Browsing by Author "Top, Ayben"

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Cation exchange (Ag+, Zn2+, Cu2+) behavior of natural zeolites
(Izmir Institute of Technology, 2001) Top, Ayben; Ülkü, Semra
In this study, clinoptilolite, most abundant zeolite present in nature, was proposed as a low cost antibacterial material. As a preliminary work, antibacterial activities of the original, Ag, and Zn forms of the clinoptilolite were investigated against several strains and compared to the commercial antibiotics. No antibacterial action was observed for the original clinoptilolite. Ag loaded clinoptilolite was found to be superior to the Zn-form against Proteus spp. and Pseudomonas aeruginosa.The original, Na, Ag, Zn, and Cu forms of the clinoptilolite samples were characterized by FTIR spectroscopy, thermal analyses (TGA, DTA, and DSC), and N2 physisorption studies. Specific attention was given in to the chemical analysis of the clinoptilolite by ICP-AES. Using the standard addition method, the respective idealized formulas of the original and Na-clinoptilolite based on 72 oxygen atoms in the unit cell were calculated as:(Na0.816 K2.070) (Ca1.060 Mg0.264) (Al5.653 Fe0.390) (Si30.084) O72. 20.023 H2O, and (Na4.763 K1.057) (Ca0.076 Mg0.094) (Al5.843 Fe0.221) (Si29.911) O72.17.049 H2O.In the FTIR spectra of the original and exchanged forms of the clinoptilolite, considerable shifts (from 3460 to 3494.8 cm-1) were observed in the band, which is formed due to the interactions of water molecules with the framework via hydrogen bonds. The positions of the other bands were not affected by cation exchange significantly. From the TGA curves, the water contents ranged between 14.31 and 11.00 % for the original and the cation-exchanged forms of the clinoptilolite. Mainly, two endotherms and one exotherm were obtained in the DTA curves. The first endotherm occurred up to about 150 oC, the second endotherm lied between 200 and 700 oC, and the exotherm was obtained at about 850 oC. Significant differences observed between the shapes of the DSC curves indicated that the cations control the dehydration behavior of the samples. N2 physisorption isotherms of five samples were all, Type IV with BET surface areas ranging between 34.97 and 46.76 m2/g.Ag, Zn and Cu ion exchange equilibria were investigated at 25 oC for both the original clinoptilolite and Na-clinoptilolite. In the former case, from the plateau of the isotherms cation exchange capacities were determined as 1.184, 0.439, 0.539 meq/g clinoptilolite for Ag+, Zn2+, and Cu2+ respectively. The major portion of the exchanges was contributed by Na+ and Ca2+. Distribution coefficient values indicated that at Ag, Zn and Cu ion exchange equilibria were investigated at 25 oC for both the original clinoptilolite and Na-clinoptilolite. In the former case, from the plateau of the isotherms cation exchange capacities were determined as 1.184, 0.439, 0.539 meq/g clinoptilolite for Ag+, Zn2+, and Cu2+ respectively. The major portion of the exchanges was contributed by Na+ and Ca2+. Distribution coefficient values indicated that at relatively low initial concentrations, the preference of the clinoptilolite for Zn2+ and Cu2+ was significant. At higher concentrations, higher distribution coefficients were obtained for Ag+ compared to Zn2+ and Cu2+. Langmuir and Freundlich models were applied for each equilibrium data. For Zn2+ and Cu2+ exchanges, Langmuir model gave better correlation and Freundlich model fitted experimental data slightly better in the case of Ag+ exchange.Equilibrium isotherms for Ag+-Na+, Zn2+-Na+, and Cu2+-Na+ pairs were investigated. Silver exchange isotherm lied above the diagonal over the whole composition range. For zinc and copper exchanges, the isotherms were above the diagonal up to equivalent fractions of exchanging ion in solution phase (As) at about 0.2. While full exchange was attained for silver, partial exchanges were obtained in the case of zinc and copper. The standard free energy of exchange values were found as .6.0, 2.03 and 3.09 kj/equiv for Ag+-Na+, Zn2+-Na+, and Cu2+-Na+ pairs respectively. From these values selectivity sequence was obtained as Ag+ > Na+ > Zn2+ > Cu2+.Consequently, by considering the preliminary antibacterial activity results, specific cation exchange capacities, and selectivity sequence of the clinoptilolite, Ag-clinoptilolite seemed to be promising antibacterial material. The results of the current study compared to the literature data pointed out that cation exchange behavior of the clinoptilolite is dependent on its original cationic composition. Therefore, it is necessary to carry out specific studies on representative samples from the deposit before any practical application.
Development of endosome disruptive peptide and PEG conjugate based doxorubicin delivery system
(Izmir Institute of Technology, 2019-07) Özkıyıcı, Selin; Top, Ayben
In this study, it was aimed to develop a drug carrier system including a TAT-derived cell penetrating peptide in order to provide fast transport of anticancer drugs from endosomal compartments to nucleus. The drug delivery system, denoted as mPEGpeptide- oxime-DOX, was based on polyethylene glycol, endosome disruptive peptide (G2RQR3QR3G2S), and doxorubicin (DOX) conjugate. Control drug delivery system, lack of the peptide (mPEG-oxime-DOX) was also synthesized to assess the effect of the peptide on the physiochemical and drug release properties of the drug carrier. As the first synthesis step, mPEG-OH was converted to mPEG-aldehyde form using DMSO-acetic anhydride oxidation reaction and aldehyde functionalization was determined by using FTIR and NMR spectroscopy. The peptide and mPEG-peptide were synthesized using solid phase synthesis protocol, and their purities were confirmed using HPLC and MALDI-TOF mass spectroscopy analyses. Prior to DOX conjugation, hydroxyl group of serine residue in the mPEG-peptide system was oxidized to aldehyde. The anticancer drug was attached to the carrier molecules via amine-aldehyde reaction forming an acid cleavable oxime bond. Drug release, size distribution, and stability of the PEG-peptideoxime- DOX system were evaluated and compared with those results of the control drug delivery system. For mPEG-oxime-DOX, a pH programmed DOX release with the respective % DOX release values of ~68 % and ~28 % at pH 5.0 and pH 7.4 was observed. For mPEG-peptide-oxime-DOX, on the other hand, quite low DOX release (~10-15 %) was obtained for both pH values suggesting possible interaction between DOX and the peptide. Mean size value of the mPEG-oxime-DOX was measured as ~24 nm. However, mPEG-peptide-oxime-DOX, had quite lower hydrodynamic diameter values (~3nm and ~6 nm at pH 5.0 and pH 7.4, respectively) possibly due to repulsions between the arginines in the peptide domain. Observation of the morphology and evaluation of the cytotoxicity of these drug delivery systems are underway.
Development of keratin based hydrogel systems
(Izmir Institute of Technology, 2022-07) Yalçın Göl, Damla; Top, Ayben
In this study, keratin proteins from Merino sheep wool were obtained via oxidative extraction (Chapter 2), sulfitolysis extraction (Chapter 3) and sulfitolysis with reductive extraction methods (Chapter 4). Keratin proteins were characterized XRD and FTIR spectroscopy and thermal analysis. In the SDS-PAGE gel results of the keratins diffusive protein bands between ~23 kDa and >170 kDa and a discrete band at about 12 kDa were observed confirming highly polydisperse nature of the protein samples. Then, keratin-based hydrogel systems were obtained via different methodologies. In Chapter 2, oxidized keratins (keratoses) were crosslinked with THPC to form keratose hydrogels. Effect of the amount of the crosslinking agent on the viscoelastic, swelling, and morphological properties of hydrogels was investigated. In Chapter 3, the keratin hydrogels were obtained via reformation of disulfide bridge and self-assembly of the keratin chains. In Chapter 4, keratins reduced with DTT were crosslinked with 2000 Da PEG-(C2H4-mal)2 and 6000 Da PEG-(C2H4-mal)2 to prepare PEG-hydrogels. Storage moduli of the hydrogels were obtained in the range of 63 ± 22 and 2613 ± 254 Pa and were shown to be tuned by the amount and chain length of the crosslinker. The highest swelling ratios were obtained for the THPC crosslinked hydrogels whereas the highest pore size was observed in PEG-keratin hydrogels. Cytocompatibility of the keratin based hydrogel systems was confirmed using L929 mouse fibroblast cells by applying CCK-8 tests. Of these hydrogels, PEG-keratin hydrogels were found to support cell proliferation with a higher rate than empty TCPS wells up to 4 days. These results demonstrate that low-cost keratin-based hydrogels can be used in a variety of biomedical applications, such as drug delivery systems for cancer therapy, and scaffolds in wound healing and soft tissue engineering.
Development of PEG and PEG-peptide based drug delivery systems
(Izmir Institute of Technology, 2016-06) Balcı, Beste; Top, Ayben
In this study, two types of drug delivery systems (DDS) were prepared; mPEG (methoxy polyethylene glycol)-HYD (hydrazide)-DOX and mPEG-peptide-(HYD)-DOX. In the design of the conjugates, mPEG was used to increase the blood circulation time. HYD provided an acid cleavable bond between the carrier molecule and DOX, whereas peptide containing histidines imparted pH responsiveness of the molecule. Doxorubicin (DOX) was selected as a model anti-cancer drug. DDS were synthesized using two steps; hydrazide functionalization of carboxylic acid of the carrier molecule followed by DOX conjugation. Hydrazide form of the carrier molecules denoted as HYD1 and HYD2 were obtained using adipic acid dihydrazide (AADH) and carbohydrazide (CH), respectively. To increase DOX conjugation, trifluoroacetic acid (TFA) and DOX amounts were changed and the reactions were carried out at the conditions giving the highest DOX conjugation (mPEG-HYD:DOX:TFA= 2.5mg:2mg:20μL per 1 mL of DMSO). The peptide (AT1=CGGGHHHHHHGGGE) was synthesized using solid phase peptide synthesis (SPPS) and PEGylated using mPEG-maleimide to obtain mPEG-AT1 conjugate. The purity of AT1 and mPEG-AT1 were confirmed using mass spectroscopy and high performance liquid chromatography (HPLC). DOX conjugation percentages were obtained as 62  7, 60  3 and 35 + 3 for mPEG-HYD1-DOX, mPEG-HYD2-DOX and mPEG-AT1-HYD1-DOX, respectively. Drug release studies indicated modest pH responsiveness of the carrier molecules obtained using AADH. On the other hand, mPEG-HYD2-DOX released  13% of drug at the end of the 72h independent of pH. For mPEG-AT1-DOX, drug release percentage values were obtained as  15% and  30% at pH 7.4 and 5.0 respectively. Cytotoxicity of the conjugates of DDS was determined using lung cancer (A-549) cell lines. DOX equivalent IC50 values were determined as 20, 40 and 5 for mPEG-HYD1-DOX, mPEG-HYD2-DOX and mPEG-AT1-DOX respectively.
Development of peg-peptide conjugate based curcumin delivery systems
(01. Izmir Institute of Technology, 2022-12) Aydoğan, Gamze; Top, Ayben
In this study, a drug delivery system based on Pluronic F127 and a peptide conjugate was proposed. The F127-peptide conjugate was prepared by the reaction between succinimidyl functionalized F127 (SC-F127) and peptide. SC-F127 was synthesized using disuccinimidyl carbonate and DMAP. Folic acid-functionalized F127 (FA-F127) was also prepared to obtain active targeting copolymers. Four peptides containing pH-responsive multiple histidines and endosome disruptive GFWFG domain were synthesized using the Fmoc procedure. H-Gly-2-ClTrt resin and Rink amide MBHA resin were used to synthesize side-chain-protected and deprotected peptides, respectively. 2-chlorotrityl resin failed in synthesizing the high-purity peptides with adjacent histidines in their sequences. Peptide-4 having a sequence of GGH6GFWFG, was prepared with acceptable purity using rink amide MBHA resin and was conjugated to SC-F127. Curcumin was loaded to F127 and F127-peptide using the thin film method with DCM solvent. Almost all curcumin was encapsulated into F127 micelles. However, the entrapment efficiency % of the F127-peptide micelles was ~86% due to the lower solubility of F127-peptide conjugate in DCM. Dynamic light scattering experiments were used to determine the stability and size distribution of the micelles. Number-based size distributions of both micelles indicated that a single peak between 10 and 30 nm was independent of pH. The peak position did not change upon incubating the micelles at 37oC up to a few days. Initially, intensity-based results of both samples indicate bidisperse populations at pH 5.0 and 7.4. Curcumin-loaded F127 micelles aggregated in the three days, as revealed by the formation of the third peak above 1000 nm independent of pH. Curcumin-loaded F127-peptide micelles, on the other hand, retain their stability for up to five days at neutral pH. For this sample, the third peak was observed only at pH 5.0 on days 2 and 5.
Effects of grid design on lead-acid battery performance
(Izmir Institute of Technology, 2017-07) İşler, Tuğçe; Ebil, Özgenç; Top, Ayben
In today’s world, approximately 88 percent of the total energy demand is supplied by fossil fuels; however, it has become clear that; other energy sources are needed due to limited fossil fuels. The demand for energy can most effectively be filled by renewable energy sources as installed energy storage capacity is growing rapidly. If renewable energy sources advance enough to fulfill the high demand, earth-friendly, clean and sustainable energy will help to protect the environment, thus ensuring a healthier life for future generations. Energy storage systems are essential in this endeavor, and in order to become more prevalent, storage systems for renewable energy sources must supply electricity without interruption as much as possible. As an electrochemical storage, a battery with a high level of performance, high energy density and life cycle could offer a viable solution for electricity storage provided that battery cost should be economically viable. This thesis aims to improve the geometry of the grid used in lead acid batteries in order to obtain a more uniform current and potential distribution, and minimize the potential drop for improved battery performance. A 3D mathematical model was developed using finite element method to evaluate the behavior of the grid under various conditions. Five different porous grid geometries were simulated under different loads and optimum grid geometry was identified. The 3D mathematical model of the lead-acid battery based on finite element method was simulated under certain conditions in order to evaluate the effect of grid geometry on battery performance.
Hydrogels and self-assemled nanostructures based on wool keratose
(Izmir Institute of Technology, 2017-07) Pakkaner, Efecan; Top, Ayben
In this study, water soluble keratose proteins were extracted from “Ovis aries” wool using peracetic acid oxidation with a yield of 35 ± 5 %. Wool samples and the extracted keratose proteins were characterized by using FT-IR, XRD, SEM and TGA techniques. α-keratose fractions (MW = 43-53 kDa) along with cleaved fragments of α-keratoses with molecular weights between 23 and 33 kDa were identified in the extracted protein mixture using SDS-PAGE analysis. DLS and AFM experiments indicated self-assembled globular nanoparticles with diameters of 20-40 nm formed at 5 and 10 mg/ml keratose concentrations. On the other hand, at 10 % w/v keratose concentration interconnected keratose hydrogels with pore sizes of 6 ± 4 and 7 ± 4 μm were obtained upon incubation at 37 and 50 °C, respectively. Storage moduli (G’) of these physical hydrogels were increased from ~100 to ~1000 Pa, as gelation temperature was increased from 37 to 50 °C. Hydrogels were also obtained at 7.5 % w/v keratose concentration by the addition of a crosslinker, THPC. Amine group:crosslinker ratio was used as 1:1, 1:2 and 1:4. As the amount of crosslinker increased, network transformed from fibrous to more planar structures exhibiting a significant decrease in average pore size from 24 to 11 μm. G’ values of the crosslinked hydrogels were obtained between ~1 and ~5 kPa tuned by the crosslinking amount. Cell interaction properties of a select physical hydrogel prepared at 37 °C was tested using CCK-8 assay. It was observed that the keratose hydrogel supported L929 mouse fibroblast cell proliferation as much as collagen, which suggests that these keratose hydrogels can be promising candidates in soft tissue engineering applications.
PEG-peptide drug carrier systems with enzymatic degradation units
(Izmir Institute of Technology, 2016-12) Yüksel, Nesligül; Top, Ayben
In this study, it was aimed to develop drug delivery systems with high drug release rate, capable of overcoming multidrug resistance of cancer cells. The first generation drug delivery system (DDS) denoted as mPEG-AT3-DOX was prepared by methoxy polyethylene glycol (mPEG) and peptide conjugation, and the model anticancer drug, DOX, was attached to the mPEG-peptide carrier system using stable amide linkage. mPEG was used to increase blood circulation time of the DDS. Designed peptide (AT3 = CG3H6R2ALALG3E) controls release of the drug via RRALAL sequence, which is a substrate for a lysosomal enzyme, cathepsin B, overexpressed in most of the tumor cells. pH responsive histidines and reactive amino acids (glutamic acid and cysteine) for drug and mPEG conjugations were also added to the peptide sequence. The peptide synthesized by using Fmoc chemistry was conjugated to mPEG-maleimide via Michael addition reaction. DOX was attached to the carboxylic acid group present in the carrier system (mPEG-AT3) via amide linkage. Mass spectroscopy and HPLC were used to assess the purity of the AT3 and mPEG-AT3. At pH 7.4, mPEG-AT3-DOX exhibited bimodal size (hydrodynamic diameter) distribution at about 15 and 30 nm independent of pH. % DOX release from mPEG-AT3-DOX was observed to be below 10 % at neutral pH and pH 5.0 in the absence of cathepsin B, and increased to 17 ± 2 % in the presence of cathepsin B. Complete degradation of AT3 peptide within three hours in the presence of cathepsin B suggests lower than expected DOX release is due to aggregation tendency of the DDS. Cytotoxicity of the conjugates was evaluated using the lung cancer (A549) and prostate cancer (PC3) cell lines. At the end of 24 hours the absolute IC50 values of free DOX and mPEG-AT3-DOX were obtained as 1.37 ± 0.05 and 1.33 ± 0.11 for the A549 cell line, 1.51 + 0.07 and 1.63 + 0.80 μg equivalent DOX / ml for the PC3 cell line, respectively. Considering, these results, the second generation DDS will be designed to have more pronounced pH sensitive property by increasing the number of histidines in the peptide domain.
Peptide hydrogels containing cell attachment molecules
(Izmir Institute of Technology, 2019-07) Uysal, Berk; Top, Ayben
In this study, peptides with sequences and notations as KLELKLELKLEL (KLEL), KLDVKLDVKLDV (KLDV), KLDVKLDVKLKV (KLKV1), KLKVKLDVKLKV (KLKV2), KLKVKLKVKLKV (KLKV3) were synthesized using solid phase peptide synthesis (SPPS) method based on Fmoc chemistry. Reverse phase HPLC and MALDI-TOF mass spectroscopy characterization methods were used to assess the purity of the peptides. Three different synthesis procedures were tested, and it was found that employing DMF:DMSO at 1:1 ratio as a solvent increased purity of the resultant peptide. FTIR results indicated the presence of expected β-sheet secondary structure, as well as an interference band from TFA salts for all of the peptides. All the peptides formed hydrogels at pH 7.4 with 1 wt% concentration in deionized water (DIW). AFM results of these hydrogels indicated that KLKV1 and KLKV2 had fibrous morphology with a width of 5-20 nm and 7-18 nm respectively. KLDV and KLKV3 peptide hydrogels, on the other hand, exhibited globular structures, having sizes with 15-50 nm and 8-15 nm, respectively. Storage moduli (G’) of these hydrogels in DIW were obtained as ~860 ± 150 Pa, ~260 ± 60 Pa, ~210 ± 30 Pa and ~1850 ± 200 Pa for KLDV, KLKV1, KLKV2 and KLKV3 respectively. Of these peptides, only HCl salt of KLDV and KLKV1 peptides more readily formed hydrogels in PBS but at 1.5 wt% concentration. G’ values of these KLDV and KLKV1 hydrogels were determined as ~1810 ± 850 Pa and ~700 ± 230 Pa, respectively. Cell proliferation tests (CCK-8 assay) of KLDV and KLKV1 hydrogels were performed by using L929 mouse fibroblast cells. Empty wells (TCPS) were used as a control group. Cell proliferation was observed to be comparable for both select hydrogels and empty wells, suggesting possible applications of these hydrogels in tissue engineering.