Person: EKENTOK ATICI, CEYDA
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EKENTOK ATICI
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CEYDA
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Publication Metadata only The cellular uptake and endosomal escape mechanisms of chitosan-protamine-siRNA nanoplexes for efficient gene transfection and silencing(2021-09-11) EKENTOK ATICI, CEYDA; ÖZBAŞ, SUNA; CÖMEZ, BİRNUR; ŞALVA E., EKENTOK C., CÖMEZ B., ÖZBAŞ S., AKBUĞA F. J.Aim: The use of antisense-based molecules in gene expression inhibition has allowed the design of a new pathway for therapeutics. In order to ensure that oligonucleotides with gene silencing potential can be used effectively in therapy, a suitable carrier system is required that can be transported to the target site. In this study, gene silencing activities of siRNA targeted to the LacZ gene were compared and the gene delivery capabilities, transfection efficiency, cellular uptake and endosomal escape mechanisms of nanoplexes prepared with siRNA and chitosan/protamine polymers were investigated. Material and methods: Nanoplex formulations were prepared by simple complexation method of chitosan/protamine polymers and oligonucleotides. The particle size and zeta potential of the prepared nanoplexes were measured, and their serum and enzyme stability were investigated. In order to determine the transfection and gene silencing activities of the selected formulations, HEK293 cells stably expressing beta-gal were prepared, inhibition of beta-gal protein was measured by enzymatic assay and suppression by X-gal method was evaluated microscopically. Cellular uptake and endosomal escape mechanisms of nanoplexes were studied. Results: Chitosan/Protamine/siLacZ nanoplexes have been observed to protect siRNA against enzymatic and serum degradation for up to 48 hours. It was observed that the transfection efficiency was the highest in the formulations prepared together with chitosan/protamine at a rate of 10/10/1. It was observed that the transfection increased significantly with the increase in the ratio of chitosan and protamine. Transfection efficiency was found to be 88.60% at a rate of 10/10/1. In the cellular uptake study, it was observed that the inhibitor that reduced cellular uptake the most was phenylarsine oxide, and the uptake of siRNA carried by nanoplexes was 56%. There was no decrease in cellular uptake when chlorpromazine hydrochloride inhibitor was administered. This indicated that the nanoplexes were not uptake by clathrin-mediated endocytosis. It was observed that cellular uptake was 75% with colchicine, this inhibitor decreases cellular uptake by inhibiting microtubules in cells. Conclusion: It has been shown that cellular uptake and transfection studies with chitosan/protamine nanoplexes can be used as an effective carrier system for siRNA transport.Publication Metadata only Hedeflendirilmiş Nanopartiküllerin transdermal taşınması ve in vitro değerlendirilmesi(Jenny Stanford Publishing, 2021-11-01) EKENTOK ATICI, CEYDA; SEZER, ALİ DEMİR; ÇELİK, AYSUN; Ekentok Atıcı C., Çelik A., Sezer A. D.Publication Open Access Generation of stable cell line by using chitosan as gene delivery system(SPRINGER, 2016-08) EKENTOK ATICI, CEYDA; 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.Publication Metadata only Administration of Ad5 chitosan PEG-aptamer vector delivering PDGF-D shRNA decreases tumor growth in rat breast cancer model(2021-10-01) EKENTOK ATICI, CEYDA; Ekentok-Atici C., Akbuga J.Publication Open Access Viral vector platforms within the gene therapy landscape(2023-01-01) EKENTOK ATICI, CEYDA; EKENTOK ATICI C.Gene therapy is the treatment of a disease through transferring genetic material (DNA/RNA) into the cells of patients. The transferred gene can act following ways: (i) enabling expression of the transferred gene, (ii) inhibiting the expression of a target gene and (iii) modifying a target gene. Gene therapy clinical trials started nearly fifty years ago with treatment of inherited monogenic disorder. Soon after that, gene therapy based clinical approaches was extended to acquired diseases such as cancer [1]. Since the first successful gene therapy clinical trial on a four-year girl who had ADA deficiency was initiated in 1990, more than 30 gene therapy products have been approved worldwide by different authorities [2]. Several approaches like physical methods (electroporation, microinjection, biolistic etc.), chemical methods (polymers, lipids, peptides etc.) and biological methods (adenoviruses, adeno-associated viruses, retroviruses etc.) can be employed to deliver the DNA inside cells. No single method works best for all applications. Factors that determine the choice of the method include cost, reproducibility, toxicity, mechanism of delivery, ease of use, and efficiency [3]. Adenoviral vectors (Ads), adeno-associated viral vector (AAVs), retroviral vectors (RVs) and lentiviral vectors (LVs) are the most common viral delivery systems for gene therapy applications. These systems have the benefits of high transfection potency and constant expression of therapeutic genes. However, limitations in large scale virus production, immunogenicity, toxicity and insertional mutagenesis are their common disadvantages. Current strategies to overcome these disadvantages are; (i) localized delivery and transcriptional targeting for toxicity and off-target effects (ii) immunosuppressive drugs and anti-inflammatory agents for immune response and (iii) biomaterial-mediated viral gene delivery [4]. The polymers can be used for viral vector modification and evolving hybrid vectors is a promising strategy for gene therapy applications. Surface modification of Ads with cationic polymers can be done by (i) non-covalent coating (physical modification) (ii) covalent coating (chemical modification). Non-covalent coating strategies gain attention because of ease of manipulation [5]. In our recent study we aimed to prepare Ad/chitosan hybrid vector to deliver shPDGF-D in breast cancer cell line MDA-MB-231. We covalently coat Ad surface with different amount and molecular weight chitosan and investigate gene silencing efficiency of vectors. In vitro cell culture studies showed that both low and high molecular weight chitosan increased PDGF-D silencing efficiency of Ad5 vector at 48 hours significantly. Also, the invasion ability of MDA-MB-231 cells decreased after treatment with coated Ad vector correlated with PDGF-D silencing results [6]. In conclusion, the results showed that non-covalent modification of Ad surface with polymers increased in vitro silencing efficiency, which may allow decrease viral dose for safer and efficient therapy.Publication Open Access A Comparison Study of Fiber Diameter's Effect on Characteristic Features of Donepezil/Curcumin-Loaded Polycaprolactone/Polylactic Acid Nanofibers(2022-05-01) EKENTOK ATICI, CEYDA; GÜNDÜZ, OĞUZHAN; ÇAM, MUHAMMET EMİN; TATAR, ESRA; YAVUZ, AYŞE NUR; Aydin S., Kabaoglu I., Guler E., Topal F., YAVUZ A. N., EKENTOK ATICI C., TATAR E., Gurbuz F., GÜNDÜZ O., ÇAM M. E.Nanofibers (NFs) offer an alternative option for the treatment of Alzheimer\"s disease (AD) by addressing unmet clinical problems. In this study, anti-AD drugs, donepezil (DO) and curcumin (CUR), are loaded in polylactic acid/polycaprolactone NFs. The effect of fiber diameter on drug release behavior is mainly observed, and the successful loading of DO and CUR to NFs is demonstrated. The tensile strength of DO/CUR-loaded NFs (DNFs) with lower fiber diameter is found to be higher. The working temperature is increased by the decrease of glass transition temperature and increase of the melting temperature after loading drugs. Furthermore, the increase in the percentage of swelling and decrease in the degradation rate for NFs are observed due to the increase of fiber diameter. Encapsulation efficiency and burst release percentages for DNFs are augmented by the increase of fiber diameter. Nevertheless, DNFs exhibit a sustained drug release manner over 2 weeks. NFs do not demonstrate a toxic effect on L929 (mouse fibroblast) cells, and additionally, they promote cell proliferation. Considering all these results, it is proven that the fiber diameter affects all characteristic features of NFs, and DNFs lead to a new and promising drug delivery system for the treatment of AD.Publication Open Access In vitro gene silencing effect of chitosan/shRNA PDGF-D nanoparticles in breast cancer(MARMARA UNIV, FAC PHARMACY, 2017-10-03) EKENTOK ATICI, CEYDA; Ekentok, Ceyda; Turan, Suna Ozbas; Akbuga, JulideBreast cancer is the most common cancer worldwide in women and it is highly malignant and fatal. PDGF-D plays role in regulation of many cellular processes such as angiogenesis. PDGF-D is overexpressed in many types of cancers and promote tumor growth and metastasis. Silencing of PDGF-D gene by using shRNA with an appropriate carrier system may decrease tumor growth and metastasis. In our study, we prepared chitosan nanoparticles loaded with five different shRNA plasmids targeting different exons of PDGF-D gene. Then, nanoparticles were characterized in vitro and transfection efficiency of these nanoparticles were investigated in breast cancer cell lines (MCF7, MDA-MB-231 and MDA-MB-435). The effects of single and multiple shRNA sequences, molecular weight of chitosan (150 kDa and 400 kDa) and the amount of shRNA (100 and 500 mu g) on the characterization and transfection efficiencies of nanoparticles have been studied. Size of nanoparticles changed between 200-400 nm and approximately 95-100% encapsulation efficiency were obtained. Release of shRNA changed with the molecular weight of chitosan. It was obtained that formulation containing shRNA plasmid targeting PDGF-D exon 6 (NP1) has the highest silencing efficiency in MDA-MB-231 cell line. It was also evaluated that chitosan can be a suitable gene delivery system for shRNA targeting PDGF-D.Publication Metadata only Investigation of antineoplastic activity of achillea nobilis subsp. Neilreichii plant in experimental breast cancer(2019-07-03) YAVUZ, AYŞE NUR; EKENTOK ATICI, CEYDA; TAŞKIN, TURGUT; ÖZBAŞ, SUNA; KABASAKAL, LEVENT; Sehlan S. S., Yavuz A. N., Ekentok Atıcı C., Taşkın T., Alan S., Özbaş S., Kabasakal L.Publication Open Access Combination therapy with chitosan/siRNA nanoplexes targeting PDGF-D and PDGFR-β reveals anticancer effect in breast cancer(2022-11-22) EKENTOK ATICI, CEYDA; Şalva E., Özbaş S., Alan S., Özkan N., Ekentok-Atıcı C., Kabasakal L., Akbuğa J.Publication Metadata only Mammaglobin gen ekspresyonunun kitozan/siRNA nanopleksleri ile baskılanması ve in vitro karakterizasyonu(2018-11-16) EKENTOK ATICI, CEYDA; ÖZBAŞ, SUNA; Özkavak Ş. B., Ekentok Atıcı C., Şalva E., Özbaş S.