Evaluation of cell penetrating peptide delivery system on HPV16E7 expression in three types of cell line

Background: the poor permeability of the plasma and nuclear membranes to DNA plasmids are two major barriers for the development of these therapeutic molecules. Therefore, success in gene therapy approaches depends on the development of efficient and safe non-viral delivery systems

Objectives: the aim of this study was to investigate the in vitro delivery of plasmid DNA encoding HPV16 E7 gene using cell penetrating peptide delivery system to achieve the best conditions for cell transfection and protein expression. For this purpose, we have used a cationic peptide delivery system, MPG which forms stable non-covalent complexes with nucleic acids for delivery of pEGFP-E7 as a model antigen in vitro

Materials and Methods: DNA construct encoding HPV16 E7 [pEGFP-E7] was prepared in large scale with high purity. MPG peptide/ DNA complexes were prepared at different N/P [nitrogen/phosphate] ratios and physicochemical characterization and stability of nanoparticles were investigated. In vitro peptide-mediated E7-GFP DNA transfection, and its expression was evaluated in three cell types. To quantify the transfection efficiency of this delivery system, transfected cells were harvested and assessed for GFP-positive cells by flow cytometry. Furthermore, E7-GFP expression was confirmed by western blot analysis

Results: the cellular uptake of MPG based nanoparticles was shown to be comparable with standard reagent PEI. The COS-7 cells transfected by MPG-based nanoparticles at an N/P ratio of 15:1 showed the highest transfection efficiency and gene expression

Conclusions: the results indicated that the efficient gene expression depends on both cell type and N/P ratio applied, in vitro. The efficient protein expression detected by western blotting and flow cytometry supports the potential of MPGbased nanoparticles as a potent gene delivery system

Targeted delivery of 5-fluorouracil with monoclonal antibody modified bovine serum albumin nanoparticles

Herein, 1F2, an anti-HER2 monoclonal antibody [mAb], was covalently coupled to the surface of 5-Fluorouracil [5-FU] loaded bovine serum albumin [BSA] nanoparticles. Concerning two different crosslinkers for conjugation of 1F2, Maleimide-poly [ethylene glycol]-Succinimidyl carbonate [Mal-PEG5000-NHS] was selected due to its higher conjugation efficiency [23 +/- 4%] obtained in comparison to N-succinimidyl 3-[2-Pyridyl Dithio] Propionate [SPDP] [8 +/- 2%]. A slight increase in the average particle size with a negligible prolongation of the 5-FU release was observed after 1F2 coupling. The 1F2-coupled 5-FU-loaded BSA nanoparticles interacted with nearly all HER2 receptors available on the surface of HER2-positive SKBR3 cells. No cellular uptake was observed for HER2-negative MCF7 cells. Physicochemical and biological properties of the mAb-modified nanoparticles did not significantly alter after three months of storage at room temperature. The in-vitro cytotoxicity evaluation by MTT assay, demonstrated lower SKBR3 viability [50.7 +/- 9 %] after 5 hours contact with 1F2-coupled 5-FU-loaded BSA nanoparticles in comparison with the other control systems due to their cell attachment and internalization after washing. In addition, no significant toxicity was observed on MCF7 cells. This novel system can efficiently be employed for targeted delivery of 5-FU to HER2-positive cancerous cells

Maximizing production of human interferon- gamma in HCDC of recombinant E. coli

Tuning recombinant protein expression is an approach which can be successfully employed for increasing the yield of recombinant protein production in high cell density cultures. On the other hand, most of the previous results reported the optimization induction conditions during batch and continuous culture of recombinant E. coli, and consequently fed-batch culture have received less attention. Hence, in this research induction conditions for the over-production of recombinant interferon- gamma including the amount of inducer, induction time and post-induction duration during chemical induction were optimized. E. coli BL21 [DE3] [pET3a-hifn gamma] was used to over-express human interferon-gamma [hIFN- gamma] in an exponential fed-batch procedure with a maximum attainable specific growth rate of 0.55 h[-1] at the beginning of feeding and 0.4 h[-1] in induction time. The factors were considered as the amount of inducer [IPTG] in the range of 0.565- 22 mg g[-1] L[-1] at seven levels, cell density at induction time as 53, 65 and 75 g [dry cell weight] L[-1], induction duration at different intervals of 3, 4, and 5 h after induction time. The final concentration of biomass and interferon gamma reached to 127 g L[-1] [DCW] and 51 g [hIFN-gamma] L[-1] after 17 h, and also the final specific yield and overall productivity were obtained 0.4 g [hIFN- gamma] g[-1] DCW and 3 g [hIFN- gamma] L[-1] h[-1], respectively, which are the highest amounts of reported specific yield and productivity for recombinant proteins production

Mathematical models for microbial kinetics in solid-state fermentation: a review

In this review, we discuss empirical and stoichiometric models, which have been developed recently in SSF processes and the influence of environmental conditions on the variables of these models. Additionally, new studies on modeling of product formation are also mentioned. Solid-state fermentation [SSF] is recognized as a cheap process for producing many valuable products like industrial enzymes and bioethanol. To develop, optimize, and scale-up this process, mathematical models are required. In this review, we collected all the papers regarding microbial growth and product formation modeling in SSF. The pros and cons of each model and confirmation with experimental data were also discussed. We discussed here the simple empirical growth kinetics models and the effect of environmental conditions on these models parameters, stoichiometric models and product formation models. Simple empirical models are used widely in the kinetic modeling of SSF processes due to their simplicity and ease of use. However, more studies should be done in this field to make them more accurate, especially; the effect of environmental conditions, like temperature and moisture, on key variables of the model must be considered. Robust modeling methods, like stoichiometric models, are in their early stages in SSF processes and require more studies. Developing models in which transport phenomena models are coupled with the growth kinetics models can help better SSF bioreactor designing. On the other hand, to use SSF for producing valuable products, product formation models, which are not developed well in SSF processes, are necessary. To use SSF for producing valuable metabolites in large scales, more attention is required for modeling the SSF processes, especially for product formation models and using modern methods like stoichiometric models.

Comparative study on silver nanoparticles properties produced by green methods

In the present study, properties of silver nanoparticles [AgNPs] such as average size, size distribution and morphology were investigated by Tollens, polysaccharide, modified polysaccharide and microbial methods. The synthesized AgNPs were characterized by UV-visible spectroscopy, scanning electron microscopy [SEM], transmission electron microscopy [TEM], dynamics light scattering [DLS] and energy dispersive X-ray [EDX] analyses. Analysis of reaction mixtures confirmed that Tollens, polysaccharide and modified polysaccharide methods generated smaller AgNPs with better size distribution as compared to that produced in microbial method. The average size of produced AgNPs by Tollens, polysaccharide and modified polysaccharide were 42, 30 and 20 nm respectively. Moreover, microbial method generated AgNPs with average size of 54 nm in the case of cell-free filtrate mediated synthesis and 84 nm in case of the supernatant mediated synthesis. Analysis of fungus-mediated synthesis of AgNPs showed that the size distribution of AgNPs produced by supernatant is narrower than that produced by filtrate. Also, cell-free filtrate resulted in the formation of smaller AgNPs with average size of 59 nm compared to the supernatant. The comparative analysis of produced AgNPs by the above mentioned methods confirmed that modified polysaccharide method led to the formation of AgNPs with smallest size and highest productivity

Evaluation of Ca-independent alpha-amylase production by Bacillus sp. KR-8104 in submerged and solid state fermentation systems

This study investigates the production of crude Caindependent and low pH active alpha-amylase by Bacillus sp. KR-8104 in submerged fermentation [SmF] and solid-state fermentation [SSF] systems. Different parameters were evaluated in each system using "one factor at a time" approach to improve the production of enzyme. The results showed that in the SmF the maximum enzyme production was achieved in culture medium that contained dextrin as a carbon source, as well as yeast extract and meat extract as nitrogen sources incubated at 37§C and 180 rpm for 48 h. While SSF of Bacillus sp. KR-8104 using wheat bran [WB] as a substrate showed that using tap water or distilled water as a moisturizing agent, a substrate-water ratio of 1:1.5 [w/v] and incubation at 37§C for 48 h gave the maximum alpha-amylase production. From different extraction medium examined in this study 0.1% [v/v] aqueous mixture of Tween 20 and distilled water illustrated maximum results [100 U/g]

[Bacterial overexpression of the human interleukin-2 in insoluble form via the pET Trx fusion system]

Selection of a system for successful recombinant protein production is important. The aim of this study was to produce high levels of human interleukin-2 [hIL-2] in soluble form. To this end, the pET32a vector in Escherichia coli BL21 [DE3] was used as an expression system, since it was previously used for the production of mouse IL-2 in soluble form. The results indicated that contrary to expectations, the expressed protein was in the form of inclusion bodies and perhaps amino acid differences between human and mouse IL-2 should be determinant. The hIL-2 protein is a small peptide, therefore its recovery as a biologically functional protein by the process of refolding may be feasible and could lead to high yields at the industrial scale

Production of recombinant human granulocyte-colony stimulating factor by Pichia pastoris

Human granulocyte-colony stimulating factor [hGCSF] cDNA was expressed in the methylotrophic yeast Pichia pastoris under the control of the alcohol oxidase [AOX1] promoter. An expression vector for hG-CSF secretion was constructed using pPIC9 vector. Higher levels of hG-CSF was obtained using a P. pastoris Mut+ [methanol utilization fast] phenotype. The effects of environmental factors such as, temperature and pH on the P. pastoris cell growth and hGCSF production during fermentation were investigated. Cell growth and hG-CSF production were found to be optimal at 28C and pH 6.0. A fed-batch fermentation process was also developed to obtain high cell density and higher levels of protein expression. Using a high cell density cultivation method, cell dry weight and hGCSF concentration reached 100 g/l and 35 mg/l, respectively

Modeling of single cell protein production from cheese why using tanks-in-series model

In this work, mathematical modeling of microbial [Trichosporon sp.] biomass production in a stirred tank bioreactor and in an external airlift bioreactor has been investigated. A model based on a tanks-in-series model without back-flow has been used to simulate the production of single cell protein in the external airlift bioreactor under an unsteady condition and without oxygen limitation, utilizing cheese whey as a substrate. The kinetic parameters of cell growth and substrate consumption including micro [m] [maximum specific growth], K [s] [growth associated parameter], Gamma [saturated onstant] and lambda [non growth associated parameter], were determinded based on experimental data derived from the batch process in the stirred tank reactor and teh kinetic model, which resulted in 0.59 h[-1], 46.84 g/l, 0.383 and 1.275, respectively. Estimated biokinetic parameters were applied to find the profiles of biomass and cactose in the airlift bioreactor. MATLAB software was used to kind kinetic parameters and solve the equations of teh tanks-in-series model. The number of stages of the tanks-in-series obtained equals 16

Evaluation of strategies for temperature and moisture control in solid state packed bed bioreactors

Different control strategies of bed temperature and moisture were investigated using various inlet air temperatures and air fluxes in both the ordinary packed bed bioreactor [without cooling water in the jacket] and the bioreactor with cooling water in jacket. The experiments were carried out within a 1-L solid-state packed bed bioreactor in which Aspergillus niger was cultivated on wheat bran. On-line measurements of oxygen quantity in the outlet air and temperature of the bed and the inlet air flux were carried out in both types of the bioreactors. Effects of certain control strategies on fungal growth rate were compared in both the bioreactors. According to experimental results, using the bioreactor with the cooling water in the jacket is a better strategy for control of bed temperature and moisture during packed bed solid state fermentation. Cumulative oxygen consumption in this bioreactor was approximately 1.7 times higher than other control strategies used in this study

Physiological and morphological changes of recombinant E. coli during over-expression of human interferon-gamma in HCDC

The objective of this research was to investigate the influence of the over-expression of recombinant interferon-gamma during high cell density cultivation on cellular characteristics of recombinant E. coli. Batch and fedbatch culture techniques were employed to grow Escherichia coli BL21 for production of human gamma-interferon in pET expression system. Final cell densities in batch and fed-batch cultivations were approximately 7 and 127 g cell dry weight [CDW] l[-1], respectively. In both systems, specific growth rate decreased and reached zero, 4 hours after the induction. It was found that high cell density and overexpression of interferon-gamma had no substantial effects on cell lysis and plasmid stability. Plasmid content of the cells was nearly similar and remained constant during the post-induction period in both batch and fed-batch cultures [60 mg plasmid per g[-1] CDW]. In both systems, time profiles of acetate and lactate production were similar, lactate concentration was lower than that of acetate and the concentrations of both were lower than the inhibitory level. Maximum extracellular cAMP concentration occurred at the start of induction in fedbatch culture and was higher than the amount produced during the batch process. The size of E. coli cells reduced significantly as cell density increased and the morphology of the cells in high cell density changed from the usual rod shape to spherical, while the expression of interferon-gamma remained almost constant

Production of single cell protein from natural gas: parameter optimization and RNA evaluation

Production of single cell protein [SCP] from natural gas in a one liter bubble column reactor and optimization of the process parameters were investigated. The medium specifications, nitrogen sources, initial inoculum volume, and inlet ratio of gas to air were considered as process parameters to be optimized. The optimum condition for highest biomass production in which the maximum quantity of protein was obtained, were the use of a certain carbon-less salt broth utilizable with methane [named as Methane Salt Broth/MSB] and sodium nitrate as medium and nitrogen source respectively. Also, 7%[v/v] inoculation size, and an inlet gas mixture of 60/40 natural gas/air were determined as the effective inoculation and appropriate volume configuration of inlet gases. Protein production in optimum condition was 69.3%[w/w] of biomass in dry basis which its structural amino acids can be in comparable with other nutrient sources. An average amount of 10 g RNA out of 100 g of cellular protein [or 6%[w/w] RNA in whole biomass] was extracted from the biomass which is extremely near to the possible minimum of RNA distribution among bacteria. Heat shock treatment was applied for reducing the RNA in the biomass bulk. Heat Treatment at 60 to 65°C for 10 to 20 min provided the best RNA reduction results [around 1 gram in 100 grams of protein]. Regarding the structural amino acids and RNA content, the properties of single cell protein resulted in this experimental work, were in a frame which it could be consumed safely