Immobilization and bioactivity of glucose oxidase in hydrogel microspheres formulated by an emulsification-internal gelation-adsorption-polyelectrolyte coating method.

The purpose of this study was to develop a novel microsphere formulation of glucose oxidase (GOX) with high drug loading, encapsulation efficiency and bioactivity. GOX was encapsulated in alginate/chitosan microspheres (ACMS) using an emulsification-internal gelation, followed by GOX adsorption and polyelectrolyte coating method. The factors influencing GOX loading, encapsulation efficiency and activity of the loaded GOX were investigated. The resultant ACMS in wet state were spherical with a mean diameter of about 138 microm. GOX loading was found to be pH dependent. High GOX loading and encapsulation efficiency were achieved when the pH of the adsorption medium was lower than the isoelectric point (pI) of GOX. GOX loading and encapsulation efficiency increased with increasing GOX concentration in the loading solution, but decreased with increasing chitosan concentration in the coating solution. The activity of loaded GOX increased and then decreased with increasing chitosan concentration. The activity of GOX in ACMS was maintained and showed sustained production of H(2)O(2) as compared to free GOX. Around 90% of the original activity of immobilized GOX remained after lyophilization and storage at -20 degrees C for a month. These results suggest that the ACMS and the fabrication method are suitable for microencapsulation of proteins like GOX.

[1]  M. Appleby,et al.  The pH dependence of the individual steps in the glucose oxidase reaction. , 1969, The Journal of biological chemistry.

[2]  Kai Zhang,et al.  Modulated insulin permeation across a glucose-sensitive polymeric composite membrane. , 2002, Journal of controlled release : official journal of the Controlled Release Society.

[3]  D. Pettit,et al.  Biodegradable polymers for protein and peptide drug delivery. , 1995, Bioconjugate chemistry.

[4]  K. Griebenow,et al.  Stabilization of alpha-chymotrypsin at the CH2Cl2/water interface and upon water-in-oil-in-water encapsulation in PLGA microspheres. , 2003, Journal of controlled release : official journal of the Controlled Release Society.

[5]  A. Fournier,et al.  Production of alginate beads by emulsification/internal gelation. II. Physicochemistry , 1995, Applied Microbiology and Biotechnology.

[6]  G. Roberts.  Analysis of Chitin and Chitosan , 1992 .

[7]  M. Suckow,et al.  Oral vaccination of animals with antigens encapsulated in alginate microspheres. , 1999, Vaccine.

[8]  K. De Yao,et al.  Controlled release of albumin from chitosan-alginate microcapsules. , 1994, Journal of pharmaceutical sciences.

[9]  Y. Ikada,et al.  Entrapment of islets into reversible disulfide hydrogels. , 1998, Journal of biomedical materials research.

[10]  V MASSEY,et al.  PURIFICATION AND PROPERTIES OF THE GLUCOSE OXIDASE FROM ASPERGILLUS NIGER. , 1965, The Journal of biological chemistry.

[11]  J. de la Noüe,et al.  Evaluation of protein release from chitosan-alginate microcapsules produced using external or internal gelation. , 2001, Journal of microencapsulation.

[12]  S. Betigeri,et al.  Immobilization of lipase using hydrophilic polymers in the form of hydrogel beads. , 2002, Biomaterials.

[13]  R. Neufeld,et al.  Poly(methylene co-guanidine) coated alginate as an encapsulation matrix for urease. , 2000 .

[14]  Ick Chan Kwon,et al.  Effects of the controlled-released TGF-beta 1 from chitosan microspheres on chondrocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold. , 2004, Biomaterials.

[15]  N. Peppas,et al.  Hydrogels in Pharmaceutical Formulations , 1999 .

[16]  F. Gu,et al.  Sustained delivery of vascular endothelial growth factor with alginate beads. , 2004, Journal of controlled release : official journal of the Controlled Release Society.

[17]  Ivar Storrø,et al.  Alginate as immobilization material: III. Diffusional properties , 1992, Biotechnology and bioengineering.

[18]  Wayne R. Gombotz,et al.  Calcium-alginate beads for the oral delivery of transforming growth factor-β1 (TGF-β1): stabilization of TGF-β1 by the addition of polyacrylic acid within acid-treated beads , 1994 .

[19]  G. Skjåk-Bræk,et al.  Microcapsules of alginate-chitosan--I. A quantitative study of the interaction between alginate and chitosan. , 1998, Biomaterials.

[20]  F. Lim,et al.  Microencapsulated islets as bioartificial endocrine pancreas. , 1980, Science.

[21]  G. Roberts,et al.  Determination of the viscometric constants for chitosan , 1982 .

[22]  T. Chandy,et al.  Chitosan/calcium–alginate beads for oral delivery of insulin , 1996 .

[23]  Trong-Ming Don,et al.  Free radical degradation of chitosan with potassium persulfate , 2002 .

[24]  T. Ohno,et al.  Controlled release of interleukin-2 for tumour immunotherapy using alginate/chitosan porous microspheres , 1997 .

[25]  M. Baca-Estrada,et al.  Intranasal immunization using biphasic lipid vesicles as delivery systems for OmlA bacterial protein antigen and CpG oligonucleotides adjuvant in a mouse model , 2005, The Journal of pharmacy and pharmacology.

[26]  V. Sinha,et al.  Biodegradable microspheres for protein delivery. , 2003, Journal of controlled release : official journal of the Controlled Release Society.

[27]  D. Poncelet,et al.  External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation. , 1998, Biotechnology and bioengineering.

[28]  Hideo Tanaka,et al.  Diffusion characteristics of substrates in Ca‐alginate gel beads , 1984, Biotechnology and bioengineering.

[29]  N. Özer,et al.  Poly(hydroxybutyrate-co-hydroxyvalerate) nanocapsules as enzyme carriers for cancer therapy: an in vitro study , 2002, Journal of microencapsulation.

[30]  Robert Langer,et al.  Coated alginate microspheres : factors influencing the controlled delivery of macromolecules , 1991 .

[31]  W. Hennink,et al.  The effect of a water/organic solvent interface on the structural stability of lysozyme. , 2000, Journal of controlled release : official journal of the Controlled Release Society.

[32]  M. Schubert,et al.  Characterisation of surface-modified solid lipid nanoparticles (SLN): influence of lecithin and nonionic emulsifier. , 2005, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[33]  A. Fournier,et al.  Production of alginate beads by emulsification/internal gelation. I. Methodology , 1992, Applied Microbiology and Biotechnology.

[34]  Xiaolin Xie,et al.  Effect of drug-loading methods on drug load, encapsulation efficiency and release properties of alginate/poly-L-arginine/chitosan ternary complex microcapsules. , 2004, Macromolecular bioscience.