Karbon nanotüp-nükleik asit konjugatlarının hazırlanması, gen aktarımında kullanım potansiyellerinin incelenmesi

Biyomoleküllerin hücre içine alınımı klinik anlamda teşhis ve tedavi açısından, moleküler biyolojik anlamda ise viral olmayan transfeksiyon ajanlarının yaygınlaştırılması açısından oldukça önemlidir. Protein, nükleik asit benzeri makromoleküllerin ve birçok küçük molekülün hücre içine taşınımı sırasında karşılaşılan adsorbsiyon sorununu aşmak için yeni stratejilerin geliştirilmesi bir zorunluluktur. Gen transferinin söz konusu olduğu durumlarda, immünolojik cevaba sebep olan viral transfeksiyon yöntemlerine alternatif yeni yöntemlere ihtiyaç duyulmaktadır İlgilenilen molekülün kovalent olarak bir taşıyıcı moleküle bağlanmasıyla oluşturulan biyokonjugatlar, hücre içine alınım sorununa yeni çözümler getirmektedir. Bu amaçla manyetik nanopartiküller dendrimerler, katyonik polimerler biyobozunur nanopartiküller ve karbon nanotüpler artan ilgiyle kullanılmaktadır. Bu proje kapsamında karbon nanotüpler kimyasal süreçlerle karboksil grup fonksiyonlu hale getirilmiş ve amino linker modifiye plazmid DNA tasarlanmıştır. Bu moleküller arasında kurulan amid bağı ile kovalent bağlı biyokonjugatlar sentezlenmiş ve elde edilen biyokonjugat ile bakterilere plazmid taşınımı ve gen ekspresyonu viral olmayan başka yöntemler ile karşılaştırılmış ve sistemin verimliliği incelenmiştir Ayrıca karbon nanotüplerin bakteriler üzerindeki sitotoksik etkileri de incelenmiştir. Yapılan bir çok optimizasyon çalışması sonunda kovalent bağlı konjugatın klasik yöntemler kullanılmadan transformasyonu sağladığı görülmüştür. Öne sürülen sistemin karbon nanotüp ile daha önce yapılmış adsorbsiyona dayalı konjugatlara alternatif olarak literatüre katkı sağlayacağı düşünülmektedir

Bağımlılıkta gelişen glutamat eksitotoksisitesine bağlı nörodejenerasyonun engellenmesi

Madde kullanımının bir sonucu olarak gelişen glutamat uyarımının artması, nörodejenerasyona yol açmakta ve sonuç olarak sinir hücrelerinde yapı-fonksiyon bozuklukları ve hücre ölümü görülmektedir. Glutamat eksitotoksisitesinden temel olarak iyonotropik glutamat reseptörlerinin aktivasyonu, hücre içi oksidatif stresin artması ve mitokondri fonksiyon bozuklukları sorumludur. Sinaptik glutamatın azaltılması, NMDA reseptör antagonistlerinin kullanılması, antioksidan bileşiklerin verilmesi ya da hücre içi antioksidan savunma mekanizmalarının güçlendirilmesi ile eksitotoksisite kaynaklı nörodejenerasyon ortadan kaldırılabilir. Tez çalışması kapsamında, eksitotoksisitenin in vitro koşullarda modellenmesi için nöroblastoma hücre hatları ve primer serebral korteks hücreleri kullanılarak hücre canlılığı ve sitotoksisite testleri yapıldı. Glutamat ile tetiklenen eksitotoksik etkinin giderilmesi için doğal antioksidan bileşikler ve bunları içeren emülsiyonlar kullanılmıştır. Ayrıca, metamfetaminin yol açtığı eksitotoksisite mekanizmalarının aydınlatılması için eksitatör amino asit taşıyıcı protein 3 (EAAT3) mercek altına alınmıştır. EAAT3 proteinin hücre membran lokalizasyonu ile metamfetamin arasındaki ilişki mikroskobik olarak incelenmiş ve mutant EAAT3 kullanılarak protein yapısında sorumlu bölge çalışılmıştır. Ayrıca, bu proteinin glutamat ve sistein taşınımı üzerinden, hücre içi antioksidan savunmalara etkisi incelenmiştir. Elde edilen bulgular ve literatür karşılaştırıldığında, hücre hatları ve glutamat kullanılarak oluşturulan in vitro eksitotoksisite modellerinde, kullanılan çözgen girişiminin ihmal edildiği ve hatalı olarak düşük konstantrasyonların etkin doz olarak verildiği görülmüştür. Yüksek doz glutamat ile devam ettiğimiz modelde, kurkumin ve kurkumin içeren nanoemülsiyonlar, glutamat eksitotoksisitesinin giderilmesinde etkili olmuştur. Bununla beraber, kurkumin içeren nanoemülsiyon, kontrol nanoemülsiyonlar ile karşılaştırıldığında daha etkin olmadığı görülmüştür. Metamfetaminin, sitotoksisiteden ziyade hücre proliferasyonu üzerinde inhibe edici bir etkisi olduğu görülmüştür. EAAT3 proteini incelendiğinde, bu proteinin metamfetamin etkisiyle hücre membranından ayrılarak hücre içine göç ettiği gösterilmiştir. Bunun sonucu olarak da hücre içi sistein alımı mekanizmaları etkilenerek, glutatyon sentezi ve indirgenmiş glutatyon miktarı azalmıştır. Çalışmalar, eksitotoksisitede oksidatif stres gideriminin hedeflenebileceğini göstermiştir. Ayrıca, EAAT3 proteininin hücre içine alımında sorumlu motifinin belirlenmesi daha sonraki çalışmalarda protein-protein etkileşimlerinin aydınlatılmasına ve ilaç etki hedeflerinin belirlenmesini sağlayabilecektir.Increased glutamate stimulation which may be result of drug abuse leads to increased neurodegeneration and as a result structural and functional disorders as well as cell death occurs in neural cells. Activation of ionotropic glutamate receptors, increased intracellular oxidative stress and mitochondrial dysfunction are mainly responsible for glutamate excitotoxicity. Excitotoxicity-induced neurodegeneration can be eliminated by reducing synaptic glutamate, using NMDA receptor antagonists, introducing antioxidant compounds or strengthening intracellular antioxidant defence mechanisms. Within the scope of the thesis, in order to model excitotoxicity in vitro, cell viability and cytotoxicity assays were performed on neuroblastoma cell lines and primary cerebral cortex cells. Natural antioxidant compounds and emulsion containing these compounds ere used to eliminate the excitotoxic effect induced by glutamate. In addition, the excitatory amino acid transporter protein 3 (EAAT3) was examined to elucidate the mechanisms of excitotoxicity caused by methamphetamine. The relationship between the cell membrane localization of EAAT3 protein and methamphetamine was examined by microscopy and the responsible region in the protein structure was studied using mutant EAAT3. Moreover, effect of this protein on intracellular antioxidant defence systems via glutamat and cysteine transport. Comparison of our findings and literature indicates that, in vitro excitotoxicity models using cell lines and glutamate neglect the intervention caused by solvent and thus, result of reporting low concentrations of glutamate as effective dose. Curcumin and curcumin-containing nanoemulsions were effective in removing glutamate excitotoxicity in the preferred model of high-dose glutamate applied in this study. However, curcumin-containing nanoemulsion was not more effective than control nanoemulsions. It was observed that, methamphetamine has an inhibitory effect on cell proliferation instead of direct cytotoxicity. When the EAAT3 protein was examined, it was shown that this protein was translocated from the cell membrane and migrated into the cell by methamphetamine. As a result, cellular uptake mechanisms of cysteine were affected and glutathione synthesis and reduced glutathione levels were decreased. Studies show that, oxidative stress can be a viable target to reduce excitotoxicity. Additionally, identification of the motif responsible for EAAT3 internalization may elucidate future protein-protein interactions and identify drug action targets

COMPOSITION ANALYSIS OF E-LIQUIDS AND THEIR EFFECTS ON HEALTHY LIVER AND PHARYNGEAL CARCINOMA CELL LINES

Electronic cigarettes have become popular worldwide in recent years although their effects on human health are still not properly known. The lack of regulations brings a problem of inconsistency between ingredients and the product label. We aimed to analyse the contents of widely used e-liquids and their effects on two different cell lines. Eleven e-liquid samples were selected according to their availability and popularity. Nicotine, propylene glycol (PG), glycerine (GLY), and volatile compounds in e-liquids were analysed by High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC). 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay was used to determine the effects of e-liquids on transformed human normal liver epithelial cell line (THLE-2) and human pharyngeal carcinoma cell line (Detroit 562). Nicotine amounts were found to be consistent with product labels. and GLY were not only different between brands but also for products within the same brand. THLE-2 cell viability was inversely correlated with e-liquid concentration. However, decreases in cell viability were not correlated with nicotine amount. Interestingly, effects of several samples on Detroit 562 cells were triphasic; decrease in viability at lower doses, cell survival in mid-concentrations and loss of viability in highest doses. The analytical composition of e-liquids differs greatly among products which corresponds to different cellular effects. Viability of cancer cells does not change in a dose-dependent manner, which suggest that cellular differences may play role in the outcome of these products.The study was supported by The Scientific and Technological Research Council of Tuerkiye (TUBITAK) with project number 114Z687 and Ege University Science and Technology Centre with the project number 15-BIL-033.Scientific and Technological Research Council of Tuerkiye (TUBITAK) [114Z687]; Ege University Science and Technology Centre [15-BIL-033

The Covalent Bioconjugate of Multiwalled Carbon Nanotube and Amino-Modified Linearized Plasmid DNA for Gene Delivery

WOS: 000331385800024PubMed ID: 24288272Carbon nanotubes (CNTs) are allotropes of carbon, which have unique physical, mechanical, and electronic properties. Among various biomedical applications, CNTs also attract interest as nonviral gene delivery systems. Functionalization of CNTs with cationic groups enables delivery of negatively charged DNA into cells. In contrast to this well-known strategy for DNA delivery, our approach included the covalent attachment of linearized plasmid DNA to carboxylated multiwalled CNTs (MWCNTs). Carboxyl groups were introduced onto MWCNTs by oxidative treatment, and then the carboxyl groups were activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The whole pQE-70 vector including the gene encoding green fluorescent protein (GFP) was subjected to polymerase chain reaction (PCR) using the modified nucleotide N6-(6-Amino)hexyl-2-deoxyadenosine-5-triphosphate. Hence, free amino groups were introduced onto the linearized plasmid. Covalent bonding between the amino-modified plasmid DNA and the carboxylated MWCNTs was achieved via EDC chemistry. The resulting bioconjugate was successfully transformed into chemically competent Escherichia coli cells, without necessity of a heat-shock step at 42 degrees C. The presence of Ca2+ in transformation medium was required to neutralize the electrostatic repulsion between DNA and negatively charged outer layer of E. coli. The transformants, which were able to express GFP were inspected manually on ampicillin agar plates. Our study represents a novelty with respect to other noncovalent CNT gene delivery systems. Considering the interest for delivery of linear DNA fragments, our study could give insights into further studies. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:224-232, 2014Research Foundation of Ege UniversityEge University [10-FEN-052]The authors acknowledge financial support from the Research Foundation of Ege University (project number: 10-FEN-052). There is no conflict of interest associated with this manuscript

Modified gold surfaces by 6-(ferrocenyl)hexanethiol/dendrimer/gold nanoparticles as a platform for the mediated biosensing applications

WOS: 000315761700010PubMed ID: 25427467An electrochemical biosensor mediated by using 6-(Ferrocenyl) hexanethiol (FcSH) was fabricated by construction of gold nanoparticles (AuNPs) on the surface of polyamidoamine dendrimer (PAMAM) modified gold electrode. Glucose oxidase (GOx) was used as a model enzyme and was immobilized onto the gold surface forming a self assembled monolayer via FcSH and cysteamine. Cyclic voltammetry and amperometry were used for the characterization of electrochemical response towards glucose substrate. Following the optimization of medium pH, enzyme loading, AuNP and FcSH amount, the linear range for the glucose was studied and found as 1.0 to 5.0 mM with the detection limit (LOD) of 0.6 mM according to S/N = 3. Finally, the proposed Au/AuNP/(FcSH + Cyst)/PAMAM/GOx biosensor was successfully applied for the glucose analysis in beverages, and the results were compared with those obtained by HPLC. (C) 2012 Elsevier B.V. All rights reserved.Ege University Research FundEge University [2011 FEN 039]The authors acknowledge the financial support from the Ege University Research Fund (project number: 2011 FEN 039)

Peptide-modified conducting polymer as a biofunctional surface: Monitoring of cell adhesion and proliferation

Here, we report the electropolymerization of 3-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)aniline monomer on indium tin oxide (ITO) glass and its use as a coating material for cell culture applications. Functional amino groups on the conducting polymer provide post-modification of the surface with the arginylglycylaspartic acid (RGD) peptide via EDC chemistry. Scanning electron microscopy, atomic force microscopy, and contact angle and surface conductivity measurements were carried out for the surface characterization. The peptide-conjugated surface was tested for adhesion and proliferation of several cell lines such as monkey kidney epithelial (Vero), human neuroblastoma (SH-SY5Y), and human immortalized skin keratinocyte (HaCaT). These cells were cultured on RGD-modified, polymer-coated ITO glass as well as conventional polystyrene surfaces for comparison. The data indicate that the RGD-modified surfaces exhibited better cell adhesion and proliferation among all surfaces compared. Cell imaging studies up to 72 h in length were performed on these surfaces using different microscopy techniques. Therefore, the novel biofunctional substrate is a promising candidate for further studies such as monitoring the effects of drugs and chemicals on cellular viability and morphology as well as cell-culture-on-a-chip applicationsHere, we report the electropolymerization of 3-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)aniline monomer on indium tin oxide (ITO) glass and its use as a coating material for cell culture applications. Functional amino groups on the conducting polymer provide post-modification of the surface with the arginylglycylaspartic acid (RGD) peptide via EDC chemistry. Scanning electron microscopy, atomic force microscopy, and contact angle and surface conductivity measurements were carried out for the surface characterization. The peptide-conjugated surface was tested for adhesion and proliferation of several cell lines such as monkey kidney epithelial (Vero), human neuroblastoma (SH-SY5Y), and human immortalized skin keratinocyte (HaCaT). These cells were cultured on RGD-modified, polymer-coated ITO glass as well as conventional polystyrene surfaces for comparison. The data indicate that the RGD-modified surfaces exhibited better cell adhesion and proliferation among all surfaces compared. Cell imaging studies up to 72 h in length were performed on these surfaces using different microscopy techniques. Therefore, the novel biofunctional substrate is a promising candidate for further studies such as monitoring the effects of drugs and chemicals on cellular viability and morphology as well as cell-culture-on-a-chip applications

Preparation of glutathione loaded nanoemulsions and testing of hepatoprotective activity on THLE-2 cells

To improve bioavailability and stability of hydrophobic and hydrophilic compounds, nanoemulsions are good alternatives as delivery systems because of their nontoxic and nonirritant nature. Glutathione (GSH) suffers from low stability in water, where its encapsulation in nanoemulsions is a powerful strategy to its stability in aqueous systems. The aim of this study was to obtain nanoemulsions from the hydrophobic/hydrophilic contents of N. sativa seed oil so as to improve GSH stability along with bioavailability of N. sativa seed oil. Then, the prepared nanoemulsions were tested for in vitro hepatoprotective activity against ethanol toxicity. To the best of our knowledge, there is no study on the test of nanoemulsions by the combination of Nigella sativa seed oils and GSH in hepatoprotective activity. Here, nanoemulsions with different contents were prepared using Nigella sativa seed oils. Content analyses and characterisation studies of prepared nanoemulsions were carried out. In order to investigate the protective effects against to ethanol exposure, THLE-2 cells were pretreated with nanoemulsions for 2 h with the maximum benign dose (0.5 mg/mL of nanoemulsions). Ethanol (400 mM) was introduced to pretreated cells and nontreated cells for 48- or 72-h periods, followed by cell viability assay was carried out. Fluorescence microscopy tests revealed the introduction of the nanoemulsions into THLE-2 cells. The findings show that nanoformulations have promising in vitro hepatoprotective effects on the THLE-2 cell line against ethanol exposure

Functional poly(p-phenylene)s as targeting and drug carrier materials

WOS: 000375958100002Polymers have a substantial attention in drug delivery systems owing to the diverse intrinsic advantages. It is important to carry the drug to the target site and release to exert its effects. Herein, poly(p-phenylene)s with amino and poly(ethylene glycol) substituents (PPP-NH2-g-PEG) were used as a carrier for doxorubicin (DOX), an anticancer drug, and haloperidol, a sigma receptor targeting ligand. Both human cervix adenocarcinoma cell line (HeLa) and human keratinocyte cell line (HaCaT) having different Sigma receptor 1 (SigmaR1) expression levels were compared. HeLa was found to express twofold SigmaR1 compared to HaCaT cells. Cell imaging studies showed that, DOX cell uptake was higher in HeLa cells when targeted with haloperidol. [GRAPHICS] .Ege UniversityEge University; Aliye USTER Foundation; Istanbul Technical University, Research FundEge University, Aliye USTER Foundation, and Istanbul Technical University, Research Fund, are acknowledged for financial support