Expression, Purification, and Biological Evaluation of XTEN-GCSF in a Neutropenic Rat Model.

Granulocyte colony-stimulating factor (GCSF) stimulates the proliferation of neutrophils but it has low serum half-life. Therefore, the present study was done to investigate the effect of XTENylation on biological activity, pharmacokinetics, and pharmacodynamics of GCSF in a neutropenic rat model. XTEN tag was genetically fused to the N-terminal region of GCSF-encoding gene fragment and subcloned into pET28a expression vector. The cytoplasmic expressed recombinant protein was characterized through intrinsic fluorescence spectroscopy (IFS), dynamic light scattering (DLS), and size exclusion chromatography (SEC). In vitro biological activity of the XTEN-GCSF protein was evaluated on NFS60 cell line. Hematopoietic properties and pharmacokinetics were also investigated in a neutropenic rat model. An approximately 140 kDa recombinant protein was detected on SDS-PAGE. Dynamic light scattering and size exclusion chromatography confirmed the increase in hydrodynamic diameter of GCSF molecule after XTENylation. GCSF derivatives showed efficacy in proliferation of NFS60 cell line among which the XTEN-GCSF represented the lowest EC50 value (100.6 pg/ml). Pharmacokinetic studies on neutropenic rats revealed that XTEN polymer could significantly increase protein serum half-life in comparison with the commercially available GCSF molecules. PEGylated and XTENylated GCSF proteins were more effective in stimulation of neutrophils compared to the GCSF molecule alone. XTENylation of GCSF represented promising results in in vitro and in vivo studies. This approach can be a potential alternative to PEGylation strategies for increasing serum half-life of protein.

Extension of human GCSF serum half-life by the fusion of albumin binding domain.

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

SN38 loaded nanostructured lipid carriers (NLCs); preparation and in vitro evaluations against glioblastoma.

SN38 is the active metabolite of irinotecan with 1000-fold greater cytotoxicity compared to the parent drug. Despite the potential, its application as a drug is still seriously limited due to its stability concerns and low solubility in acceptable pharmaceutical solvents. To address these drawbacks here nanostructured lipid carrier (NLC) containing SN38 was prepared and its cytotoxicity against U87MG glioblastoma cell line was investigated. The formulations were prepared using hot ultrasonication and solvent evaporation/emulsification methods. NLCs with a mean size of 140 nm and particle size distribution (PDI) of 0.25 were obtained. The average loading efficiency was 9.5% and its entrapment efficiency was 81%. In order to obtain an accurate determination of released amount of SN38 a novel medium and extraction method was designed, which lead to an appropriate in vitro release profile of the drug from the prepared NLCs. The MTT test results revealed the significant higher cytotoxicity of NLCs on U87MG human glioblastoma cell line compared with the free drug. The confocal microscopy images confirmed the proper penetration of the nanostructures into the cells within the first 4 h. Consequently, the results indicated promising potentials of the prepared NLCs as a novel treatment for glioblastoma.

Modified fractal iron oxide magnetic nanostructure: A novel and high performance platform for redox protein immobilization, direct electrochemistry and bioelectrocatalysis application.

A novel biosensing platform based on fractal-pattern of iron oxides magnetic nanostructures (FIOMNs) and mixed hemi/ad-micelle of sodium dodecyl sulfate (SDS) was designed for the magnetic immobilization of hemoglobin (Hb) at a screen printed carbon electrode (SPCE). The FIOMNs was successfully synthesized through hydrothermal approach and characterized by atomic force microscopy (AFM), scanning electron microscopy (SEM) and X-ray diffraction (XRD). In order to provide guidelines for the mixed hemi/ad-micelle formation, zeta-potential isotherms were investigated. The construction steps of the biosensor were evaluated by electrochemical impedance spectroscopy, cyclic voltammetry and Fourier transform infrared spectroscopy. Direct electron transfer of Hb incorporated into the biocomposite film was realized with a pair of quasi-reversible redox peak at the formal potential of -0.355V vs. Ag/AgCl attributing to heme Fe(III)/Fe(II) redox couple. The results suggested that synergistic functions regarding to the hyper-branched and multidirectional structure of FIOMNs and the dual interaction ability of mixed hemi/ad-micelle array of SDS molecules not only induce an effective electron transfer between the Hb and the underlying electrode (high heterogeneous electron transfer rate constant of 2.08s(-1)) but also provide powerful and special microenvironment for the adsorption of the redox proteins. Furthermore, the biosensor displayed an excellent performance to the electrocatalytic reduction of H2O2 with a detection limit of 0.48µM and Michaelis-Menten constant (Km) value of 44.2µM. The fabricated biosensor represented the features of sensitivity, disposable design, low sample volume, rapid and simple preparation step, and acceptable anti-interferences, which offer great perspectives for the screen-determination of H2O2 in real samples.

Determination of the role of calcium on instability of neurotoxic metabolite of ecstasy by HPTLC-mass.

BACKGROUND: Ecstasy is one of the popular illicit drugs in the world and its usage has been recently increased in Iran. This compound can destroy the serotonergic neurons and produces cognitive and psychopathology diseases. 3,4-dihydroxymethamphetamine (HHMA) which is the main metabolite of this compound, seems to be responsible for this effect. However, no consensus has been reached among the researchers about its role. This disagreement between the researches may be due to failure in determination of HHMA as free form in physiological fluids. In this study, the stability of this crucial metabolite of ecstasy was examined in different mediums. METHODS: The stability of HHMA was studied in the perfusion medium and water at 100 and 10 ng/mL concentrations. Moreover, the effect of temperature (0-25°C), pH (3-10), calcium chloride (0-150 g/L) and ethylenediaminetetraacetic acid (EDTA) on the stability of HHMA was also examined. RESULTS: Our result suggested that the free form of HHMA could be degraded in the perfusion medium. The rate of this degradation has direct proportion to temperature (at 25°C = 0.037 min-1 and at 0°C = 0.002 min-1). Calcium chloride and sodium bicarbonate are two responsible components in this instability. Moreover, the alkaline pHs and increasing the shaking time can accelerate this effect. Although, while degradation was prevented at pH=3, EDTA could only reduce this rate about 30%. CONCLUSIONS: Calcium cation can act as an accelerator of HHMA degradation. Therefore, the perfusion medium should not contain Ca2+ and the pH of medium is better to be adjusted at acidic range. Since, the internal cellular source of calcium is endoplasmic reticulum system, it can be assumed that, this cation may change HHMA and dopamine to reactive compounds that can bind covalently to the cysteinyl group of biological compounds and damage cellular components.