Purpose Genetic diseases can be the result of genetic dysfunctions that happen due to some inhibitory and/or environmental risk factors, which are mostly called mutations. One of the most promising treatments for these diseases is correcting the faulty gene. Gene delivery systems are an important issue in improving the gene therapy efficiency. Therefore, the main purpose of this study was modifying graphene oxide nanoparticles by spermine in order to optimize the gene delivery system. Methods Graphene oxide/APTES was modified by spermine (GOAS) and characterized by FT-IR, DLS, SEM and AFM techniques. Then pEGFP-p53 was loaded on GOAS, transfected into cells and evaluated by fluorescent microscopy and gene expression techniques. Results FT-IR data approved the GOAS sheet formation. Ninety percent of the particles were less than 56 nm based on DLS analysis. SEM analysis indicated that the sheets were dispersed with no aggregation. AFM results confirmed the dispersed structures with thickness of 1.25±0.87 nm. STA analysis showed that GOAS started to decompose from 400°C and was very unstable during the heating process. The first weight loss up to 200°C was due to the evaporation of absorbed water, the second one observed in the range of 200–550°C was assigned to the decomposition of labile oxygen- and nitrogen-containing functional groups, and the third one above 550°C was attributed to the removal of oxygen functionalities. In vitro release of DNA demonstrated the efficient activity of the new synthesized system. Ninety percent of the cells were transfected and showed the GFP under fluorescence microscopy, and TP53 gene was expressed 51-fold in BT-20 cells compared to β-actin as the reference gene. Flow cytometry analysis confirmed the apoptosis of the cells rather than necrosis. Conclusion It could be concluded that the new synthesized structure could transfer a high amount of the therapeutic agent into cells with best activity.

Graphical abstract Schematic representation of experimental design in different groups of mothers and offspring. CTL group: control group; Veh group: vehicle group received normal saline; Ni group: nicotine group received 1 mg/kg/day nicotine, subcutaneously; EtOH group: ethanol group received 3 g/kg/day, 20 % v/v ethanol intraperitoneally; Ni + EtOH group: ethanol plus nicotine which received both.

Purpose: Human butyrylcholinesterase (BChE) serves as a bio scavenger to counteract organophosphate poisoning. It is also a potential drug candidate in several therapeutic fields. Therefore, in the present study, we constructed a new dual-promoter plasmid consisting of Cytomegalovirus (CMV) and human elongation factor 1α (EF-1α) promoters and transfected that into HEK-293 cells using Lipofectamine to enhance the BChE secretion. Methods: The new dual-promoter construction (pBudCE dual BChE) including two copies of the BChE gene was designed and transfected into cells by liposomal structures. The cloned plasmids were evaluated by enzyme digestion and gel electrophoresis analysis. Experimental groups were categorized into the cells transfected by pBudCE dual BChE (treatment), pCMV (positive control) vectors, and nontransfected cells (negative control). BChE gene expression was evaluated by qRT-PCR and the enzyme activity was assessed using modified Ellman's method. The freeze-thaw process was carried out for analyzing the stability of the pBudCE dual BChE vector. Results: Validation examination of the cloned plasmids confirmed the successful cloning process. The gene expression level and Ellman's method value in pBudCE dual BChE was higher than the other groups. CMV promoter has also increased the enzyme activity, although the difference was not significant compared with the control group. Interestingly, freeze-thaw cycles followed by several passages did not affect the enzyme activity. Conclusion: The designed construction with CMV and EF-1α promoters could increase BChE gene expression and the activity of the BChE enzyme in HEK-293 cell line. Largescale production of BChE enzyme can be achieved by using dual-promoter plasmid construction compared to a single-promoter vector to be used in clinical trials.

BACKGROUND: Butyrylcholineesterase (BChE) is a therapeutic drug and its producing as a recombinant protein is an essential issue in biotechnology. One of the highlights in this regard is choosing the best host cells and plasmids. OBJECTIVES: The aim of this study is to evaluate the production of butyrylcholinesterase in Vero, HEK-293, and CHO cell lines using a dual promoter vector. MATERIAL AND METHODS: The dual-promoter construction (pBudCE dual BChE) was transfected into cell lines categorized in three experimental groups (pBudCE dual BChE, pCMV and negative control). BChE gene expression and enzyme activity was evaluated at different times. RESULTS: All three cell lines showed higher gene expression level in pBudCE dual BChE group. BChE enzyme activity level of this group in CHO cells decreased in sixth day and increased in ninth day. In HEK-293 cells it has a downward trend from sixth to ninth day and in Vero cells its level in the ninth day was the highest. CONCLUSION: The difference of pBudCE dual BChE and pCMV groups was more pronounced in the HEK-293 cell and the BChE gene expression level of this cells was higher than the others while, CHO cells showed higher level of BChE enzyme activity.

Green LED and three‐dimensional (3D) scaffolds have recently received extensive attentions due to their impact on cell proliferation and differentiation. Melatonin, a circadian rhythm‐regulating hormone, is involved in some physiological phenomena including testosterone biosynthesis. Lower testosterone biosynthesis results in some disorders such as puberty retarding, andropause, and muscle weakness. Therefore, our aim was to investigate the proliferation of Leydig cells and their testosterone‐related Gene expression and secretion under the influence of 3D scaffold, green light and melatonin. The experimental groups of TM3 cells embedded in the 3D scaffold, were exposed to green light, melatonin, both and all three factors. Expression of cell cycle genes including PCNA, CYCLIND1, CDC2 and CDKN1B, and testosterone related genes; GATA4 and RORα were also examined. 3D scaffold enhanced Leydig cells proliferation, and testosterone‐related genes expression. While melatonin decreased cell proliferation and testosterone‐related genes expression. Green light did not significantly change the results but slightly decreased cell proliferation and testosterone synthesis. The combination of green light with melatonin significantly reduced the proliferation rate of TM3 cells and the expression of steroidogenic genes, while the combination of green light with scaffold improved the results. In general, the use of scaffolding enhances proliferation and testosterone‐related genes expression of TM3 Leydig cells. Also, application of green light and scaffolding reduces the deleterious effects of melatonin on these cells.