N. G. Balabushevich

Fabrication and Characterization of Polyelectrolyte Microparticles with Protein

The incorporation of proteins into microparticles fabricated by layer-by-layer adsorption of oppositely charged polyelectrolytes (dextran sulfate and protamine) on protein microaggregates was studied. Microaggregates with insulin were prepared by two different techniques: 1) formation of insoluble polyelectrolyte complex consisting of insulin and dextran sulfate (aggregate size of 7-20 μm), or 2) salting out of insulin from solution by sodium chloride (aggregate size of 5-13 μm). Microparticles varying in the number of cycles (from 1 to 8) of polyelectrolyte adsorption on protein aggregates were examined and compared. Morphology of the microparticles was studied by scanning electron and optical microscopy. It was shown that polyelectrolyte microparticles retained the shape and dimensions of the initial protein aggregates used as a template. Ultrasonication of microparticles obtained using salted out protein aggregates resulted in the formation of stable nanoparticles (100-200 nm). Regulation of protein release from the microparticles of both types by varying the number of polyelectrolyte adsorption cycles and pH of the medium was demonstrated. Insulin not bound to polyelectrolytes was released from the microparticles at pH values between 6 and 8, which corresponds to the pH of the human small intestine and ileum.

Polyelectrolyte assembling for protein microencapsulation

We have developed a novel microencapsulation technique based on covering micronized protein-containing aggregates with a polyelectrolyte multilayer. Here we describe the procedure of the incorporation of pancreatic proteases or ovalbumin into microparticles by layer-by-layer adsorption of oppositely charged polyelectrolytes (dextran sulfate, chitosan) on a proteinaceous core and give an account of the impact of the microencapsulation protocol on the protein release parameters. Insoluble protein-polyanion microaggregates were used as templates. Microparticles varying in the number of cycles of polyelectrolyte adsorption on protein-polyanion aggregates were examined and compared. Polyelectrolyte microparticles were shown to retain the shape and dimensions of the initial template. The microparticles were stable at pH below 5; at pH close to the pH of small intestine a protein release started. The higher the number of polyelectrolyte adsorption cycles was, the lower was the protein release rate. The microparticles obtained seem to be a promising vehicle for oral protein delivery.

Mucoadhesive polyelectrolyte microparticles containing recombinant human insulin and its analogs aspart and lispro

Microparticles containing recombinant human insulin and its analogs aspart and lispro were prepared using an alternate adsorption of chitosan and dextran sulfate from solutions onto microaggregates of protein-dextran sulfate insoluble complex. The following properties of polyelectrolyte hormone-containing microparticles were studied: pH stability, surface charge, mucoadhesive properties, Ca2+ binding, degradation under the influence of proteases (trypsin, chymotrypsin). The influence of the self-association ability of encapsulated insulins on the form of protein releasing from microparticles was studied. Insulins aspart and lispro released from the microparticles as monomers were more liable to proteolysis than human insulin released as a hexamer. The combined effect of properties of polyelectrolyte microparticles and of encapsulated recombinant proteins on the bioavailability of insulin under peroral administration is discussed.

Use of Protease Inhibitors in Composite Polyelectrolyte Microparticles in Order to Increase the Bioavailability of Perorally Administered Encapsulated Proteins

Protein protease inhibitors (aprotinin, soybean Bowman–Birk inhibitor, and Kunitz soybean trypsin inhibitor) possessing different specificity with respect to trypsin, chymotrypsin, and elastase were encapsulated together with a cargo protein in polyelectrolyte microparticles using layer-by-layer (LbL) deposition techniques. The most efficient inclusion of the inhibitors occurred at the formation stage of the insoluble protein complex with the polyanion. Simultaneous immobilization of the inhibitor and protein did not influence the physicochemical properties of the microparticles, specifically their pH-sensitive behavior under conditions modeling the passage through various parts of the human gastrointestinal tract after peroral administration. The most effective protection against the action of proteolytic enzymes of pancreatic juice and the small intestine was achieved for simultaneous release of cargo protein and inhibitor from the microparticles. Soybean Bowman–Birk inhibitor, which is most similar to insulin with respect to physicochemical properties, in addition to the soybean extract enriched with protease inhibitors were the most suitable agents for protection of human insulin or its rapidly acting analogs (lispro and aspart). These findings suggested that simultaneous microencapsulation of both protein and protein protease inhibitor was a promising way to increase the protein bioavailability upon peroral administration of polyelectrolyte microparticles.

Protein microparticles with controlled stability prepared via layer-by-layer adsorption of biopolyelectrolytes

This review covers the experimental data on the preparation and characterization of protein microparticles with controlled stability that are formed by layer-by-layer adsorption of oppositely charged macromolecules. Variants of using proteins as adsorbed polyelectrolyes, methods of incorporating proteins into matrixes (aggregates and microspheres) for further deposition of biopolyelectrolytes, and immobilization of proteins in preformed multilayered polyelectrolyte particles due to a change in the permeability of their shells are considered. Special attention is given to biocompatible and biodegradable microparticles characterized by depot functions, that is, the ability to reliably protect biologically active compounds from aggregative media of the body and to quantitatively release protein preparations (hormones, enzymes, and peptides) into solution when a certain acidity of solution is attained. This feature is especially important for designing peroral means of protein delivery.

Encapsulation of Catalase in Polyelectrolyte Microspheres Composed of Melamine Formaldehyde, Dextran Sulfate, and Protamine

Immobilization of catalase (molecular weight 240,000 daltons) in polyelectrolyte microspheres was studied. The microspheres were obtained by alternating adsorption of dextran sulfate and protamine on commercially available melamine formaldehyde cores followed by the core hydrolysis at pH 1.7. As the interior of the microspheres was filled with homogeneous matrix, the catalase distribution inside the microspheres was uniform. The quantity of entrapped catalase was dependent on the initial concentration of the enzyme and pH of solution, and the peak value was 108-109 molecules per microsphere. It was demonstrated that catalase was entrapped in the microspheres via electrostatic and hydrophobic interactions. The catalase activity inside the microspheres increased as the quantity of enzyme decreased, which was due to the switch between diffusion and kinetic regimes of the enzymatic reaction. The microspheres could be applied for separation and concentration of high molecular weight proteins.

Protein-loaded microspheres prepared by sequential adsorption of dextran sulphate and protamine on melamine formaldehyde core.

Polyelectrolyte multilayer microspheres were fabricated by layer-by-layer self-assembly of a dextran sulphate and a protamine on melamine formaldehyde cores, followed by the partial decomposition of the core. Effects of pH on the encapsulation of proteins and enzymes with different physico-chemical properties (insulin, aprotinin, peroxidase, glucose oxidase (GOD), catalase (Cat)) in the prepared microspheres were then studied. This method of protein encapsulation demonstrated a high loading capacity and efficiency. The protein incorporation and release was regulated by the pH of the solution. Encapsulated enzymes retained a high specific activity depending on the amount of protein incorporated. Bienzyme system GOD/Cat immobilized in the microspheres was suitable for the glucose content assay.

Effects of ultra violet radiation on the soluble proteins of human hair

Exposure of hair fibers from healthy volunteers to Ultra Violet Radiation (UVR) under laboratory conditions enhanced protein elution from the hair tresses into a buffer solution (pH 10.5). At the same time the UVR decreased the intensity of tryptophan fluorescence in the eluted proteins. After mechanical homogenization of these hair samples, the increase of soluble protein was registered for UVR treated hair as well as the rise in sulfhydryl group content of these proteins. Analysis of soluble proteins from hair samples homogenized before and after protein elution has shown that mainly proteins rich in sulfhydryl groups were eluted and as a result sulfhydryl content of proteins in hair shaft decreased. The hypothesis concerning the effects of environmental factors on the properties of hair shaft proteins was examined, the proximal and distal parts of normal hair (0-5 cm and 15-20 cm from hair root) were compared. In the distal parts there was a higher quantity of soluble proteins registered after homogenization, with decreased sulfhydryl group content and tryptophan fluorescence. It could be supposed that this difference results from the steady rupture of cystine in sulfur bridges and tryptophan under exposure to environmental factors (mainly, UVR), followed by elution of the resulting peptides.

Photoinduced formation of thiols in human hair

Raman, scanning electron, and optical microscopy of hair and spectrophotometry of soluble hair proteins are used to study the effect of UV-vis radiation on white hair. The samples of a healthy subject are irradiated using a mercury lamp and compared with non-irradiated (control) hair. The cuticle damage with partial exfoliation is revealed with the aid of SEM and optical microscopy of semifine sections. Gel filtration chromatography shows that the molecular weight of soluble proteins ranges from 5 to 7kDa. Absorption spectroscopy proves an increase in amount of thiols in a heavier fraction of the soluble proteins of irradiated samples under study. Raman data indicate a decrease in the amount of SS and CS bonds in cystines and an increase in the amount of SH bonds due to irradiation. Such changes are more pronounced in peripheral regions of hair. Conformational changes of hair keratins presumably related to the cleavage of disulfide bonds, follow from variations in amide I and low-frequency Raman bands. An increase in the content of thiols in proteins revealed by both photometric data on soluble proteins and Raman microspectroscopy of hair cuts can be used to develop a protocol of the analysis of photoinduced hair modification.

Layer-by-layer adsorption of biopolyelectrolytes as a universal approach to fabrication of protein-loaded microparticles

This study was aimed at examination of microparticles formed via the layer-by-layer adsorption of dextran sulfate and chitosan onto the insoluble complexes of various proteins with polyanions. Microparticles with all tested proteins were stable at pH values of 1–5. At pH > 6 the mucoadhesivity of the microparticles changed and the encapsulated proteins were released. Microparticles were able to protect the proteins from proteases. Proteinous protease inhibitors encapsulated as well (2–3%) completely prevented protein proteolysis. The pharmacological effect of microencapsulated insulin was studied in vivo using the model of chronic diabetes in rats, which were treated by oral administration.