Cell and Tissue Journal

Abstract Introduction: Concern over fossil energy costs and environmental deterioration, along with energy security, has created a strong motivation for research and development of routes to provide sustainable, renewable fuels. In recent years, the use of biomass to produce highly valued chemicals has attracted widespread attention. lignocellulosic biomass, as a promising renewable resource for biofuel production, has distinct advantages in terms of economic and environmental aspects. The conversion of renewable raw materials to hydrocarbon fuels is an attractive alternative to fossil fuels from an economic and environmental point of view. The production process of lignocellulosic biomass mainly consists of biomass accumulation, biomass decomposition, simple sugars, and conversion of sugars to biofuel. One of the crucial steps in the economic success of lignocellulosic biofuels depends on the inhibition of competitive metabolism in microorganisms to achieve high productivity. To date, a growing focus is on the use of S. cerevisiae and E. coli as cell lines. These two cellular factories have the benefits that are well known.

Aim: The organic compound D-1,2,4-Butanetriol is a valuable chemical with wide-ranging applications in various fields. The chemical synthesis routes for BT have many drawbacks. By genetically modifying microorganisms, the metabolic pathway for producing many substances, including BT, can be engineered. The organic compound D-1,2,4-butanediol (BT) is an important intermediate chemical widely used in fields such as pharmaceuticals, paper, polymer materials, and military applications. When D-xylose sugar is provided to the bacterium, it is first converted to an intermediate compound called xylonolactone. This compound itself slowly converts into xylonate through a non-enzymatic reaction. To produce xylonate, the engineered bacteria have received xylose and initially, by a dehydrogenase reaction by the xylose dehydrogenase enzyme, that converts it into an intermediate substance: xylonolactone. The xylonolactone is converted slowly and in a nonenzymatic reaction to xylonate. Xylonate is a five-carbon organic acid. Over the past few years, xylonate has been increasingly being considered as an important chemical due to its potential as an important chemical component. Xylonate has many applications that can be used in the food, chemical, and pharmaceutical industries. Specifically, xylonate can act as a precursor for the synthesis of D-1,2,4-Butanetriol and a decrease in concrete water. E.coli due to fast growth in cheap culture medium, having two enzymes for the bto synthesis and product production in lower 24 hours of fermentation was chosen as the target strain of genetic engineering and metabolism. This study aimed to clone and express xylose dehydrogenase from Caulobacter vibrioides in E.coli. Materials and Methods: At first, to access the bacterial gene sequence, the genome of the target bacterium was extracted. Then, to create a strain expressing the enzymes xylose dehydrogenase and xylonolactonase, the genes for these proteins were amplified from Caulobacter vibrioides CB1 and transferred to E. coli. For this purpose, the target genes were amplified using specifically designed primers via the Polymerase Chain Reaction (PCR) method and initially cloned into a pTZ57cloning vector and then subcloned into pET 26b expression vector. At the final step, the expression of the enzyme was assessed by SDS-PAGE, and the other confirmation was the reduction of NAD+ to NADH, which was used as an activity indicator of the enzyme, as investigated by a change in NADH absorbance at 340 nm. Results: Confirmatory tests were performed to ensure the presence of the gene in the vectors (using restriction enzymes and colony PCR for gene amplification). The expression and activity of the enzyme were analysed. The recombinant protein's presence was confirmed by SDS-PAGE for the xylose dehydrogenase gene, with a molecular weight of 52.2 kDa. The estimated recombinant protein expression levels were approximately 25%. Conclusion: The objective of this research was solely to establish the metabolic pathway for xylonate production in E. coli by surface expression of enzymes in this pathway (xylose dehydrogenase). The results obtained in this study confirm that half of the pathway is active at the cell surface, but further experiments are required to determine the precise production levels and complete the pathway. This study aimed to create a metabolic pathway for producing xylonate in E.coli.

Abstract Introduction: Gene therapy involves transferring genetic material into target cells to correct mutations or introduce new biological functions. Among delivery systems, lentiviral vectors are considered efficient and reliable tools due to their ability to integrate stably into the host genome and transduce both dividing and non-dividing cells. This property provides long-term gene expression, which is highly valuable for therapeutic and experimental applications. The PDX1 (Pancreatic and Duodenal Homeobox 1) gene plays a central role in pancreatic organogenesis and the regulation of insulin-producing beta cells. It acts as a transcription factor controlling genes critical for endocrine differentiation and insulin secretion. Chick embryos are a useful experimental model due to their accessibility, rapid development, and the responsiveness of their fibroblast and germ cells to gene transfer systems. These features make them suitable for studying gene delivery efficiency and expression stability.
Aim: The aim of this study was to construct and produce recombinant lentiviral vectors carrying the PDX1 gene in HEK293T-LentiX cells and evaluate their transfer efficiency in chick embryonic fibroblast and germ cells. This work was conducted to assess the potential of lentiviral systems for stable gene delivery in avian cells.
Materials and Methods: HEK293T-LentiX cells were selected as producer cells due to their high transfection efficiency and viral packaging capability. They were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin under standard incubation conditions (37°C, 5% CO₂). Lentiviral particles were generated using a three-plasmid packaging system, including a transfer vector containing the PDX1 gene and two helper plasmids. Transfection was carried out using the calcium phosphate method. After 48–72 hours, the viral-containing supernatant was collected, filtered, and concentrated. Viral titers were determined by evaluating GFP expression in target cells through fluorescence microscopy and flow cytometry. Chick embryonic fibroblast and primordial germ cells were isolated and infected with various viral concentrations in the presence of 8 µg/ml polybrene to enhance infection. After incubation, cells were examined for GFP signal as evidence of successful gene transfer.
Results: High-titer recombinant lentiviral particles were successfully produced in HEK293T cells. Fluorescence microscopy revealed strong GFP expression, confirming the presence of functional viral particles. Flow cytometry analysis provided quantitative confirmation of high viral titers. Following transduction, chick embryonic fibroblast and germ cells exhibited clear GFP expression, indicating efficient infection and gene transfer. The PDX1 gene was successfully delivered and expressed within target cells. Although transduction efficiency varied slightly between cell types, the overall results demonstrated that the lentiviral system provided stable and effective gene delivery to chick embryo-derived cells.
Discussion: The study confirmed that lentiviral vectors carrying the PDX1 gene could be efficiently produced and used to achieve stable gene transfer in chick embryonic cells. This system’s ability to integrate permanently into the host genome ensures consistent gene expression over time without repeated transfection. For functional genes like PDX1, this stability is crucial for maintaining insulin-related pathways and pancreatic cell differentiation. Chick embryos serve as an advantageous model because their cells are easily accessible, grow rapidly, and respond well to viral vectors. Such characteristics make them ideal for investigating genetic regulation during early development. Evaluation of viral titers using fluorescence microscopy and flow cytometry provided reliable data confirming efficient vector production. The integration of new tools such as CRISPR/Cas9 can further enhance lentiviral design precision, allowing targeted modification of specific genes. Combining these technologies may open promising avenues for studying metabolic disorders and for gene-based therapies.
Conclusion: Recombinant lentiviral vectors carrying the PDX1 gene were successfully generated and used to transduce chick embryonic fibroblast and germ cells. The system exhibited high production efficiency, stable gene expression, and suitability for in vitro studies. These findings demonstrate that lentiviral vectors represent a powerful and versatile platform for gene transfer and experimental modeling in avian systems. Moreover, coupling lentiviral vectors with genome editing technologies could expand future applications in regenerative medicine and genetic engineering.

Abstract Introduction: Cancer is one of the most significant diseases of the present century and ranks as the second leading cause of death worldwide. Its incidence, particularly breast cancer in women, is steadily increasing. This disease arises from uncontrolled cell proliferation and the ability of cancer cells to migrate to healthy tissues, leading to tumor formation and metastasis. The genetic and biological heterogeneity of cancer cells, especially in breast cancer, complicates effective treatment. Conventional therapies such as surgery, chemotherapy, and radiotherapy, although effective in reducing tumor size, are associated with limitations, including damage to healthy cells, drug resistance, and systemic side effects. Therefore, the development of targeted and innovative therapeutic strategies is a priority in cancer research. One promising approach is the use of nanoparticles for targeted drug delivery. Due to their small size, high surface-to-volume ratio, and modifiable surfaces, nanoparticles can efficiently carry drugs and deliver them to target cells. Controlled drug release from nanoparticles reduces uptake by healthy cells and minimizes systemic toxicity. Iron oxide nanoparticles (Fe₃O₄) possess unique magnetic and chemical properties that enable precise guidance to tumor sites and allow real-time monitoring of drug distribution via MRI. Surface modification with biocompatible polymers such as polyethylene glycol (PEG) enhances nanoparticle stability, prolongs systemic circulation, reduces immune clearance, and provides sites for conjugating targeting ligands such as folic acid and glucose. Polylactic acid (PLA), a biodegradable and biocompatible polymer, increases drug-loading capacity and enables sustained and controlled drug release. Combining PLA with PEG and targeting ligands creates multifunctional nanoparticles that are stable, biocompatible, and capable of selectively recognizing cancer cells while minimizing side effects on healthy tissues. Targeting ligands such as folic acid and glucose facilitate selective cellular uptake; folic acid binds to overexpressed receptors on many cancer cells, promoting intracellular drug delivery, while glucose exploits the high metabolic demand of cancer cells, improving the delivery of therapeutic agents or genes.
Aims: Dual-ligand nanoparticles enable multi-pathway targeting, enhancing therapeutic efficacy and potentially overcoming drug resistance. In this study, multifunctional PLA-PEG nanoparticles functionalized with folic acid and glucose and incorporating Fe₃O₄ were developed for targeted drug delivery to triple-negative breast cancer cells (Hs-578T). This system combines magnetic guidance, biocompatibility, controlled drug release, and selective targeting, providing a promising platform for effective and safe breast cancer treatment with reduced side effects compared to conventional therapies.
Materials and Methods: Various chemicals and reagents, including MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)), Ethanol, HCl, NaCl, KH₂PO₄, Chloroform, NH2-PEG-FA, NH2-PEG-Glu, NaOH, and paraformaldehyde were procured from commercial suppliers, while FBS, DMEM, and trypsin-EDTA were used for in vitro studies with Hs-578T breast cancer cells. Multifunctional nanoparticles PPF (PLA-PEG-FA), PPG (PLA-PEG-Glu), and PPGF (PLA-PEG-Glu/FA) were synthesized via the reaction of PLA-acrylate with the respective NH2-PEG derivatives in chloroform under mechanical stirring and mild heating, followed by purification through dialysis and freeze-drying. Methanolic extract of Silybum marianum was prepared by ultrasonication of powdered aerial parts in 80% methanol, followed by centrifugation and filtration. Fe₃O₄ nanoparticles were synthesized via co-precipitation of Fe (III) and Fe (II) salts under nitrogen, followed by gradual addition of Silybum marianum plant extract and NaOH, leading to nanoparticle formation. The particles were collected magnetically, washed, freeze-dried, and stored in dark, dry conditions. Targeted delivery of Curcumin (CUR) was achieved using folic acid and glucose ligands. CUR and Fe₃O₄-OA were incorporated into PPF, PPG, or PPGF matrices using sonication and emulsification in PVA solutions, followed by rotary evaporation to remove chloroform, washing, and filtration to remove unencapsulated drug and large particles. Structural and chemical composition were analyzed using 1H-NMR and FTIR spectroscopy. Morphology was observed via TEM, and average size and zeta potential were measured using dynamic light scattering (DLS). Drug encapsulation efficiency was determined spectrophotometrically by measuring unencapsulated CUR in the supernatant. CUR release kinetics were evaluated in two pH environments: pH=7.4 (normal cells), and pH=4.5 (tumor-like), with periodic sampling and replacement with fresh medium over three days. Cytotoxicity was assessed via the MTT assay, with IC₅₀ values determined after treating Hs-578T cells with varying concentrations of CUR, nanoparticles with CUR, or blank nanoparticles. Apoptosis was analyzed using Annexin V/propidium iodide staining and flow cytometry to distinguish apoptotic and necrotic cells. All experiments were performed in triplicate. Data were analyzed using one-way ANOVA followed by Duncan’s test at a 5% significance level, and normality was verified with the Kolmogorov–Smirnov test. Results are reported as mean ± standard deviation.
Results: Targeted drug delivery has emerged as a promising strategy for treating difficult-to-treat diseases, including cancer, by directing therapeutic agents specifically to diseased tissues while minimizing uptake by normal cells. Biocompatible and biodegradable polymers are essential for efficient delivery systems. PLA, a widely used biodegradable polymer, allows loading and controlled release of hydrophobic drugs like curcumin. However, PLA alone can aggregate in serum due to hydrophobic interactions, leading to immune recognition and clearance. To overcome this, PLA was conjugated with PEG, which enhances nanoparticle circulation time and provides a stealth effect, reducing recognition by the immune system. PEG also improves drug loading and stability, although it is not biodegradable and may accumulate over repeated doses. Magnetic Fe₃O₄ nanoparticles were incorporated to increase targeting efficiency, while folic acid and glucose ligands were used for selective recognition of cancer cells. The synthesized PLA-based nanoparticles demonstrated good biocompatibility with Hs-578T cells, showing no significant cytotoxicity. Drug encapsulation within PLA-PEG nanoparticles allowed controlled and sustained release of curcumin, which was particularly enhanced under acidic conditions mimicking the tumor microenvironment. Notably, nanoparticles containing both folic acid and PEG showed higher drug release compared to those with PEG alone, highlighting the role of folic acid in facilitating curcumin release. This pH-sensitive release profile minimizes drug exposure to normal tissues, potentially reducing side effects. Cytotoxicity assay revealed that curcumin-loaded nanoparticles significantly inhibited Hs-578T cell proliferation compared to free curcumin, with the dual-ligand nanoparticles exhibiting the lowest IC₅₀ values. Flow cytometry analysis demonstrated that these nanoparticles primarily induced apoptosis rather than necrosis, suggesting selective activation of programmed cell death pathways. The enhanced apoptotic effect is likely due to increased cellular uptake and targeted delivery, which facilitates higher intracellular concentrations of curcumin.
Discussion: Overall, the results indicate that PLA-PEG-based nanoparticles, functionalized with magnetic Fe₃O₄ and surface ligands, provide an effective platform for targeted curcumin delivery. These nanoparticles combine biocompatibility, controlled release, tumor-specific accumulation, and apoptosis induction, which collectively enhance therapeutic efficacy while minimizing systemic toxicity. The study underscores the potential of multifunctional nanocarriers as a promising approach for improving the effectiveness of anticancer therapies and offers a foundation for the further development of targeted, stimuli-responsive drug delivery systems.
Conclusion: As a final conclusion of this study, it can be stated that the development of novel drug delivery systems based on PLA-PEG copolymer nanoparticles is a promising and strategic step toward overcoming the therapeutic challenges of intractable diseases such as cancer. This study clearly demonstrated that the design of such nanocarriers not only offers high biocompatibility and safety for normal cells, but also, by taking advantage of the camouflage effect caused by PEG, increases blood circulation time and prevents premature elimination by the immune system. The outstanding feature of this system is the ability to control and intelligently release the drug in response to specific stimuli of the tumor microenvironment, especially higher acidity, which allows for maximum drug delivery to the target tissue and, subsequently, a significant reduction in systemic side effects. In this study, the effect of adding targeting ligands such as folic acid and glucose on the accuracy and efficiency of these nanoparticles in recognizing and binding to cancer cells was also clearly observed. In addition, the results of this study showed that the designed nanoparticles were successfully able to increase the induction of cell apoptosis in Hs-578T cancer cells. These findings strongly support the superiority of this nanotechnology compared to conventional drug formulations, both in increasing therapeutic efficacy and reducing toxicity. Overall, it can be concluded that PLA-PEG-based smart nanoparticles, with their multimodal capabilities, have great potential to become a comprehensive and reliable approach in the next generation of targeted cancer therapies, paving the way for further studies and ultimately effective clinical applications.