting of specific genes responsible from onset and progression of kidney diseases offer a new therapeutic strategy in the field of renal gene therapy. The altered expression of platelet derived growth factor (PDGF) is an important marker of renal diseases. In this study, we investigated in vitro gene silencing efficiency of chitosan nanoplexes containing PDGF-B and PDGFR-beta targeted siRNAs in the kidney cell lines including HEK-293 and MDCK and delivery to the kidney as an in vivo delivery system. As a result, PDGF-B expression was significantly inhibited by co-delivery of chitosan/siPDGF-B+siPDGFR-beta nanoplexes prepared using in the different weight ratios (10/1, 20/1 and 50/1). When 20/1 and 50/1 weight ratios of chitosan nanoplexes were i.v. injected to rats, chitosan/FITC-siPDGFB nanoplexes were reached to kidney tissue at 4 h after intravenous injection. These results suggest that delivery of siRNA using chitosan nanoplexes may be effective for the therapy of kidney diseases.Öğe Inhibition of Glomerular Mesangial Cell Proliferation by siPDGF-B- and siPDGFR-?-Containing Chitosan Nanoplexes(Springer, 2017) Salva, Emine; Turan, Suna Ozbas; Akbuga, JulideMesangioproliferative glomerulonephritis is a disease that has a high incidence in humans. In this disease, the proliferation of glomerular mesangial cells and the production of extracellular matrix are important. In recent years, the RNAi technology has been widely used in the treatment of various diseases due to its capability to inhibit the gene expression with high specificity and targeting. The objective of this study was to decrease mesangial cell proliferation by knocking down PDGF-B and its receptor, PDGFR-beta. To be able to use small interfering RNAs (siRNAs) in the treatment of this disease successfully, it is necessary to develop appropriate delivery systems. Chitosan, which is a biopolymer, is used as a siRNA delivery system in kidney drug targeting. In order to deliver siRNA molecules targeted at PDGF-B and PDGFR-beta, chitosan/siRNA nanoplexes were prepared. The in vitro characterization, transfection studies, and knockdown efficiencies were studied in immortalized and primary rat mesangial cells. In addition, the effects of chitosan nanoplexes on mesangial cell proliferation and migration were investigated. After in vitro transfection, the PDGF-B and PDGFR-beta gene silencing efficiencies of PDGF-B and PDGFR-beta targeting siRNA-containing chitosan nanoplexes were 74 and 71% in immortalized rat mesangial cells and 66 and 62% in primary rat mesangial cells, respectively. siPDGF-B- and siPDGFR-beta-containing nanoplexes indicated a significant decrease in mesangial cell migration and proliferation. These results suggested that mesangial cell proliferation may be inhibited by silencing of the PDGF-B signaling pathway. Gene silencing approaches with chitosan-based gene delivery systems have promise for the efficient treatment of renal disease.Öğe Investigation of the Therapeutic Efficacy of Codelivery of psiRNA-Vascular Endothelial Growth Factor and pIL-4 into Chitosan Nanoparticles in the Breast Tumor Model(Elsevier Science Inc, 2014) Salva, Emine; Turan, Suna O.; Kabasakal, Levent; Alan, Saadet; Ozkan, Naziye; Eren, Fatih; Akbuga, JulideAngiogenesis has been known to increase tumor growth and for its metastatic potential in human tumors. Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis and is a promising therapeutic target for breast cancer. VEGF is an essential target for RNAi-based gene therapy of breast cancer. Interleukin-4 (IL-4) may act as an anti-angiogenic molecule that inhibits tumor growth and migration in rats. The purpose of the present study was to improve therapeutic efficacy in breast cancer with the codelivery of siRNA-expressing plasmid targeting VEGF and IL-4-expressing plasmid encapsulating into chitosan nanoparticles (NPs). The codelivery of psiVEGF and pIL-4 plasmids greatly enhanced in vitro and in vivo gene-silencing efficiency. For the in vitro study, when psiVEGF and pIL-4 into chitosan NPs were combined (81%), the gene-silencing effect was higher than psiVEGF and pIL-4 NPs alone. The in vivo study breast tumor model demonstrated that the administration of coencapsulation of psiVEGF and pIL-4 into chitosan NPs caused an additive effect on breast tumor growth inhibition (97%), compared with containing NPs psiVEGF or pIL-4 alone. These results indicate that chitosan NPs can be effectively used for the codelivery of pIL-4 and siVEGF-expressing plasmid in a combination therapy against breast cancer. (c) 2013 Wiley Periodicals, Inc.Öğe Investigation of therapeutic effects in the wound healing of chitosan/pGM-CSF complexes(Univ Sao Paulo, Conjunto Quimicas, 2022) Salva, Emine; Alan, Saadet; Karakoyun, Berna; Cakalagaoglu, Fulya; Ozbas, Suna; Akbuga, JulideGranulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to promote the growth, proliferation, and migration of endothelial and keratinocyte cells. Chitosan has been widely used as a biopolymer in wound-healing studies. The aim of this study was to investigate the in vitro proliferative effects of chitosan/pGM-CSF complexes as well as the therapeutic role of the complexes in an in vivo rat wound model. The effect of complexes on cell proliferation and migration was examined. Wounds were made in Wistar-albino rats, and examined histopathologically. The cell proliferation and migration were increased weight ratio- and time-dependently in HaCaT and NIH-3T3 cell lines. Wound healing was significantly accelerated in rats treated with the complexes. These results showed that the delivery of pGM-CSF using chitosan complexes could play an accelerating role in the cell proliferation, migration, and wound-healing process.Öğe Modulation of the dual-faced effects of miR-141 with chitosan/miR-141 nanoplexes in breast cancer cells(Wiley, 2019) Kaban, Kubra; Salva, Emine; Akbuga, JulideBackground miR-141, known as a tumor suppressive microRNA, is downregulated in breast cancer. However, recent contrasting studies report that it also acts as oncogene when it is upregulated. The present study aimed to investigate whether miR-141 is a tumor suppressor or oncogenic when it reaches normal levels in chitosan/miR-141 nanoplexes. Methods Chitosan nanoplexes were prepared using simple complexation method. Nanoplexes were characterized by a gel retardation assay and zeta potential and particle size measurements. To determine the expression level of miR-141, a quantitative real-time polymerase chain reaction was performed. The effects of miR-141 mimics were investigated with respect to angiogenesis by vascular endothelial growth factor (VEGF), epithelial-mesenchymal transition (EMT) by E-cadherin, metastasis by Igfbp-4 and Tinagl1 enzyme-linked immunosorbent assays, invasion by an invasion chamber, and apoptosis by Annexin V. Results The miR-141 expression levels of MDA-MB-231 and MDA-MB-435 cells by administration of chitosan/mimic miR-141 nanoplexes reached endogenous miR-141 levels of a non-tumorigenic epithelial breast cell line, MCF-10A. According to our results, metastasis, VEGF, EMT and invasion in breast cancer cells were diminished, whereas apoptosis increased by 1.5- and 2.4-fold in breast cancer cell lines as a result of the miR-141 mimics. Conclusions In conclusion, we have demonstrated that administration of miR-141 mimics at the determined doses to breast cancer cells revealed a tumor suppressor effect, and not the oncogenic face. The delivery of miR-141 by chitosan nanoplexes presents a promising approach for the suppression of breast cancer.Öğe Tumor Inhibition by Using Chitosan: siRNA PDGFR-? in Breast Cancer Model of Rat(Nature Publishing Group, 2016) Akbuga, Julide; Turan, Suna Ozbas; Salva, Emine; Ozkan, Naziye; Kabasakal, Levent; Alan, Saadet[Abstract Not Available]
