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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 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 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 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]
