N. I. Larionova

Entrapment of alpha-chymotrypsin into hollow polyelectrolyte microcapsules.

Stable hollow polyelectrolyte capsules were produced by the layer by layer assembling of non-biodegradable polyelectrolytes - poly(allylamine) and poly(styrenesulfonate) on melamine formaldehyde micro cores followed by the core decomposition at low pH. A proteolytic enzyme, a-chymotrypsin, was encapsulated into these microcapsules with high yields of up to 100%. The encapsulation procedure was based on the protein adsorption onto the capsule shells and on the pH-depend ent opening and closing of capsule wall pores. The protein in the capsules retained a high activity, and thermo- and storage stability. The nanostructured polyelectrolyte shell protected the proteinase from a high molecular weight inhibitor. Such enzyme-loaded capsules can be used as microreactors for biocatalysis.

Mucoadhesive drug delivery systems (Review)

This review addresses contemporary mucoadhesive drug delivery systems. The use of hydrophilic polymers increases the retention time of the delivery system on mucosal tissues, leading to the gradual release of the active ingredient and better tolerance by the patient. The mucoadhesive interaction is explained in relation to the structural characteristics of mucosal tissues and the properties of the polymers. A separate section addresses the advantages and disadvantages of various mucoadhesive drug delivery systems (tablets, films, gels, microcapsules, and nanocarriers) and developed and commercially available medicinal formulations based on mucoadhesive polymers.

Inclusion of Proteins into Polyelectrolyte Microparticles by Alternative Adsorption of Polyelectrolytes on Protein Aggregates

A new method of protein immobilization into polyelectrolyte microparticles by alternative adsorption of the oppositely charged polyelectrolytes on the aggregates obtained by salting out of protein is proposed. The model protein α-chymotrypsin (ChT) was included in the polyelectrolyte microparticles obtained by various number of polyelectrolyte adsorption steps (from 1 to 11). The main parameters of ChT inclusion into microparticles were calculated. Scanning electron and optical microscopy were used for characterization of morphology and determination of particle size which was from 1 to 10 μm in most cases. It was shown that the size and shape of protein-containing particles and protein aggregates used as a matrix were similar. Change in ChT enzymatic activity during entrapment into polyelectrolyte particles and activity of released protein were studied. The effect of pH on release of incorporated proteins was investigated; it was shown that change in pH and the number of polyelectrolyte adsorption steps allows protein release to be manipulated.

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.

New protease inhibitors from buckwheat seeds: properties, partial amino acid sequences and possible biological role.

Abstract Preparations of new low molecular weight protein inhibitors of serine proteinases have been obtained from buckwheat Fagopyrum esculentum seeds by chromatography of seed extracts on trypsinSepharose 4B, MonoQ and MonoS ionexchangers. Their molecular masses, determined by mass spectrometry, were equal to 5203 (BWI-1c), 5347 (BWI-2c), 7760 (BWI-3c) and 6031 daltons (BWI-4c). All inhibitors possessed high pHstability in the pH range 212 and thermostability. In addition to trypsin, BWI-3c and BWI-4c inhibitors inhibited chymotrypsin and subtilisinlike proteases. The inhibition constants (K) for trypsin, chymotrypsin and subtilisin by the studied inhibitors were determined. The Nterminal sequences of all inhibitors were established: BWI-1c (23 residues), BWI-2c (33 residues), BWI-3c (18 residues) and BWI-4c (20 residues). According to the physicochemical properties and Nterminal amino acid sequences, buckwheat seed protease inhibitors BWI-3c and BWI-4c are suggested to belong to the potato proteinase inhibitor I family.

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.