Shan-Jing Yao

Preparation and characterization of biocompatible microcapsules of sodium cellulose sulfate/chitosan by means of layer-by-layer self-assembly.

A novel microcapsule system composed of sodium cellulose sulfate (NaCS) and chitosan was prepared by layer-by-layer (LbL) self-assembly technique. As a potential drug delivery system, it had several advantages in biocompatibility and biodegradation due to its use of natural polysaccharides. Some parameters in the preparation of the microcapsule, such as layers in the LbL process (up to 20 layers), chitosan viscosities (50, 100, and 200 mPas), outmost layer materials (NaCS or chitosan), and NaCl concentrations (0 M, 0.5 M, 1 M), were investigated to better understand their effects on the LbL process. A novel method for removing the core templates PLA and CaCO3 from the formed core-shell structure was proposed by addition of N-methyl pyrrolidone or EDTA-Na. This preparation process was well observed by a microscope-camera system. The hollow microcapsules were tested by scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and scanning probe microscopy (SPM) to characterize their size, morphology, and surface. The results showed that the shell thickness of microcapsules was about 6 nm, and the average thickness of one layer of NaCS/chitosan polyelectrolyte complex was 1.5 nm. The molecular weight cutoff in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed about 70 kDa by using of diffusion test with the cell lysate of Escherichia coli.

Purification and Characterization of Glutamate Decarboxylase of Lactobacillus brevis CGMCC 1306 Isolated from Fresh Milk

Abstract A Lactobacillus brevis CGMCC 1306 isolated from fresh milk without pasteurization was found to have higher glutamate decarboxylase (GAD) activity. An effective isolation and purification procedure of GAD from a cell-free extract of Lactobacillus brevis was developed, and the procedure included four steps: 30%—90% saturation (NH 4 ) 2 SO 4 fractional precipitation, Q sepharose FF anion-exchange chromatography, sephacryl S-200 gel filtration, and resource Q anion-exchange chromatography. Using this protocol, the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purification fold and activity recovery of GAD were 43.78 and 16.95%, respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37°C, respectively. The purified GAD had a half-life of 50min at 45°C and the K m value of the enzyme from Lineweaver-Burk plot was found to be 8.22. 5′-pyridoxal phosphate (PLP) had little effect on the regulation of its activity.

An improved process for the preparation of sodium cellulose sulphate

Abstract Based on the reaction mechanism of cellulose sulphation, an improved process for the preparation of sodium cellulose sulphate (NaCS) was developed, in which the reaction solution could be regenerated by adding fuming H 2 SO 4 (oleum) and/or normal H 2 SO 4 and used many times. The NaCS prepared in this process showed the same qualities as that obtained with fresh reaction solution. This regeneration method successfully improved the process of production of NaCS, i.e. the costs of production and corrosive waste were greatly reduced.

Molecular insight into the ligand-IgG interactions for 4-mercaptoethyl-pyridine based hydrophobic charge-induction chromatography.

Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the ligand is a novel technology for antibody purification. In the present work, the molecular simulation methods were used to investigate the interactions between MEP ligand and Fc fragment of IgG (Fc-A). Six ligands with different structures of spacer arm were studied with molecular docking and dynamics simulation at neutral and acidic pH. The binding modes and the interaction energies were analyzed. The results indicated that all ligands tested could bind into the selected pocket on the C(H2) domain of Fc-A at neutral pH. The pyridine ring on the top of MEP ligands acts as a major role to provide the hydrophobic association and hydrogen bond for the ligand-IgG binding; meanwhile, the sulfone group on the spacer arm might form the additional hydrogen bond and enhance the binding of ligand onto the surface of IgG. The replacements of thioether sulfur atom on the spacer arm with either nitrogen or oxygen atom seem to have little influence on the binding. The influences of pH on the ligand-IgG interactions were also studied with the molecular dynamics simulation. It was found that MEP ligands would departed from the surface of Fc-A at low pH due to the electrostatic repulsion. The ligands with a sulfone group on the spacer arm would weaken the electrostatic repulsion and need more acidic conditions for the departing of ligand. The molecular simulation results were in agreement with some experimental observations, which would be useful to elucidate the molecular mechanism of HCIC and design a novel ligand to improve the efficiency of antibody separation.

Enhancing IgG purification from serum albumin containing feedstock with hydrophobic charge-induction chromatography.

Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the ligand is a novel technology for antibody purification, however, the separation selectivity still needs to be improved for the applications, especially for the impurity of serum albumin. In this study, with bovine serum immunoglobulin G (IgG) as the model, the purification of IgG from the serum albumin containing feedstock was developed with the commercial HCIC resin MEP HyperCel, focusing on the optimization of operation pH and salt addition. The adsorption isotherms of IgG and bovine serum albumin (BSA) were investigated at different pHs, and the binding and elution behaviors of two proteins in the column were also studied at varying pHs. In addition, the protein-ligand interactions were investigated with some additives in the buffer. It was found that the conditions of pH 6 with 0.1 M NaCl or pH 8 could be used to effectively remove BSA from the MEP resin without the influence on IgG adsorption. Two modes with control of loading or washing buffer were tested to enhance the purification of IgG from BSA containing feedstock, and the purity of IgG was improved to about 95% compared with 62.9% for the control. The results demonstrated that the control of loading pH or the addition of NaCl in the buffer might be an effective method to improve the purification of antibody with the HCIC process.

Modeling the protein partitioning in aqueous polymer two-phase systems: influence of polymer concentration and molecular weight

Aqueous polymer two-phase system provides a powerful method for separation and purification of biomaterials. Among various factors, polymer concentration and polymer molecular weight are essential and have strong impact on the protein partitioning in these systems. Based on the modified Pitzers model, a simple expression has been obtained for correlating protein partitioning in aqueous polymer two-phase systems with varying polymer concentration and different polymer molecular weights. Using only one group of parameters for each target protein, the partition coefficients of five proteins (lysozyme, chymotrypsinogen-A, bovine serum albumin, transferrin and catalase) in 16 sets of polyethylene glycol (PEG)/dextran systems were correlated. In addition, partition behavior of lactate dehydrogenase in PEG/hydroxypropyl starch systems were measured, correlated and partially predicted. A comparison of calculated and experimental data indicated that the model provides good correlation and prediction abilities on the protein partitioning in aqueous polymer two-phase systems with a wide range of polymer concentration and molecular weight.

INVESTIGATION OF PHASE EQUILIBRIUM FOR TERNARY SYSTEMS CONTAINING ETHANOL,WATER AND CARBON DIOXIDE AT ELEVATED PRESSURES

Abstract Using a two-phase circulation apparatus, phase equilibrium data at elevated pressures for ternary systems consisting of CO 2 , C 2 H 5 OH and H 2 O have been measured. A new method for the calculation of liquid phase compositions from P, T , and supercritical fluid phase compositions by the modified Peng-Robinson equation is proposed. The applicability of this method is examined for our experimental data and literature data. As a result, it is confirmed that the method can represent the experimental data with good accuracy.

Evaluation of immunoglobulin adsorption on the hydrophobic charge-induction resins with different ligand densities and pore sizes

Hydrophobic charge-induction chromatography (HCIC) is a novel technology for antibody purification. The ligand densities and pore properties of HCIC resins have significant effects on the separation behavior of protein, however, the understandings are quite limited. In the present work, new HCIC ligand, 2-mercapto-1-methylimidazole (MMI) was coupled to three agarose matrices with different pore sizes. A series of MMI resins with different ligand density and pore size was prepared by the control of ligand coupling. The adsorption isotherms and kinetics on the series of MMI resins were investigated with bovine serum immunoglobulin as the model IgG, and the effects of salt addition were studied. The Langmuir equation and pore diffusion model were used to fit the experimental data, and the influences of ligand density, pore size and salt addition on the saturated adsorption capacity, the dissociation constant and the effective diffusivity were discussed. It was found that the adsorption capacities and the effective pore diffusion coefficient increased with the increase of ligand density and pore size. The effects of salt addition on the adsorption behaviors were dependent on the ligand density. For low ligand density the IgG adsorption was salt-promoted, while the resins with high ligand density showed a salt-independent property. The results indicated that for a given protein the ligand density and pore size of HCIC resins should be optimized for improving the protein adsorption.

Preparation of ibuprofen-loaded chitosan films for oral mucosal drug delivery using supercritical solution impregnation.

Drug-loaded chitosan films suitable for oral mucosal drug delivery were prepared using supercritical solution impregnation (SSI) technology. Firstly, chitosan films were obtained via casting method, and the film properties including water-uptake, erosion and mucoadhesive were characterized. SSI process was then employed to load the drug of ibuprofen onto the prepared chitosan films, and the effects of impregnation pressure and temperature on morphologies of the ibuprofen-loaded chitosan films and drug loading capacity (DLC) were studied. The SEM and X-ray diffraction patterns suggested that distinct ibuprofen shapes such as microparticles, flake, rod-like and needle-like occurred after impregnation at different pressures, and DLC varied from 7.9% to 130.4% during the SSI process. The ex vivo release profiles showed that ibuprofen-loaded chitosan films could deliver the drug across the rabbit buccal mucosa, and up to 70% of the ibuprofen was released from the matrix in 460 min. SSI process is a promising method to prepare drug-loaded film formulations for oral mucosal drug delivery, which provides the advantages of low solvent residual and sustained- and controlled- release behavior.

On-column refolding of recombinant human interferon-γ with an immobilized chaperone fragment

The chaperone mini‐GroEL is a soluble recombinant fragment containing the 191–345 amino acid sequence of GroEL with a 6×His tag. The refolding protocol assisted with mini‐GroEL was studied for the activity recovery of rhIFN‐γ inclusion bodies. In a suspended system, mini‐GroEL showed significant enhancement of the activity recovery of rhIFN‐γ, applyed with a 1–5:1 stoichiometry of mini‐GroEL to rhIFN‐γ at 25 °C. Moreover, 1 M urea in the renaturation buffer had a synergistic effect on suppressing the aggregation and improving the activity recovery. Finally, a novel chromatographic column, containing 1 cm height of Sephadex G 200 at the top of column and packed with immobilized mini‐GroEL to promote refolding, was devised. The total activity recovered per milligram of denatured rhIFN‐γ was up to 3.93 × 106 IU with the immobilized mini‐GroEL column, which was reused four times without evident loss of renaturation ability. A convenient technique with the integrated process of chaperon preparation and rhIFN‐γ folding in vitro was developed.