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A Silver Compound with Potential Applications as an Anticancer, Antibacterial, Antifungal, and Anti-Alzheimer's Agent
(Wiley-V C H Verlag Gmbh, 2023) Korkmaz, Nesrin; Kisa, Dursun; Kaplan, Ozlem; Tosun, Nazan Goksen; Ozgur, Aykut; Imamoglu, Rizvan; Karadag, Ahmet
This study investigated the biological activity of the silver coordination compound K-22. The IC50 values of K-22 on cancer cell lines range from 0.797 mu g/mL to 3.426 mu g/mL, indicating that K-22 might preferably inhibit A549, Saos-2, MCF-7, and HT-29 cell proliferation and thus have better therapeutic activity. Furthermore, K-22 stimulated apoptosis via up-regulation of the mRNA and protein expression level of Bax/Bcl-2 ratio in A549, Saos-2, MCF-7, and HT-29. K-22 exhibited antimicrobial activity against S. aureus, E. faecalis, K. pneumonia, P. aeruginosa, C. utilis, and C. albicans. Experimental results show that the compound has inhibitory potential with an IC50 value of 178.10 mu M for the BChE (butyrylcholinesterase) enzyme, which has a vital role in the progression of Alzheimer's disease. As a result, compound K-22 exhibits a strong potential for medical use due to its anticancer, antibacterial, antifungal, and anti-Alzheimer properties.
Biosynthesis of Iron, Copper and Silver Nanoparticles Using Polygonum Cognatum and Tragopogon Porrifolius Extracts and Evaluation of Their Antimicrobial Potentials
(2023) Kaplan, Ozlem; Tosun, Nazan Gökşen
In this study, the biosynthesis of iron (Fe), copper (Cu) and silver (Ag) nanoparticles was aimed using aqueous extracts of Polygonum cognatum (P. cognatum) and Tragopogon porrifolius (T. porrifolius). The synthesized nanoparticles were characterized using UV/Vis spectroscopy, fourier transform infrared spectroscopy (FTIR) and dynamic light scattering technique (DLS). The antibacterial activity of the nanoparticles was analyzed against well-known pathogens Klebsiella pneumonia, Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus aureus. In addition, the anti-fungal activity of the nanoparticles against Candida albicans and Candida utilis strains was evaluated. The obtained results showed that the synthesized nanoparticles have a moderate antimicrobial effect.
Development of recombinant protein-based nanoparticle systems for inducing tumor cell apoptosis: In vitro evaluation of their cytotoxic and apoptotic effects on cancer cells
(Elsevier, 2024) Kaplan, Ozlem; Gok, Mehmet Koray; Pekmez, Murat; Tayhan, Secil Erden; Ozguemues, Saadet; Gokce, Isa; Arda, Nazli
Myeloid leukemia cell differentiation protein (Mcl-1) is a B -cell lymphoma 2 (Bcl-2) family antiapoptotic protein that regulates cellular apoptosis. Mcl-1 expression is important for cancer cell survival and a prospective target in cancer treatment. Bcl-2 homology 3 (BH3) mimetic peptides, such as the Mcl-1-specific MS1 peptide that targets conserved BH3 domains, are highly specific Mcl-1 inhibitors. However, the application of these peptides is limited due to their metabolic instability, degradation by cellular proteases, and low cell permeability. In this study, it was aimed to form nanostructures to increase the cellular uptake and stability of the Mcl-1 inhibitor MS1 peptide. The Bim BH3 mimetic MS1 peptide was produced recombinantly in fusion with mNeonGreen fluorescent protein and polyhistidine (6xHisTag). Self -assembled protein nanoparticles were fabricated from the recombinant MS1-mNeonGreen protein. Furthermore, the MS1-mNeonGreen protein was encapsulated into polymeric nanoparticles. The cytotoxic effects of these nanoparticles on cancer cells [cervical cancer cell line (HeLa), lung cancer cell line (A549) and breast cancer cell line (MDA-MB-231)] and healthy cell line [Human umbilical vein endothelial cells (HUVEC)] was analyzed by MTT test. The apoptotic effect of nanoparticles on these cells was investigated by determining the antiapoptotic and proapoptotic protein levels. As a result, it was revealed that the nanoparticles have a specific cytotoxic effect against the cells and induce apoptosis.
Dual targeting of HSP90 and BCL-2 in breast cancer cells using inhibitors BIIB021 and ABT-263
(Springer, 2025) Tosun, Nazan Goksen; Kaplan, Ozlem
Purpose:The incidence of breast cancer has been increasing in recent years, and monotherapy approaches are not sufficient alone in the treatment of breast cancer. In the combined therapy approach, combining two or three different agents in lower doses can mitigate the side effects on living cells and tissues caused by high doses of chemical agents used alone. ABT-263 (navitoclax), a clinically tested Bcl-2 family protein inhibitor, has shown limited success in clinical trials due to the development of resistance to monotherapy in breast cancer cells. This resistance shows that monotherapy approaches are inadequate and more effective treatment strategies are needed. It is the ability of HSP90 inhibitors to destabilize many oncoproteins that are critical for the survival of cancer cells. This study aimed to examine the anticancer activity of the combination of ABT-263 with BIIB021, a new generation HSP90 inhibitor, on two widely used breast cancer cell lines: MCF-7 (ER-positive) and MDA-MB-231 (triple-negative breast cancer, TNBC). These cell lines were selected to represent distinct breast cancer subtypes with different molecular characteristics and clinical behaviors.Methods: Single and combined cytotoxic effects of this agents on MCF-7 and MDA-MB-231 breast cancer cell lines were determined using the MTT cell viability test. The combined use of these two agents showed a synergistic effect, and this effect was assigned using the Chou and Talalay method. mRNA and protein levels of apoptosis-related genes Bax, Bcl-2, Casp9, and Heat Shock Proteins HSP27, HSP70, and HSP90 were analyzed using Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western Blotting, respectively.Results:The cytotoxicity analysis, combined with the application of the Chou-Talalay method, demonstrated that the BIIB021 and ABT-263 combination exhibited significantly greater anticancer activity compared to the individual effects of either BIIB021 or ABT-263 in breast cancer cell lines. The analysis of mRNA and protein levels indicated that the BIIB021+ABT-263 combination may have triggered the intrinsic apoptotic pathway in breast cancer cells.Conclusion: This study showed that co-administration of ABT-263 and BIIB021 agents exhibited synergistic cytotoxic effects and increased the expression of apoptosis-related genes in breast cancer cell lines
Efficacy of the greenly synthesized silver, copper, and nickel nanoparticles using Allium tuncelianum extract against Acanthamoeba castellanii
(Elsevier, 2023) Aykur, Mehmet; Tosun, Nazan Goksen; Kaplan, Ozlem; Ozgur, Aykut
Acanthamoeba is a common protozoan in many environments, leading to infection in humans and animals. Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE) are caused by Acanthamoeba. AK is an infection in the eye that can lead to vision loss and does not have a fully effective treatment. Therefore, there is an urgent need to develop new therapies against Acanthamoeba for the treatment of AK and GAE. Green nanotechnology synthesis approaches have been recently reported to be more environmentally friendly and effective in antimicrobial, antiviral, antifungal, and antiprotozoal activities. Therefore, they might be a good strategy for developing anti-Acanthamoeba substances. This study aimed to use the microwave-assisted method to prepare AgNPs, CuNPs, and NiNPs using the crude extract of Allium tuncelianum (AT). Moreover, the synthesized AT-AgNPs, AT-CuNPs, and AT-NiNPs were characterized using UV-Visible spectroscopy, DLS, and FTIR tech-niques. The first time anti-amoebic activity of AT-AgNPs, AT-CuNPs, and AT-NiNPs was evaluated against Acanthamoeba castellanii. Anti-amoebic activity as IC50 value of AT-AgNPs, AT-CuNPs, and AT-NiNPs was observed 1556.56 +/- 7.36 mu g/ml, 1826.44 +/- 17.84 mu g/ml, and 2014.23 +/- 7.04 mu g/ml after 24 h, respectively. After 24 h, AT-AgNPs were shown to be superior to other NPs in killing Acanthamoeba trophozoites at a 2000 mu g/ ml concentration. AT-AgNPs' IC50 value was determined to be effective against Acanthamoeba trophozoites at a concentration almost twice as low as PVP-I's IC50 value after 48 h. At doses of 500 mu g/ml, 1000 mu g/ml, and 2000 mu g/ml, the impact of AT-AgNPs on the viability of 50% Acanthamoeba trophozoites was assessed after 48 h. The conclusion of the present study demonstrates the most effective of AT-AgNPs among the nanoparticles when used against the treatment of infections caused by A. castellanii. These agents show the potential to create new, efficient, and secure treatment options.
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.
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.
Escherichia coli as a versatile cell factory: Advances and challenges in recombinant protein production
(Academic Press Inc Elsevier Science, 2024) Incir, Ibrahim; Kaplan, Ozlem
E. coli plays a substantial role in recombinant protein production. Its importance increased with the discovery of recombinant DNA technology and the subsequent production of the first recombinant insulin in E. coli. E. coli is a widely used and cost-effective host to produce recombinant proteins. It is also noteworthy that a significant portion of the approved therapeutic proteins have been produced in this organism. Despite these advantages, it has some disadvantages, such as toxicity and lack of eukaryotic post-translational modifications that can lead to the production of misfolded, insoluble, or dysfunctional proteins. This study focused on the challenges and engineering approaches for improved expression and solubility in recombinant protein production in E. coli. In this context, solution strategies such as strain and vector selection, codon usage, mRNA stability, expression conditions, translocation to the periplasmic region and addition of fusion tags in E. coli were discussed.
Escherichia coli in the production of biopharmaceuticals
(Wiley, 2024) Incir, Ibrahim; Kaplan, Ozlem
Escherichia coli has shouldered a massive workload with the discovery of recombinant DNA technology. A new era began in the biopharmaceutical industry with the production of insulin, the first recombinant protein, in E. coli and its use in treating diabetes. After insulin, many biopharmaceuticals produced from E. coli have been approved by the US Food and Drug Administration and the European Medicines Agency to treat various human diseases. Although E. coli has some disadvantages, such as lack of post-translational modifications and toxicity, it is an important host with advantages such as being a well-known bacterium in recombinant protein production, cheap, simple production system, and high yield. This study examined biopharmaceuticals produced and approved in E. coli under the headings of peptides, hormones, enzymes, fusion proteins, antibody fragments, vaccines, and other pharmaceuticals. The topics on which these biopharmaceuticals were approved for treating human diseases, when and by which company they were produced, and their use and development in the field are included.
Evaluation of Anticancer and Antimicrobial Potentials of Tarantula cubensis Venom (Theranekron® D6)
(2024) Tosun, Nazan Gökşen; Kaplan, Ozlem; Ozgur, Aykut
Tarantula cubensis venom (Theranekron®D6) is widely used in veterinary medicine as a drug with anti-tumor, wound healing, anti-inflammatory, antiphlogistic properties. The purpose of the study was to explore Theranekron®D6 (TD6)'s antibacterial activity and its impact on apoptotic cell death in human colon and liver cancer cell lines. TD6 showed a dose and time dependent cytotoxic effect at 12h and 24h in HT-29 and HUH-7 cancer cell lines. The IC50 values of TD6 were calculated as 12.18 µg/mL and 25.10 µg/mL in HT-29 and HUH-7 cell lines at 24h, respectively. TD6 induced apoptosis in HT-29 and HUH-7 cell lines. In these cells exposed to TD6, while the BAX/BCL-2 ratio and CASP-3 mRNA level increased, the HSP90 mRNA and protein level decreased. Also, TD6 exhibited antimicrobial activities against Pseudomonas aeruginosa, Enterococcus faecalis, Klebsiella pneumonia, Staphylococcus aureus, Candida albicans and Candida utilis. Obtained results demonstrated that TD6 has great potentials as alternative therapeutic for cancer and infectious diseases as apoptosis inducer and antimicrobial agent.
Evaluation of combined use of hsp90 inhibitor mpc-3100 and traditional cancer drug 5-fu on liver cancer cell lines
(2023) Kaplan, Ozlem
Hepatocellular carcinoma (HCC), which constitutes an important part of the global cancer burden, poses an important problem in the field of medicine. Combination therapy targets multiple mechanisms simultaneously using different therapeutic agents together. Heat shock protein 90 (HSP90) inhibitors are emerging as interesting targets in this area, since they play a vital role in the control of cellular processes and impact malignant cell survival and resistance mechanisms. This study evaluated the combined effect of the HSP90 inhibitor MPC-3100 and the traditional chemotherapy drug 5-fluorouracil (5-FU) on HCC. MTT assay was performed to evaluate the individual and combined cytotoxic effects of 5-FU and MPC-3100 on HUH-7 and HepG2 liver cancer cell lines. To assess the effectiveness of combination therapy, the Chou and Talalay method was applied. Both 5-FU and MPC-3100 and 5-FU+ MPC-3100 exhibited dose- and time-dependent cytotoxic effects. Combined administration of the two drugs showed an antagonistic impact on the cell lines. The findings demonstrated that combining 5-FU with MPC-3100 was less effective in inducing cytotoxicity in liver cancer cell lines compared to the use of each drug separately. In this context, the combination of these two drugs in liver cancer is not an appropriate strategy for effective treatment. Current research findings will help design more effective and targeted therapies for HCC and other cancers.
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.
Investigation of the Usability of Polymeric Nanoparticle-Based Transfection Agent for CRISPR/CAS9 in HUVEC Cell Line
(2025) Imamoglu, Rızvan; Kaplan, Ozlem; Gök, Mehmet Koray; Gokce, Isa
Genome editing technology is a promising, popular technology for diagnosing and treating many diseases. One of the most popular techniques utilized in genome editing research nowadays is the CRISPR/Cas system. The CRISPR/Cas system holds considerable potential for diagnostic and therapeutic applications due to its ease of use, speed, and affordability. The safe and effective delivery of the CRISPR-based genome editing system to the intended human body cells is one of the system's problems. This study investigated the silencing of the COX-2 gene via the CRISPR/Cas system using a P?AE-based transfection agent in the HUVEC cell line. Western blot analysis was performed to confirm COX-2 gene silencing in the HUVEC cell line, and the effects of COX-2 gene silencing on cell migration in these cells were examined. As a result, the transfection efficiency of the P?AE-based transfection agent for the HUVEC cell line was determined as 90-95%, and the CRISPR efficiency was calculated as 57.12% based on the Western blot results. It was revealed that the P?AE-based nanoparticles used in this study can be used as a good transfection agent in CRISPR/Cas-mediated genome editing in the HUVEC cell line, especially in plasmid transfection.
Molecular pathway of anticancer effect of next-generation HSP90 inhibitors XL-888 and Debio0932 in neuroblastoma cell line
(Humana Press Inc, 2024) Kaplan, Ozlem; Tosun, Nazan Goksen
Neuroblastoma is a common nervous system tumor in childhood, and current treatments are not adequate. HSP90 is a molecular chaperone protein that plays a critical role in the regulation of cancer-related proteins. HSP90 inhibition may exert anticancer effects by targeting cancer-related processes such as tumor growth, cell proliferation, metastasis, and apoptosis. Therefore, HSP90 inhibition is a promising strategy in the treatment of various types of cancer, and the development of next-generation inhibitors could potentially lead to more effective and safer treatments. XL-888 and Debio0932 is a next-generation HSP90 inhibitor and can inhibit the correct folding and stabilization of client proteins that cancer-associated HSP90 helps to fold correctly. In this study, we aimed to investigate the comprehensive molecular pathways of the anticancer activity of XL-888 and Debio0932 in human neuroblastoma cells SH-SY5Y. The cytotoxic effects of XL-888 and Debio0932 on the neuroblastoma cell line SH-SY5Y cells were evaluated by MTT assay. Then, the effect of these HSP90 inhibitors on the expression of important genes in cancer was revealed by Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) method. The qRT-PCR data were evaluated using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) biological process tools. Finally, the effect of HSP90 inhibitors on HSP27, HSP70 and HSP90 protein expression was investigated by Western blotting analysis. The results revealed that XL-888 and Debio0932 had a role in regulating many cancer-related pathways such as migration, invasion, metastasis, angiogenesis, and apoptosis in SH-SY5Y cells. In conclusion, it shows that HSP90 inhibitors can be considered as a promising candidate in the treatment of neuroblastoma and resistance to chemotherapy.
Recent progress in chitosan-based nanoparticles for drug delivery: a review on modifications and therapeutic potential
(Taylor & Francis Ltd, 2025) Bal, Kevser; Celik, Sibel Kucukertugrul; Senturk, Sema; Kaplan, Ozlem; Eker, Emine Busra; Gok, Mehmet Koray
Chitosan, obtained from chitin by deacetylation, is a versatile biopolymer known for its biocompatibility, biodegradability and environmental friendliness. Combined with its chemical and physical modifiability, these properties have made chitosan an important material in biomedical and pharmaceutical fields, especially in drug delivery systems. Chitosan-based nanomaterials exhibit enhanced functions through various chemical modifications such as thiolation, acetylation, carboxylation and phosphorylation, as well as through physical and enzymatic approaches. These modifications address inherent limitations such as poor solubility, limited acid resistance and insufficient mechanical strength, expanding the applications of chitosan in tissue engineering, gene therapy, vaccine delivery, wound healing and bioimaging. This review provides an in-depth analysis of the chemical structure, physicochemical properties and modification strategies of chitosan. It also explores current methodologies for preparing chitosan nanoparticles, along with drug loading and release techniques. Various targeting strategies employed in chitosan-based delivery systems are examined in detail. To illustrate the clinical relevance of these approaches, representative examples from recent therapeutic studies are included. Moreover, it highlights future research directions and the innovation potential of chitosan-based materials.
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.
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.
Soluble Expression of Human Granulocyte Colony Stimulating Factor (hG-CSF) in Escherichia coli Expression System
(2025) Songur, Mustafa; Kaplan, Ozlem; Imamoglu, Rızvan; Gokce, Isa
Human granulocyte colony stimulating factor (hG-CSF) is a hematological growth factor that plays a crucial role in neutrophil production and differentiation. Some foreign biomolecules, especially of human origin, such as hG-CSF, sometimes aggregate because of different factors during expression and create inclusion bodies in Escherichia coli (E. coli). Refolding process is commonly used to recover these very valuable molecules, but still significant amounts of protein remain unusable. Refolding procedures are frequently costly, time-consuming, and not fully efficient. Therefore, the use of molecular chaperones to improve soluble expression of proteins was evaluated in the study. In this context, hG-CSF was co-expressed with five chaperone plasmid systems (pGro7, pG-KJE8, pG-Tf2, pKJE7, pTf16) to ensure the expression of hG-CSF in soluble form. Among these, the pKJE7 plasmid was found to be the most effective in obtaining hG-CSF in soluble form, yielding 92% purity after Ni-NTA affinity chromatography purification. The total yield of hG-CSF obtained was 1.6 mg per 1 L bacterial culture. The biological activity of the soluble hG-CSF was evaluated in human umbilical vein endothelial cells (HUVECs). A 24-hour interaction of hG-CSF with HUVECs resulted in a significant increase in cell viability at all applied doses, demonstrating its bioactivity. As a result, hG-CSF, which previously aggregated as an inclusion body in the E. coli expression system, was correctly folded by co-expression with chaperone proteins were obtained as more efficient and purer.
Synergistic induction of apoptosis in liver cancer cells: exploring the combined potential of doxorubicin and XL-888
(Humana Press Inc, 2023) Kaplan, Ozlem
Combination therapy has been frequently preferred in treating various types of cancer in recent years. Targeted cancer therapy has also become one of the remarkable treatment modalities. Therefore, the aim of the study to investigate its cytotoxic and apoptotic effects on liver cancer cell lines by combining the classical chemotherapeutic drug doxorubicin (DOX) and a targeted agent, the new generation HSP90 inhibitor XL-888. The molecular docking method was used to predict the binding conformation of XL-888 on the human Hsp90. The single and combined cytotoxic effects of DOX and XL-888 on liver cancer cell lines HepG2 and HUH-7 were determined by MTT assay. The effect of the combined use of two drugs was evaluated using Chou and Talalay method. The levels of apoptotic genes and heat shock proteins gene and protein expression levels were investigated by quantitative real-time polymerase chain reaction and western blotting, respectively. Molecular docking results showed that XL-888 selectively binds to the ATP binding pocket of HSP90 with an estimated free binding energy of - 7.8 kcal/mol. DOX and XL-888 and their combination showed dose-dependent cytotoxic effect. The combination of drugs showed a synergistic effect on both cell lines. The results revealed that the combination of DOX and XL-888 potently induced apoptosis in liver cancer cell lines rather than using drugs alone. The combined treatment of DOX and XL-888 demonstrated synergistic cytotoxic and apoptotic effects on liver cancer cell lines, presenting a promising approach for combination therapy in liver cancer.
Thiolated ?-cyclodextrin: The likely smallest drug carrier providing enhanced cellular uptake and endosomal escape
(Elsevier Sci Ltd, 2023) Kaplan, Ozlem; Truszkowska, Martyna; Kali, Gergely; Knoll, Patrick; Massani, Mariana Blanco; Braun, Doris Elfriede; Bernkop-Schnuerch, Andreas
This study aimed to evaluate the effect of thiolated & alpha;-cyclodextrin (& alpha;-CD-SH) on the cellular uptake of its payload.For this purpose, & alpha;-CD was thiolated using phosphorous pentasulfide. Thiolated & alpha;-CD was characterized by FT -IR and 1H NMR spectroscopy, differential scanning calorimetry (DSC), and powder X-ray diffractometry (PXRD). Cytotoxicity of & alpha;-CD-SH was evaluated on Caco-2, HEK 293, and MC3T3 cells. Dilauryl fluorescein (DLF) and coumarin-6 (Cou) serving as surrogates for a pharmaceutical payload were incorporated in & alpha;-CD-SH, and cellular uptake was analyzed by flow cytometry and confocal microscopy. Endosomal escape was investigated by confocal microscopy and hemolysis assay.Results showed no cytotoxic effect within 3 h, while dose-dependent cytotoxicity was observed within 24 h. The cellular uptake of DLF and Cou was up to 20-and 11-fold enhanced by & alpha;-CD-SH compared to native & alpha;-CD, respectively. Furthermore, & alpha;-CD-SH provided an endosomal escape.According to these results, & alpha;-CD-SH is a promising carrier to shuttle drugs into the cytoplasm of target cells.

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