Site Directed Disulfide PEGylation of Interferon-ß-1b with Fork Peptide Linker.

The attachment of PEG to biopharmaceuticals has been applied for enhancement of bioavailability and improved stability. The PEG polymer is highly hydrated; thus effective attachment to inaccessible sites could be hindered. We have devised a scheme to address this issue by introducing a considerable distance between PEG and protein by addition of a linear peptide, appended to long chained reactive linkers. Second, the position of PEG conjugation directly affects biological activity. Accordingly, a disulfide bond could be considered as an ideal choice for site directed PEGylation; but reactivity of both thiol moieties to bridging reagent is critical for maintenance of protein structure. In our design, a forked structure with two arms provides essential flexibility to account for dissociation of reduced cysteines. An efficient yield for disulfide PEGylation of IFN-ß1b was attained and specificity, biophysical characterization, biological activity, and pharmacokinetics were surveyed.

Effect of glycated insulin on the blood-brain barrier permeability: An in vitro study.

Altered blood-brain barrier (BBB) permeability may contribute to pathogenesis of diabetes-related central nervous system disorders. Considering the presence of glycated insulin in plasma of type 2 diabetic patients, we hypothesized that glycated insulin could induce changes in paracellular permeability in BBB. Therefore, the authors decided to study the effect of glycated insulin on paracellular permeability in a BBB model and the change induced in insulin conformation upon glycation. In this study, the structural modification was examined by fluorescence and circular dichroism spectroscopies and dynamic light scattering. Cell proliferation and production of ROS in astrocytes and HUVEC cells were analyzed by MTT and spectrofluorometric assays, respectively. Apoptosis induction was determined and confirmed by flow cytometry and western blot analyses, respectively. The permeability was measured Lucifer yellow and FITC-Dextran. According to our results, glycated insulin presented altered conformation and more exposed hydrophobic patches than insulin. Formation of oligomeric species and advanced glycated end products (AGEs) were determined. Lower cell viability, higher apoptosis, and more ROS were detected upon treatment of cells with glycated insulin. Finally, glycated insulin led to increased Lucifer yellow and FITC-dextran transportation across the BBB model which could result from ROS producing and apoptosis-inducing activities of AGE-insulin.

Size Control in the Nanoprecipitation Process of Stable Iodine (¹²7I) Using Microchannel Reactor-Optimization by Artificial Neural Networks.

In this study, nanosuspension of stable iodine ((127)I) was prepared by nanoprecipitation process in microfluidic devices. Then, size of particles was optimized using artificial neural networks (ANNs) modeling. The size of prepared particles was evaluated by dynamic light scattering. The response surfaces obtained from ANNs model illustrated the determining effect of input variables (solvent and antisolvent flow rate, surfactant concentration, and solvent temperature) on the output variable (nanoparticle size). Comparing the 3D graphs revealed that solvent and antisolvent flow rate had reverse relation with size of nanoparticles. Also, those graphs indicated that the solvent temperature at low values had an indirect relation with size of stable iodine ((127)I) nanoparticles, while at the high values, a direct relation was observed. In addition, it was found that the effect of surfactant concentration on particle size in the nanosuspension of stable iodine ((127)I) was depended on the solvent temperature. Nanoprecipitation process of stable iodine (127I) and optimization of particle size using ANNs modeling.

Use of artificial neural networks for analysis of the factors affecting particle size in mebudipine nanoemulsion.

In this study, a nanoemulsion containing mebudipine [composed of ethyl oleate (oil phase), Tween 80 (T80), Span 80 (S80) (surfactants), polyethylene glycol 400, ethanol (cosurfactants), and deionized water] was prepared with the aim of improving its bioavailability for an effective antihypertensive therapy. Particle size of the formulation was measured by dynamic light scattering. Then, artificial neural networks were used in identifying factors that influence the particle size of the nanoemulsion. Three variables, namely, amount of surfactant system (T80 + S80), amount of polyethylene glycol, and amount of ethanol as cosurfactants, were considered as input values and the particle size was used as output. The developed model showed that all the three inputs had some degrees of effect on particles size: increasing the value of each input decreased the size. Furthermore, amount of surfactant was found to be the dominant factor in controlling the final particle size of nanoemulsion. Communicated by Ramaswamy H. Sarma.

Investigation of Factors Affecting Aerodynamic Performance of Nebulized Nanoemulsion.

This work aimed to prepare a nanoemulsion preparation containing budesonide and assess its aerodynamic behavior in comparison with suspension of budesonide. In-vitro aerodynamic performance of the corresponding micellar solution (ie. nanoemulsion preparation without oil) was investigated too. Nanoemulsions of almond oil containing budesonide, as a hydrophobic model drug molecule, were prepared and optimized. Then, the effect of variation of surfactant/co-surfactant concentration on the aerodynamic properties of the nebulized aerosol was studied. The results indicated that the most physically stable formulation makes the smallest aerodynamic size. The concentration of co-surfactant was also shown to be critical in determination of aerodynamic size. Furthermore, the optimized sample, with 3% w/w almond oil, 20% w/w Tween 80+Span 80 and 2% w/w ethanol showed a smaller MMAD in comparison with the commercially available suspension and the micellar solution.

Development and optimization of N-Acetylcysteine-loaded poly (lactic-co-glycolic acid) nanoparticles by electrospray.

N-Acetylcysteine (NAC) loaded PLGA nanoparticles were prepared by electrospray method. The influence of independent parameters such as concentration, flow rate and nozzle to collector distance was studied on particle size and size distribution of generated nanoparticles using a Box-Behnken experimental design. Smallest size was found to be obtained at minimum value for both flow rate and concentration of polymer, regardless of collecting distance value in the ranges studied. Additionally, the minimum value of size distribution was observed at lowest values of both concentration of polymer and collecting distance, regardless of flow rate value. In total, a sample with minimum size and polydispersity was predicted to have flow rate, polymer concentration and collecting distance values of 0.06(ml/h), 0.5(%w/w) and 9.28(cm), respectively. The experimentally prepared nanoparticles with lowest size and size distribution values, had a size of 122(nm) and size distribution of 24. Zeta potential, drug loading and encapsulation efficiency of optimized nanoparticles were -6.58, 5% and 54.5%, respectively.

Synaptosomal acetylcholinesterase activity variation pattern in the presence of electromagnetic fields.

Acetylcholinesterase (AChE) is the enzyme that controls the acetylcholine (ACh) concentrations in cholinergic synaptic clefts by hydrolyzing ACh to choline and acetate. Cholinergic synapses are involved in important functions such as learning, memory and cognition. In this study, we investigated the effects of a wide range of extremely low frequency electromagnetic fields (ELF-EMFs) on synaptic ACh concentrations through AChE enzyme activity assay. Synaptosome suspensions were prepared as a neural terminus from cerebral cortex of sheep brain. Prepared synaptosomes were exposed to ELF-EMFs with frequency ranging from 50 Hz to 230 Hz for duration between 15 and 120 min and flux intensity between 0.1 mT and 1.7 mT. Consequently, AChE activity was measured by Ellman method. Raw data were analyzed by neural network based software, Inform 4.02, to predict AChE activity pattern through nine 3D curves. These curves showed that AChE activity decreases when exposed to ELF-EMFs of 1.2 mT to 1.7 mT intensity and 50 Hz to 90 Hz frequency. Thus, it is proposed that exposure to fields of in this range of frequency-intensity would be effective in clinical treatments of cholinergic disorders to increase synaptic ACh concentration. However, more in vivo experiments are needed to develop this suggested treatment.