Immobilization of laccase on itaconic acid grafted and Cu(II) ion chelated chitosan membrane for bioremediation of hazardous materials

Background: Chitosan membranes were formed through a phase inversion technique and then cross-linked with epichlorohydrin (CHX). Heterogeneous graft copolymerization of itaconic acid (IA) onto membrane was carried out with different monomer concentrations (CHX-g-p(IA)). The membrane properties such as equilibrium swelling ratio, porosity, and contact angle were measured, together with analysis by scanning electron microscopy (SEM), energy dispersive analysis of X-rays (EDAX), atomic force microscopy (AFM), and Fourier transform infrared (FTIR) spectroscopy. Results: The Cu(II) ion incorporated membranes (i.e. CHX-g-p(IA)-Cu(II)) were used for reversible immobilization of laccase using CHX-g-p(IA) membrane as a control system. Maximum laccase adsorption capacities of the CHX-g-p(IA) and CHX-g-p(IA)-Cu(II) membranes (with 9.7% grafting yield) were found to be 6.3 and 17.6 mg mL -1 membrane at pH 4.0 and 6.0, respectively. The K m value for immobilized laccase on CHX-g-p(IA)-Cu(II) (4.16 × 10 -2 mmol L -1) was 2.11-fold higher than that of free enzyme (1.97 × 10 -2 mmol L -1). Finally, the immobilized laccase was used in a batch system for degradation of three different dyes (Reactive Black 5, RB5; Cibacron Blue F3GA, CB; and Methyl Orange, MO). The immobilized laccase on CHX-g-p(IA)-Cu(II) membrane was more effective for removal of MO dye than removal of CB and RB5 dyes. CONCLUSION: Flexibility of the enzyme immobilized grafted polymer chains is expected to provide easy reaction conditions without diffusion limitation for substrate dye molecules and their products. The support described, prepared from green chemicals, can be used for the immobilization of industrially important enzymes. © 2012 Society of Chemical Industry

Poly (hydroxyethyl methacrylate-glycidyl methacrylate) films modified with different functional groups: In vitro interactions with platelets and rat stem cells

Copolymerization of 2-hydroxyethylmethacrylate (HEMA) with glycidylmethacrylate (GMA) in the presence of α-α′- azoisobisbutyronitrile (AIBN) resulted in the formation of hydrogel films carrying reactive epoxy groups. Thirteen kinds of different molecules with pendant NH2 group were used for modifications of the p(HEMA-GMA) films. The NH2 group served as anchor binding site for immobilization of functional groups on the hydrogel film via direct epoxy ring opening reaction. The modified hydrogel films were characterized by FTIR, and contact angle studies. In addition, mechanical properties of the hydrogel films were studied, and modified hydrogel films showed improved mechanical properties compared with the non-modified film, but they are less elastic than the non-modified film. The biological activities of these films such as platelet adhesion, red blood cells hemolysis, and swelling behavior were studied. The effect of modified hydrogel films, including NH2, (using different aliphatic CH2 chain lengths) CH3, SO3H, aromatic groups with substituted OH and COOH groups, and amino acids were also investigated on the adhesion, morphology and survival of rat mesenchymal stem cells (MSCs). The MTT colorimetric assay reveals that the p(HEMA-GMA)-GA-AB, p(HEMA-GMA)-GA-Phe, p(HEMA-GMA)-GA-Trp, p(HEMA-GMA)-GA-Glu formulations have an excellent biocompatibility to promote the cell adhesion and growth. We anticipate that the fabricated p(HEMA-GMA) based hydrogel films with controllable surface chemistry and good stable swelling ratio may find extensive applications in future development of tissue engineering scaffold materials, and in various biotechnological areas. © 2012 Elsevier B.V

Examination of fabrication conditions of acrylate-based hydrogel formulations for doxorubicin release and efficacy test for hepatocellular carcinoma cell

The objective of the present study was to develop 2-hydroxypropyl methacrylate-co-polyethylene methacrylate [p(HPMA-co-PEG-MEMA)] hydrogels that are able to efficiently entrap doxorubicin for the application of loco-regional control of the cancer disease. Systemic chemotherapy provides low clinical benefit while localized chemotherapy might provide a therapeutic advantage. In this study, effects of hydrogel properties such as PEG chains length, cross-linking density, biocompatibility, drug loading efficiency, and drug release kinetics were evaluated in vitro for targeted and controlled drug delivery. In addition, the characterization of the hydrogel formulations was conducted with swelling experiments, permeability tests, Fourier transform infrared, SEM, and contact angle studies. In these drug-hydrogel systems, doxorubicin contains amine group that can be expected a strong Lewis acid-base interaction between drug and polar groups of PEG chains, thus the drug was released in a timely fashion with an electrostatic interaction mechanism. It was observed that doxorubicin release from the hydrogel formulations decreased when the density of cross-linking, and drug/polymer ratio were increased while an increase in the PEG chains length of the macro-monomer (i.e. PEG-MEMA) in the hydrogel system was associated with an increase in water content and doxorubicin release. The biocompatibility of the hydrogel formulations has been investigated using two measures: cytotoxicity test (using lactate dehydrogenase assay) and major serum proteins adsorption studies. Antitumor activity of the released doxorubicin was assessed using a human SNU398 human hepatocellular carcinoma cell line. It was observed that doxorubicin released from all of our hydrogel formulations which remained biologically active and had the capability to kill the tested cancer cells. © 2014 Taylor and Francis

Synthesis and spectroscopic characterization of superparamagnetic beads of copolymers of methacrylic acid, methyl methacrylate and ethylene glycol dimethacrylate and their application to protein separation

Novel magnetic beads were prepared from methacrylic acid, methyl methacrylate and ethylene glycol dimethacrylate in the presence of ferric ions via suspension polymerization. After polymerization, thermal treatment of the Fe(III)-ion containing beads with Fe(II) ions under alkaline condition resulted encapsulation of Fe3O4 crystals within the polymer matrix. The magnetic beads were characterized by surface area measurement, swelling tests, electron spin resonance (ESR), differential scanning calorimetry (DSC), differential thermal analysis (DTA) and scanning electron microscopy (SEM). To evaluate the magnetic properties of the composite beads, Mössbauer spectra measurements were carried out. ESR and Mössbauer spectroscopy data revealed that the beads were highly superparamagnetic. The DSC data of the magnetic beads also showed an enhanced glass transition temperature owing to the presence of the Fe3O4 crystals within the polymer network. In addition, the presence of functional carboxyl groups on the surface permits further modifications in future applications. The magnetic beads were used for separation of human serum albumin and cytochrome c from aqueous solution in a batch system. © 2007 Society of Chemical lndustry

Affinity dye-ligand poly(hydroxyethyl methacrylate)/chitosan composite membrane for adsorption lysozyme and kinetic properties (Letter)

[No abstract available

Polyethylenimine-grafted and HSA-immobilized poly(GMA-MMA) affinity adsorbents for bilirubin removal

The epoxy-group-containing microspheres from cross-linked glycidyl methacrylate and methyl methacrylate, poly(GAM-MMA), were prepared by suspension polymerisation. The epoxy groups of the poly(GMA-MMA) microspheres were used for grafting with an anionic polymer polyethylenimine (PEI) to prepare non-specific affinity adsorbents (poly(GMA-MMA)-PEI) for bilirubin removal. The specificity of the poly(GMA-MMA)-PEI adsorbent to bilirubin was further increased by immobilization of human serum albumin (HSA) via adsorption onto PEI-grafted poly(GMA-MMA) adsorbent. Various amounts of HSA were immobilized on the poly(GMA-MMA)-PEI adsorbent by changing the medium pH and initial HSA concentration. The maximum HSA content was obtained at 68.3mg g-1 microspheres. The effects of pH, ionic strength, temperature and initial bilirubin concentration on the adsorption capacity of both adsorbents were investigated in a batch system. Separation of bilirubin from human serum was also investigated in a continuous-flow system. The bilirubin adsorption on the poly(GMA-MMA)-PEI and poly(GMA-MMA)-PEI-HSA was not well described by the Langmuir model, but obeyed the Freundlich isotherm model. The poly(GMA-MMA)-PEI affinity microspheres are stable when subjected to sanitization with sodium hydroxide after repeated adsorption-desorption cycles. © 2004 Society of Chemical Industry

Characterization of β-galactosidase immobilized into poly(hydroxyethyl methacrylate) membranes

β-galactosidase was immobilized into pHEMA membranes with the highest specific activity yield of 9.5%. The specific activity of the entrapped enzyme was found to be decreased as the enzyme loading increased in pHEMA membranes. The optimum pH and temperature for maximum activity of the immobilized βgalactosidase was found to be at pH 7.5 and 50°C, respectively, and were the same as native enzyme. K(m) and V(max) values for the free enzyme were found to be 0.256 mM and 26.6 μmole/min/mg, respectively. K(m) value of immobilized βgalactosidase was found to be increased about 3 folds upon immobilization. Operational, thermal and storage stability of β-galactosidase were found to increase with immobilization. Immobilized enzyme preparation was reused in 15 cycles without significant loss in activity

Affinity microspheres and their application to lysozynia adsorption: Cibacron Blue F3GA and Cu(II) with poly(HEMA-EGDMA)

Lysozyme adsorption onto Cibacron Blue F3GA attached and Cu(II) incorporated poly(2-hydroxyethyl methacrylate-ethylene glycol dimethacrylate) [poly(HEMA-EGDMA)] microspheres was investigated. The microspheres were prepared by suspension polymerization. Various amounts of Cibacron Blue F3GA were attached covalently onto the microspheres by changing the initial concentration of dye in the reaction medium. The microspheres with a swelling ratio of 65%, and carrying different amounts of dye (between 1.4 and 22.5?mol/g-1) were used in the lysozyme adsorption studies. Lysozyme adsorption on these microspheres from aqueous solutions containing different amounts of lysozyme at different pH values was investigated in batch reactors. The lysozyme adsorption capacity of the dye-metal chelated microspheres (238.2mgg-1) was greater than that of the dye-attached microspheres (175.1mgg-1). The maximum lyzozyme adsorption capacities (qm) and the dissociation constant (kd) values were found to be 204.9mgg-1 and 0.0715mgml-1 with dye-attached and 270.7mgg-1 and 0.0583mgml-1 with dye-metal chelated microspheres, respectively. More than 90% of the adsorbed lysozyme were desorbed in 60 min in the desorption medium containing 0.5M KSCN at pH 8.0 or 25mM EDTA at pH 4.9. © 1999 Society of Chemical Industry

Low-molecular-weight heparin-conjugated liposomes with improved stability and hemocompatibility

Multilamellar vesicles (MLV) containing phosphatidyl choline (PC), cholestrol (CHOL), and stearylamine (SA) in the molar ratio of 7:2:0.2 were prepared by the thin film hydration method. Low-molecular-weight heparin (LMWH, MW: 3000) was conjugated with the MLV using carbodiimide (EDC). Infrared, Raman, and nuclear magnetic resonance spectra and DSC of each sample (MLV, LMWH, and MLV-LMWH) were obtained, enabling the authors to determine the chemical changes that occurred in the MLV structure at the end of the conjugation step. In addition, the changes in the chemical structures of the conjugated samples were revealed by the use of elemental analysis. Particle size analysis was used to determine the difference between the sizes of MLV and MLV-LMWH. In order to study the effect of LMWH on the behavior of MLV-LMWH in blood, osmotic fragility (in saline and plasma), hemolytic activity, and plasma recalcification time tests were carried out. These tests showed that it was possible to construct liposomes that would not induce reactions in the blood and would have potentially longer half-lives in the circulation