Smart redox-sensitive micelles based on chitosan for dasatinib delivery in suppressing inflammatory diseases.

Dasatinib (DAS) exhibits anti-inflammatory effects by retrieving the balance between inflammatory and anti-inflammatory cytokines secreted by macrophages. The aim of this study was the development of redox-responsive micelles with the potential of passive targeting and on-demand drug release for DAS delivery to macrophages. For this purpose, two molecular weights of chitosan (CHIT) were conjugated to DAS at different molar ratios using 3,3'-dithiodipropionic anhydride (DTDPA) as disulfide bond containing linker to synthesize a series of CHIT-S-S-DAS amphiphilic conjugates. Micelles obtained by the sonication method had particle sizes of 129.3-172.2 nm, zeta potentials of +17.5 to +20.9 mV, drug contents of 0.90-7.20 %, CMC values of 35.3-96.6 µg/ml, and exhibited redox-responsive in vitro drug release. Optimized micelles were non-toxic and dramatically more efficient than non-redox responsive micelles in reducing TNF-α and IL-6 and increasing IL-10 secretion from LPS-stimulated RAW264.7 cells. Furthermore, the redox-responsive micelles were able to reduce the mice paw edema, reduce the plasma levels of pro-inflammatory cytokines and increase plasma level of IL-10, considerably more than free DAS and non-redox responsive micelles in carrageenan-induced mice paw edema model of inflammation.

Rational design of a new mutant of tobacco etch virus protease in order to increase the in vitro solubility.

BACKGROUND AND PURPOSE: Tobacco etch virus (TEV) protease is a protease with high sequence specificity which is useful for the cleavage of fusion proteins. A major limitation of this enzyme is its relatively poor solubility. This study aimed to investigate the effects of some suggested mutations by online tools and molecular dynamics simulation to improve the solubility of TEV protease in vitro. EXPERIMENTAL APPROACH: We designed a rational multi-stage process to determine the solubilizing mutations of TEV protease. At the first stage, all the possible mutations were predicted using online tools such as PoPMuSiC and Eris servers, in which five mutations include N23F, N23L, Q74L, Q74V, and Q74I were suggested for further studies. In the next step, the three dimensional structure of the wild type (WT) and the best mutations were subjected to molecular dynamic simulations to evaluate the dynamic behaviour of the obtained structures. The selected mutation was introduced into the structure using site-directed mutagenesis and expressed in Escherichia coli BL21DE3. After purification, solubility and activity of the purified mutant and WT-TEV proteases were assayed. FINDINGS /RESULTS: By considering the analysis of various factors such as structural and solubility properties, one mutant, N23F, was selected for in vitro studies which led to a 1.5 times increase in the solubility compared to the WT while its activity was decreased somewhat. CONCLUSION AND IMPLICATIONS: We propose N23F mutation, according to computational and experimental analyses for TEV proteases which resulted in a 150% increase in solubility compared to the WT.

Shear sensitive injectable hydrogels of cross-linked tragacanthic acid for ocular drug delivery: Rheological and biological evaluation.

Drug delivery to posterior segment of eye has always been challenging. The aim of the present study was to provide a novel injectable, shear sensitive hydrogel based on tragacanthic acid (TA) with three kinds of acetate salts as cross-linker. Rheological properties by strain and shear stress sweep measurements and also dynamic rheological experiments including frequency and time sweep measurements were studied. Biological studies comprising, cell culture, Draize test on rabbit eyes and histopathological tests were done. The results showed the optimized hydrogel was biocompatible, injectable and owning acceptable firmness in rest state after injection. Healing time of the hydrogel was 46 s and was shear-sensitive. It showed no cytotoxicity on HUVEC cells. No allergic reaction was seen in Draize test and histological examination showed integrity of the retinal layers with no evidence of pathological changes, such as deformations, degeneration, or inflammation. TA hydrogel is promising in ocular drug delivery.

Poly(glycerol sebacate) nanoparticles for ocular delivery of sunitinib: physicochemical, cytotoxic and allergic studies.

Poly(glycerol sebacate) (PGS) is a new biodegradable polymer with good biocompatibility used in many fields of biomedicine and drug delivery. Sunitinib-loaded PGS/gelatine nanoparticles were prepared by the de-solvation method for retinal delivery and treatment of diabetic retinopathy. The nanoparticles were characterised by Fourier-transform infrared and differential scanning calorimetry. The effects of different formulation variables including drug-to-carrier ratio, gelatine-to-PGS ratio, and glycerine-to-sebacate ratio were assessed on the encapsulation efficiency (EE%), particle size, release efficiency (RE), and zeta potential of the nanoparticles. The in vitro cytotoxicity of PGS/gelatine nanoparticles was studied on L929 cells. Draize test on rabbit eyes was also done to investigate the possible allergic reactions caused by the polymer. Glycerine/sebacic acid was the most effective parameter on the EE and RE. Gelatine-to-PGS ratio had the most considerable effect on the particle size while the RE was more affected by the glycerine/sebacic acid ratio. The optimised formulation (S1G0.7D21.2) exhibited a particle size of 282 nm, 34.6% EE, zeta potential of -8.9 mV, and RE% of about 27.3% for drug over 228 h. The 3-(4,5-dimethylthuazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated PGS/gelatine nanoparticles were not cytotoxic and sunitinib-loaded nanoparticles were not toxic at concentrations <36 nM.

Generation and Characterization of a Functional Nanobody Against Inflammatory Chemokine CXCL10, as a Novel Strategy for the Treatment of Multiple Sclerosis.

BACKGROUND & OBJECTIVE: Chemokines and their receptors play a pivotal role in the pathogenesis of various autoimmune diseases such as multiple sclerosis, infectious diseases, and also in cancer metastasis via attraction of the pathogenic immune cells into the inflammation sites. METHODS: Inflammatory chemokine CXCL10 as a T helper (Th)1-chemokine directs chemotaxis of many cell subsets especially Th1 into the central nervous system (CNS) via its receptor CXCR3 and it has been put forward as a potential therapeutic target in the treatment of multiple sclerosis. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies occurring in camelids with unique biochemical and biophysical features which render them superior to conventional antibodies or antibody fragments. Here, we describe the generation, selection, and characterization of CXCL10-specific Nanobodies from camel immunized with CXCL10. The obtained Nanobodies displayed high affinity towards CXCL10 about 10-11-10-8 M. RESULTS: Then a Nanobody with the highest affinity named 3Nb12 was selected and investigated as a migration inhibitor of CXCR3+ cells. Chemotaxis assay results showed that 3Nb12 blocked CXCL10- CXCR3 binding and potently inhibited chemotaxis of CXCR3-transfected HEK293T cells. CONCLUSION: The nanobody 3Nb12 might be a promising specific and powerful blocking agent of CXCL10 function, which can be used for diagnostic, therapeutic and research purposes in MS.

Effect of Twine-arginine Translocation-signaling Fusion System and Chaperones Co-expression on Secretory Expression of Somatropin.

BACKGROUND: Twine-arginine translocation (TAT) system is one of the exporting systems in Escherichia coli which could transport fully/semi-correctly folded proteins outside the reductive cytoplasmic space. In combination with co-expression with a chaperone system, the correctly folded proteins could be transported to oxidative periplasmic space and culture media to pass the main limitations in E. coli expression system such as misfolding and inclusion body formation. MATERIALS AND METHODS: To study the effectiveness of signaling sequences and chaperone co-expression on the translocation of expressed protein, somatropin was selected as the target. Two common signal sequences in TAT system (TorA and SufI) were added at the N-terminal of somatropin and the cassettes were co-expressed in E. coli BL21 (DE3) by a chaperone team including DnaK/J-GrpeE. RESULTS: The expression pattern studies including Western blotting and sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that somatropin is expressed in two cassettes. However, the pattern was different for two signaling sequences. CONCLUSION: The results confirmed that the approach of using TAT-signaling sequences and co-expression with the chaperone team could enhance translocation of protein to periplasmic space and culture media compared to control groups. Western blotting results showed that the signal sequence TorA could transport more expressed proteins to the periplasmic space and culture media in comparison with SufI. However, there was a considerable amount of human growth hormone in the cytoplasm which could not be transported outside the cytoplasmic space.

Twin arginine translocation system in secretory expression of recombinant human growth hormone.

Recombinant protein production in E. coli has several advantages over other expression systems. Misfolding, inclusion body formation, and lack of eukaryotic post translational modification are the most disadvantages of this system. Exporting of correctly folded proteins to the outside of reductive cytoplasmic environment through twin-arginine system could help to pass these limiting steps. Two signal sequences, TorA and SufI are used at N-terminal of human growth hormone (hGH) bearing DsbA gene sequence at C-terminal to enhance folding. The synthetic cassettes including the signal sequence, hGH and DsbA were transformed into E. coli BL21 (DE3) to study the effect of signal sequence and DsbA chaperone on translocation and folding of the protein. The results confirmed using signal sequence at N-terminal of targeted protein and coexpression with DsbA could transport proteins to the periplasmic space and culture media compared to control groups. Although there is no protein band of somatropin in SDS-Page of culture media samples when using SufI as signaling sequence, the study demonstrated TorA signal sequence could transport the target protein to the culture media. However, there was a considerable amount of hGH in periplasmic space when using SufI compared to control.

A rapid and sensitive method for EphB4 identification as a diagnostic and therapeutic biomarker in invasive breast cancer.

BACKGROUND: In the roadmap to design diagnostic and therapeutic markers for breast cancer, EphB4 is of special interest due to its multiple roles in tumor initiation, progression and invasion. The aim of present study was to characterize a rapid and sensitive ELISA-based method to measure EphB4 level and its phosphorylation status following stimulation with its ligand, ephrinB2, in an invasive breast cancer cell line. MATERIALS AND METHODS: MDA-MB-231 breast cancer cells were lysed and EphB4 level was measured using ELISA. EphB4 level was measured in sub- and post-confluent states in culture dishes. Receptor phosphorylation was also detected by ELISA assay, using various concentrations of pre-clustered ephrinB2 for 20 minutes. RESULTS: Expression of EphB4 receptor was detected by ELISA in all samples. EphB4 level was significantly higher in post.confluent than sub.confluent cells. Phosphorylated receptor was also detectable with this method when cells were exogenously stimulated. CONCLUSIONS: Quantitative data from ELISA manifested a difference between levels of EphB4 in two states of different invasive properties. Moreover, ELISA method may be considered rapid and sensitive enough to detect even low levels of total and phosphorylated EphB4 Cost-effectiveness of this method for the detection of differential expression of EphB4 proteins in clinics is also noticeable.

Association of KCNJ11 (E23K) gene polymorphism with susceptibility to type 2 diabetes in Iranian patients.

BACKGROUND: Type 2 diabetes (T2D) is a multifactorial disease with susceptibility of several genes that are related to T2D. Insulin secretion pathway starts with potassium channels in pancreatic beta cells. KCNJ11 gene encodes ATP-sensitive potassium channel subunits. Some studies suggested that KCNJ11 (E23K) mutation increases the risk of T2D. Therefore, present study was designed to investigate the association between E23K polymorphism of KCNJ11 gene and type 2 diabetes mellitus (T2DM) in the Iranian population. MATERIALS AND METHODS: The type of study was case-control and 40 unrelated subjects, including 20 healthy controls and 20 diabetic patients were recruited (diagnosed based on American Diabetes Association criteria). Blood samples were used for isolation of genomic deoxyribonucleic acid (DNA). Having extracted the genomic DNA from human blood leukocytes by means of High Pure PCR Template Preparation Kit, PCR-restriction fragment length polymorphism method was used to detect KCNJ11 E23K gene polymorphism. BanII restriction enzyme was used for digestion. Data were analyzed using Chi-square or Fisher exact test or independent t-test, as appropriate. P < 0.05 was considered. RESULTS: We found that the carrier homozygous for KK genotype are susceptible to T2D (0.049) and in patients the frequency of K allele was higher than control subjects (0.048). CONCLUSION: The present study suggests that KCNJ11 (E23K) gene polymorphism is associated with T2DM.

Identification and cloning of putative water clarification genes of Moringa peregrina (Forssk.) Fiori in E. coli Xl1 blue cells.

BACKGROUND: Water purification processes include the use of chemical compounds despite the concern that they may induce diseases. An ecological solution to this dilemma can come from the use of plant seeds for this purpose. Moringa peregrina (Forssk.) Fiori seeds have water clarification ability. Therefore, the aim of this work was to look for certain water clarification genes in M. peregrina. MATERIALS AND METHODS: After preparation of M. peregrina callus, mRNA was extracted from these cells. After application of reverse transcriptase, the obtained cDNA (s) were used for PCR amplification of the desired genes using primers based on MO2.1 gene of Moringa oleifera. DNA amplification products were cloned in E. coli Xl1 blue cells and DNA sequences were compared with Mo1,2 gene in M. oleifera. RESULTS: We obtained 3 PCR products (approximately 200, 300, and 400 bps). CONCLUSION: After comparison of the sequences of 300bp band obtained from M. peregrina with Mo1,2 gene in M. oleifera, it seems that 300bp band is a good candidate to investigate regarding its potential flocculent activity.

Isolation and characterization of novel thermophilic lipase-secreting bacteria

The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.

Isolation and characterization of novel thermophilic lipase-secreting bacteria.

The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.

Influence of the timing of cardiac catheterization and amount of contrast media on acute renal failure after cardiac surgery.

BACKGROUND: There is limited data about the influence of timing of cardiac surgery in relation to diagnostic angiography and/or the impact of the amount of contrast media used during angiography on the occurance of acute renal failure (ARF). Therefore, in the present study the effect of the time interval between diagnostic angiography and cardiac surgery and also the amount of contrast media used during the diagnostic procedure on the incidence of ARF after cardiac surgery was investigated. METHODS: Data of 1177 patients who underwent different types of cardiac surgeries after cardiac catheterization were prospectively examined. The influence of time interval between cardiac catheterization and surgery as well as the amount of contrast agent on postoperative ARF were assessed using multivariable logistic regression. RESULTS: The patients who progressed to ARF were more likely to have received a higher dose of contrast agent compared to the mean dose. However, the time interval between cardiac surgery and last catheterization was not significantly different between the patients with and without ARF (p = 0.05). Overall, postoperative peak creatinine was highest on day 0, then decreased and remained significantly unchanged after this period. Overall prevalence of acute renal failure during follow-up period had a changeable trend and had the highest rates in days 1 (53.57%) and 6 (52.17%) after surgery. Combined coronary bypass and valve surgery were the strongest predictor of postoperative ARF (OR: 4.976, CI = 1.613-15.355 and p = 0.002), followed by intra-aortic balloon pump insertion (OR: 6.890, CI = 1.482-32.032 and p = 0.009) and usage of higher doses of contrast media agent (OR: 1.446, CI = 1.033-2.025 and p = 0.031). CONCLUSIONS: Minimizing the amount of contrast agent has a potential role in reducing the incidence of postoperative ARF in patients undergoing cardiac surgery, but delaying cardiac surgery after exposure to these agents might not have this protective effect.

Polymorphism of Apo lipoprotein E gene and the risk of multiple sclerosis.

BACKGROUND: Apolipoprotein E (ApoE) gene encodes an important protein in reforming injuries of central nervous system (CNS). It is assumed that various ApoE alleles may be functionally different. The purpose of this study was to investigate the distribution of ApoE genotypes in multiple sclerosis (MS) patients in a small cohort of Iranians. METHODS: In this case-control study, blood samples of patients and healthy volunteers were collected (n = 40) from Neurology Clinic of Alzahra Medical Complex. The ApoE genotypes were determined using DNA extracted from the samples by polymerase chain reaction (PCR) techniques followed by digestion with HhaI restriction enzyme. The results were adjusted for age of MS onset, sex, expanded disability status scale (EDSS), and type of MS (primary or secondary progressive). Results were statistically analyzed using chi-square test. RESULTS: The ApoE3/E3 genotype was detected in the majority of MS patients and the control group. Frequency distribution of E4 allele did not differ significantly between the two groups. There was no difference between ApoE allele and age of disease onset, sex, expanded disability status, or type of multiple sclerosis. CONCLUSIONS: We found no significant differences in genotype frequency between patients with multiple sclerosis and the control group. Despite the fact that small sample size was a limitation for our study, it seems that ApoE polymorphism may not be useful as a marker for screening patients with multiple sclerosis.

Embryonic stem cell-derived cardiomyocytes as a model system to study cardioprotective effects of dexamethasone in doxorubicin cardiotoxicity.

Embryonic stem cell (ESC)-derived beating cardiomyocytes may be considered as a suitable model for in vitro assessment of pharmacological and toxicological studies. In this model, laboratory animals are not required. In addition, physiological functions, such as heart beat, are assessed rather than single parameters such as cell viability. Here we report that doxorubicin (DOX) cardiotoxicity on mouse ESC-derived beating cardiomyocytes can be ameliorated by treatment with dexamethasone (DEX) when DEX is administrated only before DOX and not in combination with DOX. DEX effect appears to be mediated via glucocorticoid receptor and increases cardiomyocyte-specific gene expression. Cardiotoxicity of DOX can be augmented by calcium channel blocker, verapamil (VER) which also decreases the expression of cardiac gene markers. This model provides us with a clinical suggestion which proposes that the beneficial effect of DEX is obtained when DEX was added before DOX administration.