Electromagnetic field as a possible inhibitor of tumor invasion by declining E-cadherin/N-cadherin switching in triple negative breast cancer.

Breast cancer has been recognized as the most common cancer affecting women. Extremely low-frequency electromagnetic field (ELF-EMF) exposure can influence cellular activities such as cell-cell junctions and metastasis. However, more research is required to determine these fields' underlying mechanisms of action. Since cadherin switching is an important process during EMT (epithelial-mesenchymal transition), in this study, cadherin switching was regarded as one of the probable mechanisms of the effect of ELF-EMFs on metastasis suppression. For five days, breast cells received a 1 Hz, 100mT ELF-EMF (2 h/day). Cell invasion and migration were assessed in vitro by the Scratch wound healing assay and Transwell culture chambers. The expression of E- and N-cadherin was assessed using real-time PCR, western blotting, and Immunocytochemistry. ELF-EMF dramatically reduced the migration and invasion of MDA-MB 231 malignant cells compared to sham exposure, according to the results of the scratch test and the Transwell invasion test. The mRNA and protein expression levels of E-cadherin showed an increase, while the N-cadherin expression was found with a decrease, in MDA-MB231 cells receiving 1 Hz EMF compared to sham exposure. E-cadherin's mRNA and protein expression levels were enhanced in MCF10A cells receiving 1 Hz EMF compared to sham exposure. ELF-EMF can be used as a method for the multifaceted treatments of invasive breast cancer.

Breast cancer (BC) is one of the main reasons for cancer-related mortality among the female population. Despite the huge efforts made to treat BC, metastasis has remained one of the most complicated and hardly manageable problems. Extremely low-frequency electromagnetic fields (ELF-EMFs) are low-energy non-ionizing radiation with several biological effects whose influence on living organisms has been recently explored. However, the mechanisms of action of these electromagnetic fields have not been fully understood. For five days, breast cells received a 1Hz, 100mT ELF-EMF (2h/day). Cell invasion and migration were assessed in vitro by the Scratch wound healing assay and Transwell culture chambers. The expression of E- and N-cadherin was assessed using real-time PCR, western blotting, and Immunocytochemistry. ELF-EMF dramatically reduced the migration and invasion of MDA-MB 231 malignant cells compared to sham exposure, according to the scratch test results and the Transwell invasion test. The mRNA and protein expression levels of E-cadherin showed an increase, while the N-cadherin expression was decreased, in MDA-MB231 cells receiving 1Hz EMF compared to sham exposure. The present report can be considered as a preliminary study for future investigations on ELF-EMF that recommends ELF-EMF can be used as a method for the multifaceted treatment of invasive BC which requires further studies.

Signal amplification of a quartz crystal microbalance immunosensor by gold nanoparticles-polyethyleneimine for hepatitis B biomarker detection.

The procedures currently used for hepatitis B (HB) detection are not suitable for screening, clinical diagnosis, and point-of-care testing (POCT). Therefore, we developed and tested a QCM-based immunosensor by surface modification with AuNP-PEIs to amplify the signal and provide an oriented-immobilization surface. The AuNP-PEIs were characterized by ICP-Mass, UV/Vis, DLS, FE-SEM, and ATR-FTIR. After coating AuNP-PEIs on the gold electrode surface, anti-HBsAg antibodies were immobilized using NHS/EDC chemistry based on response surface methodology (RSM) optimization. The efficiency of the immunosensor was assessed by human sera and data were compared to gold-standard ELISA using receiver-operating-characteristic (ROC) analysis. FE-SEM, AFM, EDS, and EDS mapping confirmed AuNP-PEIs are homogeneously distributed on the surface with a high density and purity. After antibody immobilization, the immunosensor exhibited good recognition of HBsAg with a calibration curve of ∆F = - 6.910e-7x + 10(R2 = 0.9905), a LOD of 1.49 ng/mL, and a LOQ of 4.52 ng/mL. The immunosensor yielded reliable and accurate results with a specificity of 100% (95% CI 47.8-100.0) and sensitivity of 100% (95% CI 96.2-100.0). In conclusion, the fabricated immunosensor has the potential as an analytic tool with high sensitivity and specificity. However, further investigations are needed to convert it to a tiny lab-on-chip for HB diagnosis in clinical samples.

Improved anti-biofilm activity and long-lasting effects of novel serratiopeptidase immobilized on cellulose nanofibers.

Today, enzymatic treatment is a progressive field in combating biofilm producing pathogens. In this regard, serratiopeptidase, a medicinally important metalloprotease, has been recently highlighted as an enzyme with proved anti-biofilm activity. In the present study, in order to increase the long-lasting effects of the enzyme, serratiopeptidase and the novel engineered forms with enhanced anti-biofilm activity were immobilized on the surface of cellulose nanofibers (CNFs) as a natural polymer with eminent properties. For this, recombinant serratiopeptidases including the native and previously designed enzymes were produced, purified and conjugated to the CNF by chemical and physical methods. Immobilization was confirmed using different scanning and microscopic methods. The enzyme activity was assessed using casein hydrolysis test. Enzyme release analysis was performed using dialysis tube method. Anti-biofilm activity of free and immobilized enzymes has been examined on Staphylococcus aureus and Pseudomonas aeruginosa strains. Finally, cytotoxicity of enzyme-conjugated CNFs was performed by MTT assay. The casein hydrolysis results confirmed fixation of all recombinant enzymes on CNFs by chemical method; however, inadequate fixation of these enzymes was found using cold atmospheric plasma (CAP). The AFM, FTIR, and SEM analysis confirmed appropriate conjugation of enzymes on the surface of CNFs. Immobilization of enzymes on CNFs improved the anti-biofilm activity of serratiopeptidase enzymes. Interestingly, the novel engineered serratiopeptidase (T344 [8-339ss]) exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms. In conclusion, incorporation of serratiopeptidases into CNFs improves their anti-biofilm activities without baring any cytotoxicity. KEY POINTS: • Enzymes were successfully immobilized on cellulose nanofibers using chemical method. • Immobilization of enzymes on CNFs improved their anti-biofilm activity. • T344 [8-339ss] exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms.

A quartz crystal microbalance biosensor based on polyethylenimine-modified gold electrode to detect hepatitis B biomarker.

Biomarkers-based QCM-biosensors are suitable tools for the label-free detection of infectious diseases. In the current study, a QCM-biosensor was developed for the detection of HBsAg. Briefly, anti-HBsAg antibodies were covalently bound to the primary amines after PEI and thiolated-PEI surface modifications of gold-electrode. After RSM optimization, the statistical analysis revealed no significant difference between the immobilization yields of modified layers. Therefore, the PEI-modified QCM-biosensor was selected for further analysis. The PEI-surface was evaluated by FESEM, AFM, ATR-FTIR, and CA measurement. The surface hydrophilicity and its roughness were increased after PEI-coating. Also, FTIR confirmed the PEI-layering on the gold-surface. RSM optimization increased the antibody immobilization yield up to 80%. The QCM-biosensor showed noteworthy results with a wide dynamic range of 1-1 × 103 ng/mL, LOD of 3.14 ng/mL, LOQ of 9.52 ng/mL, and detection capability in human-sera, which were comparable with the ELISA. The mean accuracy of the QCM-biosensor was obtained at 91% when measured by the spike recovery test using human-sera. The biosensor was completely regenerated using 50 mM NaOH and 1% SDS. The benefits provided by the developed biosensor such as broad dynamic range, sensitivity, selectivity, stability, regenerate ability, and low cost suggest its potential application for the non-invasive and timely monitoring of HBV-biomarker.

Hyaluronic acid production and characterization by novel Bacillus subtilis harboring truncated Hyaluronan Synthase.

Hyaluronic Acid (HA) is a natural biopolymer that has important physiological and industrial applications due to its viscoelastic and hydrophilic characteristics. The responsible enzyme for HA production is Hyaluronan synthase (HAS). Although in vitro structure-function of intact HAS enzyme has been partly identified, there is no data on in vivo function of truncated HAS forms. In the current study, novel recombinant Bacillus subtilis strains harboring full length (RBSFA) and truncated forms of SeHAS (RBSTr4 and RBSTr3) were developed and HA production was studied in terms of titer, production rate and molecular weight (Mw). The maximum HA titer for RBSFA, RBSTr4 and RBSTr3 was 602 ± 16.6, 503 ± 19.4 and 728 ± 22.9 mg/L, respectively. Also, the HA production rate was 20.02, 15.90 and 24.42 mg/L.h-1, respectively. The findings revealed that RBSTr3 produced 121% and 137% more HA rather than RBSFA and RBSTr4, respectively. More interestingly, the HA Mw was about 60 kDa for all strains which is much smaller than those obtained in prior studies.

Novel serratiopeptidase exhibits different affinities to the substrates and inhibitors.

The clinical application of serratiopeptidase as an anti-biofilm and anti-inflammatory agent is restricted due to the enzyme sensitivity to the environmental conditions. In our previous study, six enzyme variants were designed by introducing different mutations and truncations that exhibited higher thermal stability. In the present study, the interaction pattern and affinity of variants to substrates and inhibitors were studied using molecular docking and in vitro studies. CABS-dock and Swiss-dock servers were used for substrate (Bradykinin and Substance-P) and inhibitor (Lisinopril and EDTA) docking, respectively. The interactions were analyzed using LigPlot, UCSF Chimera, and visual molecular dynamics packages. Free energy calculations were performed using PRODIGY. Finally, the native enzyme and the best variant in terms of interaction pattern and binding score were selected for in-vitro affinity analysis toward Bradykinin and EDTA using HPLC and casein hydrolysis test, respectively. Molecular docking revealed that T344 [8-339ss] variant showed a different pattern for both substrates and inhibitors in the way that none of the native active site residues were involved in the receptor binding. As revealed by in vitro studies, T344 [8-339ss] displayed the highest number of hydrogen bond formation in docking with Bradykinin and remarkable decrement in the binding affinity for EDTA. This was the first report on the design of novel serratiopeptidase with higher activity to Bradykinin and improved resistance to EDTA as an inhibitor.

Enhancing proteolytic activity of Lysobacter enzymogenes using cold atmospheric plasma.

Cold atmospheric plasma (CAP) is being used recently as a modern technique for microbial random mutagenesis. In the present study, CAP was used to induce mutagenesis in L. enzymogenes which is the bacteria known for producing proteolytic enzymes especially lysyl endopeptidase (Lys C). Enhanced proteolytic activity was the main criteria to select mutant strains. Therefore, the cell suspension of L. enzymogenes strain (ATCC 29487), was exposed to CAP for 30, 45, 90, and 150 s. The proteolytic activity of mutant strains was screened initially by radial caseinolytic assay and then by Ansons method in different phases of bacterial growth in the selected mutants. The purification process of Lysyl endopeptidase as the target enzyme was optimized and for enlightening molecular aspect of CAP mutagenesis, the sequences of the upstream and coding regions of lys C gene from 10 selected mutant strains were determined. The bacterial survival assessment showed that the more CAP treatment time, the less survival rate, however, in all exposure times, a number of survived mutants showed enhanced proteolytic activity. Among 38 out of 100 examined mutants which showed higher proteolytic activity than that of wild type, the M1-30 s mutant exhibited the highest increment to 1.94 fold. The SDS-PAGE analysis showed expected size of purified Lys C from M1-30 s. The Lys C gene from M14-150 s mutant strain (1.4-fold increment) harbored three point mutations which can be effective in enhancing protease activity. In conclusion, the results highlighted the role of CAP for strain improvement process to obtain industrial strains.

Label-Free Real-Time Detection of HBsAg Using a QCM Immunosensor.

BACKGROUND: Hepatitis B virus surface antigen (HBsAg) is an important protein in both diagnosis and prevention of hepatitis B infection. In the current study, a piezoelectric immunosensor based on antibody-antigen interaction was designed to detect HBsAg. A quartz crystal microbalance system was employed to detect antibody-antigen interaction. METHODS: At first, an oscillator was designed to measure the resonant frequency affected by the reactants using IC 74LVC1GX04. Antibody against HBsAg was immobilized on 10 MHz AT-cut quartz crystal. The surface modifications were monitored by atomic force microscopy (AFM) and contact angle measurements. Different concentrations of antibody were used for surface immobilization and the frequency shifts were assessed. The system stability was studied by evaluating the stability of the crystal and the immobilized antibody. The adsorption of antibody onto the crystal was analyzed using AFM and changes in the resonance frequency. Further, a direct immunoassay was performed with this immobilized antibody to identify HBsAg solutions at different concentrations. Finally, specific and non-specific responses were investigated using hepatitis B (HBsAg) and hepatitis C (HCV Ag) antigens, respectively. RESULTS: Antibodies against HBsAg were successfully immobilized on 10 MHz AT-cut quartz crystal. The stability tests of crystal immobilized with antibody and unimmobilized crystal revealed that both forms of crystals were stable. Theoretical and experimental frequency assays were compared. A decrease in the contact angle indicated the hydrophilicity of surface after modifications. AFM images illustrated a more uniform surface after antibody adsorption and the surface roughness (RMS) reduced from 1.13 to 0.99 nm. Changes in the frequency were detected after the physical adsorption of HBsAb on the designed chip. The standard curve of antigen revealed the frequency changes depend on concentration of antigen. Finally, the specificity test confirmed the specificity of the designed biosensor for the detection of HBsAg from HCV Ag. The quantization of immobilized antibody was characterized by the frequency shift of the QCM. The obtained results were compared with ELISA assay. The correlation coefficients of HBsAg dilution between QCM and ELISA was 0.9821. CONCLUSIONS: This study is a new step to meet the challenges regarding HBsAg detection. Physical adsorption used in this study was effective as the simplest immobilization method to design a QCM-based immunosensor for HBsAg detection. Facilitated, fast, and simple detection of HBsAg by an antibody-based QCM biosensor is our main objective.

Immobilization of Urease onto Modified Egg Shell Membrane through Cross Linking

Background: Immobilization is an approach in industry to improve stability and reusability of urease. The efficiency of this technique depends on the type of membrane and the method of stabilization. Methods: The PEI-modified egg shell membrane was used to immobilize urease by absorption and glutaraldehyde cross-linking methods. The membranes were characterized by Fourier-transform infrared spectroscopy (FTIR) and AFM, and Nessler method was applied to measure the kinetic of the immobilized enzymes. Finally, the storage stability (6 °C for 21 days) and reusability (until enzyme activity reached to zero) of the immobilized enzymes were investigated. Results: Based on FTIR, three new peaks were observed in both the absorption- (at 1389.7, 1230.8, and 1074.2 cm-1) and the cross-linking (at 1615-1690, 1392.7, 1450 cm-1) immobilized enzymes. The surface roughness of the native membrane was altered after PEI treatment and enzyme immobilization. The optimal pH of cross-linking immobilized enzymes was shifted to a more neutral pH, while it was alkaline in adsorption-immobilized and free enzymes. The reaction time decreased in all immobilized enzymes (100 min for free enzyme vs. 60 and 30 min after immobilizing by adsorption and cross-linking methods, respectively). The optimal temperature for all enzymes was 70 °C and they had a higher Km and a lower Vmax than free enzyme. The stability and reusability of urease were improved by both methods. Conclusion: Our findings propose these approaches as promising ways to enhance the urease efficiency for its applications in industries and medicines.

Rapid screening of high expressing Escherichia coli colonies using a novel dicistronic-autoinducible system.

BACKGROUND: Identification of high-expressing colonies is one of the main concerns in the upstream process of recombinant protein development. The common method to screen high-producing colonies is SDS-PAGE, a laborious and time-consuming process, which is based on a random and qualitative way. The current study describes the design and development of a rapid screening system composed of a dicistronic expression system containing a reporter (enhanced green fluorescent protein, eGFP), protein model (staphylokinase, SAK), and a self-inducible system containing heat shock protein 27 (Hsp27). RESULTS: Dicistronic-autoinducible system expressed eGFP and SAK successfully in 5-ml and 1-L culture volumes. High expressing colonies were identified during 6 h via fluorescent signals. In addition, the biological activity of the protein model was confirmed semi-quantitatively and quantitatively through radial caseinolytic and chromogenic methods, respectively. There was a direct correlation between eGFP fluorescent intensity and SAK activity. The correlation and linearity of expression between the two genes were respectively confirmed with Pearson correlation and linear regression. Additionally, the precision, limit of detection (LOD), and limit of quantification (LOQ) were determined. The expression of eGFP and SAK was stable during four freeze-thaw cycles. In addition, the developed protocol showed that the transformants can be inoculated directly to the culture, saving time and reducing the error-prone step of colony picking. CONCLUSION: The developed system is applicable for rapid screening of high-expressing colonies in most research laboratories. This system can be investigated for other recombinant proteins expressed in E. coli with a potential capability for automation and use at larger scales.

Design, expression and functional assessment of novel engineered serratiopeptidase analogs with enhanced protease activity and thermal stability.

Serratiopeptidase is a bacterial protease that has been used medicinally in variety of applications. Though, some drawbacks like sensitivity to environmental conditions and low penetration into cells limited its usage as a potent pharmaceutical agent. This study aimed to produce four novel truncated serratiopeptidase analogs with different lengths and possessing one disulfide bridge, in order to enhance protease activity and thermal stability of this enzyme. Mutagenesis and truncation were performed using specific primers by conventional and overlap PCR. The recombinant proteins were expressed in E. coli cells then purified and their protease activity and stability were checked at different pH and temperatures in comparison to the native form of the enzyme, Serra473. Enzyme activity assay showed that T306 [12-302 ss] was not further active which could be due to the large truncation. However, T344 [8-339 ss], T380 [8-339 ss] and T380 [12-302 ss] proteins showed higher proteolytic activity comparing to Serra473. These analogs were active at temperatures of 25-90 °C and pH 6-9.5. Interestingly, remaining enzyme activity of T344 [8-339 ss], T380 [8-339 ss] and T380 [12-302 ss] forms at 90 °C calculated as 87, 83 and 86 percent, respectively, comparing to the activity at room temperature. However, residual activity at the same conditions was 50% for the full length enzyme. Formation of disulfide bond in engineered serratiopeptidases could be the main reason for higher thermal stability compared to Serra473. Thermostability of T344 [8-339 ss], as the most thermostable designed serratiopeptidase, was additionally confirmed using differential scanning calorimetry.

Immobilization of Candida rugosa lipase for resolution of racimic ibuprofen.

AIM: Due to lipases' regio-selectivity and ability to catalyze different reactions such as hydrolysis, esterification, and transesterification, the enzyme is attractive in biotransformation technology. Besides, another technology, namely enzyme immobilization, has attracted scientists/technologists' attention to employ immobilized lipase in such a field. Thus lipase of Candida rugosa was immobilized onto silica nanoparticles through adsorption. Furthermore, the immobilized biocatalyst was characterized and used to esterify ibuprofen enantioselectively. METHODS: To characterize immobilized lipase onto silica nanoparticles scanning electron microscopy (SEM) and dynamic light scattering (DLS) were used. RESULTS: The catalytic properties of both immobilized and free lipases such as optima pH and temperature were not different. According to the results, the immobilized lipase on silica nanoparticles showed 45% and 96% conversion (C) and enantioselectivity (ees), respectively. In comparison to free lipase, the immobilized enzyme came with better catalytic activity. CONCLUSION: Silica nanoparticles as one of the most promising materials for the immobilization of lipase in enantioselective esterification of ibuprofen, were introduced in this work.

Comparison of E. coli based self-inducible expression systems containing different human heat shock proteins.

IPTG-inducible promoter is popularly used for the expression of recombinant proteins. However, it is not suitable at the industrial scale due to the high cost and toxicity on the producing cells. Recently, a Self-Inducible Expression (SILEX) system has developed to bypass such problems using Hsp70 as an autoinducer. Herein, the effect of other heat shock proteins on the autoinduction of green fluorescent protein (EGFP), romiplostim, and interleukin-2 was investigated. For quantitative measurements, EGFP expression was monitored after double-transformation of pET28a-EGFP and pET21a-(Hsp27/Hsp40/Hsp70) plasmids into E. coli using fluorimetry. Moreover, the expression level, bacterial growth curve, and plasmid and expression stability were compared to an IPTG- inducible system using EGFP. Statistical analysis revealed a significant difference in EGFP expression between autoinducible and IPTG-inducible systems. The expression level was higher in Hsp27 system than Hsp70/Hsp40 systems. However, the highest amount of expression was observed for the inducible system. IPTG-inducible and Hsp70 systems showed more lag-time in the bacterial growth curve than Hsp27/Hsp40 systems. A relatively stable EGFP expression was observed in SILEX systems after several freeze-thaw cycles within 90 days, while, IPTG-inducible system showed a decreasing trend compared to the newly transformed bacteria. Moreover, the inducible system showed more variation in the EGFP expression among different clones than clones obtained by SILEX systems. All designed SILEX systems successfully self-induced the expression of protein models. In conclusion, Hsp27 system could be considered as a suitable autoinducible system for protein expression due to less metabolic burden, lower variation in the expression level, suitable plasmid and expression stability, and a higher expression level.

Challenges and advancements in the pharmacokinetic enhancement of therapeutic proteins.

Nowadays, proteins are frequently administered as therapeutic agents in human diseases. However, the main challenge regarding the clinical application of therapeutic proteins is short circulating plasma half-life that leads to more frequent injections for maintaining therapeutic plasma levels, increased therapy costs, immunogenic reactions, and low patient compliance. So, the development of novel strategies to enhance the pharmacokinetic profile of therapeutic proteins has attracted great attention in pharmaceuticals. So far, several techniques, each with their pros and cons, have been developed including chemical bonding to polymers, hyper glycosylation, Fc fusion, human serum albumin fusion, and recombinant PEG mimetics. These techniques mainly classify into three strategies; (i) the endosomal recycling of neonatal Fc receptor which is observed for immunoglobulins and albumin, (ii) decrease in receptor-mediated clearance, and (iii) increase in hydrodynamic radius through chemical and genetic modifications. Recently, novel PEG mimetic peptides like proline/alanine/serine repeat sequences are designed to overcome pitfalls associated with the previous technologies. Biodegradability, lack of or low immunogenicity, product homogeneity, and a simple production process, currently make these polypeptides as the preferred technology for plasma half-life extension of therapeutic proteins. In this review, challenges and pitfalls in the pharmacokinetic enhancement of therapeutic proteins using PEG-mimetic peptides will be discussed in detail.

A Comparative Analysis of Different Enzyme Immobilization Nanomaterials: Progress, Constraints and Recent Trends.

Immobilization techniques have been popularly used to preserve the operational stability of the enzymes for industrial applications. The three main components of an immobilized enzyme system are the enzyme, the matrix/support, and the technique of immobilization. So far, different supports have been developed to improve the efficiency of the immobilized enzymes. But in the recent decade, nanotechnology has been of considerable research interest in the field of immobilized enzyme carriers. The materials at the nano-scale, due to their unique physicochemical properties, including; specific surface area, mass transfer limitation, and effective enzyme loading, are considered interesting matrices for enzyme immobilization. This review describes techniques employed to immobilize enzymes, and provides an integrated focus on the most common nanoparticles for enzyme conjugation. Additionally, the pros and cons of nanoparticles as immobilization matrices are also discussed. Depending on the type of enzyme and its application, in this review, the researchers are directed to select an appropriate method and support for enzyme immobilization in terms of enzyme stability and functionality.

Immobilization of recombinant lysostaphin on nanoparticle through biotin-streptavidin conjugation technology as a geometrical progressed confrontation against Staphylococcus aureus infection.

Antibiotic resistance and the colonization of resistant bacteria such as Staphylococcus aureus on surfaces, often in the form of biofilms, prolong hospitalization periods and increase mortality, thus is a significant concern for healthcare providers. To prevent biofilm formation, the inadequate concentration of using nanoparticles as antibacterial coating agents is one of the major obstacles. This study aimed to design a hypervalency TiO2 nanocomposite as a reserved base to carry a high amount of active antibacterial agents such as lysostaphin via a biotin-streptavidin-biotin bridge. The utilization of the streptavidin-biotin system could increase the abundance of lysostaphin. Lysostaphin was expressed in Escherichia coli and purified. Both recombinant lysostaphin and titanium oxide nanocomposite were conjugated with biotin and linked to a streptavidin bridge. The kinetics and activity of the enzyme were examined after each step utilizing N-acetylhexaglycine as a substrate. Physical characteristics of nanoparticles containing lysostaphin were determined using AFM, SEM, FTIR, and zeta potential. The results showed changes in size, charge, and morphology of the nanoparticles following the lysostaphin attachment. Also, the stability and kinetics of the active biological enzymes on nanoparticles were reexamined following 8 months of storage. Exploiting this approach, various biotinylated antibacterial agents could be prepared and rapidly immobilized on a nanoparticle as an active net against related infectious agents.

Optimization, physicochemical characterization, and antimicrobial activity of a novel simvastatin nano-niosomal gel against E. coli and S. aureus.

Niosomes, as a kind of drug delivery system, is widely used for the topical delivery of lipophilic drugs. Optimization of niosomes plays an essential role in enhancing their therapeutic efficiencies. This study aims to prepare an optimized niosomal formulation of simvastatin (nSIM), a lipophilic member of statins, through the experiment (Response Surface methodology). Optimized niosomes were characterized in size, polydispersity index (PDI), entrapment efficiency (EE), stability, releasing pattern, and antimicrobial activity. The different molar ratio of surfactant and cholesterol were applied to prepare various formulation of simvastatin loaded niosome. Mean particle size and size distribution were analyzed by dynamic light scattering. Antibacterial activity was determined by MIC and MBC tests against Staphylococcus aureus and Escherichia coli. The release rate of simvastatin from noisome nanoparticles was studied by the Franz diffusion cell method. The release pattern was studied through zero order, first order, Higuchi, Korsmeyer-Peppas, and Hixson-Crowell kinetics models. Optimized niosomes were obtained by span 80, drug to cholesterol ratio of 0.4 with 7 min sonication time. Mean particle size, PDI, zeta potential, and entrapment efficiency (EE%) of optimized nSIM were obtained about 168 nm, 0.34, -32.40, and 96 %, respectively. The niosomes significantly decreased the drug's releasing rate and enhanced antibacterial activity against S. aureus and E. Coli. It was found that the release pattern of drug followed the Higuchi kinetic model which means drug release is by diffusion. Overall, our findings indicated that the prepared simvastatin loaded niosomes showed good stability and biological properties than free drug. Our study suggests that niosomal formulation could be considered as a promising strategy for the delivery of poor water-soluble drugs that enhance antibacterial activity.

Lipopolysaccharide removal affinity matrices based on novel cationic amphiphilic peptides.

Lipopolysaccharide (LPS) is one of the most challenging contaminants in biopharmaceutical industries. Cationic amphiphilic peptides (CAPs) -based affinity matrices can be potent tools for LPS removal in such situations. In this study, two newly designed CAPs derived from the LPS binding site of factor C of horseshoe crab S3E3 and S3E3A were immobilized chemo-selectively on diaminodipropylamine (DADPA) and iodoacetyl functionalized Sepharose beads. Both peptides were immobilized via their carboxyl or sulfhydryl (thiol) groups by amide or thioether bonds, respectively. The generated four affinity matrices were used to remove LPS from bovine serum albumin (BSA). The effects of different influential factors including pH, NaCl, Ethylenediaminetetraacetic acid (EDTA), and LPS concentrations on LPS removal efficiency and protein recovery were investigated by Plackett Burman (PB) method.Statistical analysis revealed that immobilized S3E3 removed LPS more effectively than immobilized S3E3A. Increasing pH and LPS concentration had negative effects on LPS removal efficiency and protein recovery. Increasing NaCl concentration improved protein recovery but reduced LPS removal efficiency. Other factors such as EDTA and type of buffer had no significant effect on the measured responses.

Production and Expression Optimization of Heterologous Serratiopeptidase.

BACKGROUND: Serratiopeptidase is a bacterial metalloprotease, which is useful for the treatment of pain and inflammation. It breaks down fibrin, thins the fluids formed during inflammation and acts as an anti-biofilm agent. Because of medicinally important role of the enzyme, we aimed to study the cloning and the expression optimization of serratiopeptidase. METHODS: The heat-stable serratiopeptidase (5d7w) was selected as the template. Cloning into pET28a expression vector was performed and confirmed by colony PCR and double restriction enzyme digestion. The recombinant protein was expressed in Esherichia coli BL21 and confirmed by SDS-PAGE and Western blot analysis. Different parameters such as expression vector, culture media, post-induction incubation temperature, inducer concentration, and post-induction incubation time were altered to obtain the highest amount of the recombinant protein. RESULTS: Serratiopeptidase was successfully cloned and expressed under optimized conditions in E. coli which confirmed by western blot analysis. The optimal conditions of expression were determined using pQE30 as vector, cultivating the host bacteria in Terrific Broth (TB) medium, at 37° C, induction by IPTG concentration equal to 0.5 mM, and cells were harvested 4 h after induction. CONCLUSION: As serratiopeptidase is a multi-potent enzyme, the expressed recombinant protein can be considered as a valuable agent for pharmaceutical applications in further studies.

Enhanced antitumor activity of bovine lactoferrin through immobilization onto functionalized nano graphene oxide: an in vitro/in vivo study.

This study aims to improve the anticancer activity of bovine lactoferrin through enhancing its stability by immobilization onto graphene oxide. Bovine lactoferrin was conjugated onto graphene oxide and the conjugation process was confirmed by FT-IR, SDS-PAGE, and UV spectrophotometry. Physical characterization was performed by DLS analysis and atomic force microscopy. The cytotoxicity and cellular uptake of the final construct (CGO-PEG-bLF) was inspected on lung cancer TC-1 cells by MTT assay and flow cytometry/confocal microscopy. The anticancer mechanism of the CGO-PEG-bLF was studied by cell cycle analysis, apoptosis assay, and western blot technique. Finally, the anticancer activity of CGO-PEG-bLF was assessed in an animal model of lung cancer. Size and zeta potential of CGO-PEG-bLF was obtained in the optimum range. Compared with free bLF, more cytotoxic activity, cellular uptake and more survival time was obtained for CGO-PEG-bLF. CGO-PEG-bLF significantly inhibited tumor growth in the animal model. Cell cycle arrest and apoptosis were more induced by CGO-PEG-bLF. Moreover, exposure to CGO-PEG-bLF decreased the phospho-AKT and pro-Caspase 3 levels and increased the amount of cleaved caspase 3 in the treated cells. This study revealed the potential of CGO-PEG as a promising nanocarrier for enhancing the therapeutic efficacy of anticancer agents.