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    Enhancing gene delivery efficiency with amphiphilic chitosan modified by myristic acid and tertiary amino groups
    (Elsevier, 2024) Fidan, Emine Busra Eker; Bal, Kevser; Senturk, Sema; Kaplan, Ozlem; Demir, Kamber; Gok, Mehmet Koray
    The aim of this study is to synthesize new amphiphilic chitosan containing myristic acid as the hydrophobic tail and tertiary amine groups as the hydrophilic head and to evaluate the gene delivery efficiency. In this context, the primary amine groups of chitosan were first modified with myristic acid (Chi-M), followed by the modification of the methylol groups with 3-dimethylamino-1-propyl chloride hydrochloride. The chemical characterization of this chitosan formulation (Chi-MA) was determined using nuclear magnetic resonance (NMR), Fouriertransform infrared spectroscopy (FTIR) analysis and gel permeation chromatography-size exclusion chromatography. Chi-MA nanoparticles were prepared via ionic gelation, and particle size, polydispersity and zeta potential were determined. The nanoparticles were evaluated for their proton buffering capacity and gene complexing capacity. Additionally, the cytotoxicity of Chi-MA on HEK293T cells was determined via MTT assay, and the transfection efficiency of Chi-MA was analyzed by a flow cytometer. The results indicate a significant increase in gene complexing capacity (8-fold) and nanoparticle formation ability of Chi-MA compared to other chitosan formulations. Chi-MA nanoparticles showed no toxicity against HEK293T cells and exhibited the highest transfection efficiency with significantly lower nanoparticle: gene ratios compared to previous studies. These findings demonstrate the effective use of amphiphilic Chi-MA as a gene carrier.
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    Enhancing transfection efficiency of primary cell lines using different terminated PBAE structures without endcapping reaction
    (Springer, 2025) Demir, Irmak; Celik, Sibel Kucukertugrul; Bal, Kevser; Kaplan, Ozlem; Senturk, Sema; Demir, Kamber; Gok, Mehmet Koray
    Gene therapy holds promise for a wide range of diseases, including Alzheimer's, diabetes, and cancer, and requires the efficient transfer of nucleic acids into cells. However, transfection in primary cells is still problematic and requires the development of new transfection agents. Poly (beta-amino ester) (PBAE) has attracted great attention in transfection research due to their low toxicity, high gene loading capacity, endosomal escape ability, and biodegradability properties. In this study, two new PBAEs with different molecular weights are synthesized that could provide high viability and transfection efficiency in primary cells. They are characterized using FTIR and 1H NMR analysis. GPC-SEC system is also used to calculate the average molecular weight (Mw) and polydispersity index. PBAE nanoparticle preparation is carried out using the nanoprecipitation technique. The gene loading capacity, protective ability against nuclease degradation, and proton buffering capacity of nanoparticles are determined. Additionally, the morphology of PBAEA:pEGFN1 complexes was investigated by STEM analysis. Finally, their cytotoxicity and transfection efficiency in primary ovine fibroblast (POF) cells are also investigated. The results reveal that the new PBAE with higher Mw achieves quite high transfection efficiency of about 87% and did not show any cytotoxic effects on these cells. These findings suggest that PBAE is a promising option to achieve high transfection efficiency in primary cells.
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    Improving physiological solubility and gene transfer efficiency of chitosan via 3-nitrobenzaldehyde and amino acid conjugation
    (Elsevier, 2025) Bal, Kevser; Kaplan, Ozlem; Senturk, Sema; Celik, Sibel Kucukertugrul; Demir, Kamber; Gok, Mehmet Koray
    In this study, chitosan was chemically modified with 3-nitrobenzaldehyde (3NBA) and three amino acids (arginine, cysteine, and histidine) to enhance its gene delivery performance. 3-NBA was selected for its known DNA binding properties, while the amino acids were chosen based on their functional groups, which can improve solubility, facilitate cellular uptake, and contribute to endosomal escape. The modified chitosan polymers were characterized using Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance Spectroscopy (NMR). Nanoparticles were prepared using the ionotropic gelation method, and their particle size, polydispersity index (PDI), zeta potential were analyzed by dynamic light scattering (DLS). The particle sizes ranged from 105.07 f 3.45 to 206.15 f 10.39 nm, with PDI values between 0.29 f 0.01 and 0.39 f 0.02. Zeta potentials were measured between 32.05 f 0.49 mV and 51.95 f 0.35 mV. The cysteine-modified chitosan (Chi3NBACys) exhibited approximately 8.4-fold higher solubility than unmodified chitosan. In vitro studies demonstrated that the modified chitosan nanoparticles exhibited low cytotoxicity in HEK293T cells. Among the tested formulations, Chi-3NBACys showed the highest transfection efficiency, comparable to commercial agent LipofectamineTM 2000. These findings suggest that chitosan nanoparticles modified with 3-NBA and amino acids can be safe and efficient non-viral gene delivery vectors.
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    Redox-responsive lipoic acid-modified poly ((3-amino ester) nanoparticles for enhanced gene delivery
    (Elsevier, 2025) Kaplan, Ozlem; Bal, Kevser; Senturk, Sema; Demir, Kamber; Gok, Mehmet Koray
    Efficient and safe delivery of genetic material into cells is a critical step in gene therapy applications. Poly ((3-amino ester) (P(3AE) polymers have garnered significant attention among non-viral gene delivery systems due to their biocompatibility, low toxicity, and structural versatility for modifications. However, the hydrolysisdependent nucleic acid release mechanism of P(3AE often results in uncontrolled release, thereby limiting transfection efficiency. In this study, two P(3AE polymers were modified with lipoic acid to develop redoxresponsive drug delivery systems, and their transfection efficiency was evaluated in cervical cancer cells (HeLa) and human embryonic kidney (HEK293T) cells. The modifications were confirmed via Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) analyses. The proton buffering capacities of P(3AE and lipoic acid-modified P(3AE within the pH range of 5.0-7.4 exhibited similar profiles. Nanoparticles were prepared from these polymers using the nanoprecipitation technique, yielding particle sizes ranging from 102.0 +/- 2.5 nm to 212.2 +/- 8.8 nm, with polydispersity index (PDI) values between 0.166 +/- 0.043 and 0.280 +/- 0.019. The zeta potential of all nanoparticles ranged from +27.3 +/- 1.1 mV to +47.4 +/- 1.7 mV. While the particle sizes remained stable over six weeks, an increasing trend in PDI values was observed. A decrease in zeta potential was recorded, attributed to the hydrolysis of P(3AE. Redox sensitivity analyses using dithiothreitol (DTT) confirmed the redox responsiveness of the nanoparticles and validated their rapid degradation under reductive conditions. In vitro studies revealed that lipoic acid modifications enhanced transfection efficiency in HeLa cells. These findings suggest that lipoic acid-modified P(3AE polymers hold significant potential for developing more effective gene delivery systems targeting cancer cells.
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    Silane and amino acid functionalized PBAEs for enhanced gene Delivery: Synthesis, characterization, and transfection efficiency
    (Elsevier, 2025) Kaplan, Ozlem; Bal, Kevser; Senturk, Sema; Demir, Kamber; Gok, Mehmet Koray
    This study aimed to synthesize, characterize, and evaluate the transfection efficiency of end group-modified branched poly (R-amino ester) (PBAE) based polymers containing silane groups to be used as gene carriers. Branched PBAE polymers were synthesized using bisphenol A ethoxylate diacrylate, diethylenetriamine, and N-[3-(trimethoxysilyl)propyl] ethylenediamine, and various amino acids (histidine, isoleucine, serine, methionine, phenylalanine, arginine) were added for end group modification. The structures of the polymers were confirmed by nuclear magnetic resonance (NMR) and Fourier-transform infrared spectroscopy (FTIR) analysis. Nano-particles were prepared using the nanoprecipitation technique, and their plasmid DNA complexing and protection capacity and endosomal escape abilities were determined. Finally, the cytotoxicity of the nanoparticles on HeLa and HEK293T cell lines was tested by the MTT test, and their transfection efficiency was tested by flow cytometry and fluorescence microscopy. All the tested nanoparticles showed lower toxicity in HeLa cells than in HEK293T cells. Transfection efficiency followed an increasing order, starting from the linear PBAE, progressing to silane modification, and subsequently with modifications by histidine, isoleucine, serine, methionine, phenylalanine, and arginine. This study highlights the potential of silane and amino acid-functionalized PBAE polymers as gene carriers and the impact of these modifications on transfection efficiency.
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    Tricine-modified chitosan as a strategy for enhancing hydrophilicity and gene delivery
    (Elsevier Sci Ltd, 2025) Tantan, Yasemin; Kaplan, Ozlem; Bala, Kevser; Senturk, Sema; Fidan, Emine Buera Eker; Celik, Sibel; Demir, Kamber
    In this study, we investigated the effect of chitosan modification with tricine on transfection efficiency by preserving its ability to form complexes with plasmid DNA (pDNA) and increasing its hydrophilicity. The inherent limitations of chitosan, such as poor solubility at physiological pH, insufficient cellular uptake, and strong ionic interactions with pDNA, typically result in low transfection efficiency. To overcome these challenges, Tricine, a hydrophilic molecule containing a secondary amine group, was conjugated to chitosan. Chitosan of three different molecular weights (low, medium, and high) was modified with tricine. Structural characterization of the modified chitosan was conducted using Fourier Transformed Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR) analyses. The effects of tricine modification were assessed in terms of hydrophilicity/hydrophobicity, proton buffering capacity, particle size, PDI and zeta potential. Tricine modified low molecular weight chitosan nanoparticles (nLMWChiTri), which exhibit suitable properties for gene transfer studies, were evaluated regarding pDNA complexation ability, cytotoxicity and in vitro transfection efficiency. The results demonstrated that tricine modification enhanced the gene transfer potential of chitosan, making it competitive with the commercial transfection agent LipofectamineTM 2000 and offering a promising strategy for non-viral gene therapy applications. Furthermore, the biocompatibility and biodegradability of chitosan, combined with the improved hydrophilicity provided by tricine, makes nLMWChiTri a safer and more sustainable option for repeated use in gene delivery, overcoming the major limitations associated with other synthetic vectors such as LipofectamineTM 2000.