Suspensions of insoluble polyelectrolyte complexes of dextran sulfate? (DS) of different molecular masses with lactoferrin (LF) have been fabricated and characterized. The encapsulation efficiency of LF and DS in a complex at pH 3.0 and 4.0 was assessed, and particles were characterized by their sizes and zeta-potential. The complexes formed at pH 3.0 differed by a higher stability level. The interaction with DS resulted in a twofold decrease in the antioxidant activity of LF, although the formation of complexes was not accompanied by conformational changes in LF molecules according to IR-spectrometry data. Microencapsulation was carried out by treating the suspensions with negatively charged LF-DS complexes with protamine and chitosane solutions with different molecular masses. The composition, size, and the zeta-potential of interaction products were assessed which allowed us to select the conditions for the preparation of pH-sensitive polyelectrolyte microparticles loaded with LF which would be able to gradually release glycoprotein under conditions that model the passage through the gastrointestinal tract of humans. These data indicate that this approach is promising for the creation of pH-sensitive biopolyelectrolytes suitable for oral administration of LF to target cells.

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.

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

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.

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.

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, Ca(2+) 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.

Thorough investigation and comparative study were conducted for insulin-loaded microparticles fabricated by consecutive adsorption of polyanions (dextran sulfate and chitosan sulfate) and polycations (chitosan and protamine) onto protein microaggregates. The possible regulation of insulin release from the particles by variation in polyelectrolyte pairs, in the number of their adsorption cycles and in pH of media was demonstrated. For all studied cases the microparticles showed protective action towards insulin inactivation at acid pH values and protein release at pH > 5, corresponding to human gastro-intestinal conditions.

The interactions of zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine with protein proteinase inhibitors aprotinin and Bowman-Birk soybean proteinase inhibitor have been investigated. An increase in the hydrophobicity of the liposome surface was shown to be an important factor for the formation of proteoliposomes. According to (31)P-NMR spectra, incorporation of the proteins into the liposomes does not influence the structural organization of the surface of the liposomes. Increasing the ionic strength does not inhibit the process of proteoliposome formation. Fluorescence assay of the complexes of anthracene-labeled phospholipids with the rhodamine B-labeled protein showed that after the encapsulation into the liposomes, the protein is located inside the particles and between the bilayers. Also, the effect of phospholipids with saturated fatty acid residues on the protein-lipid interaction was studied by differential scanning calorimetry. The results indicate that water-soluble proteins efficiently interact with zwitterionic phospholipids, and the encapsulation of the proteins into the liposomes is provided by electrostatic and hydrophobic forces (in the case of aprotinin) or predominantly by hydrophobic forces (Bowman-Birk soybean proteinase inhibitor).

Possibility of encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)) was investigated. The influence of the PC/PE ratio (w/w) on efficiency of incorporation of the Bowman-Birk soybean proteinase inhibitor (BBI) and aprotinin (BPTI) into liposomes was studied. Protein encapsulation did not affect liposome sizes. Confocal laser scanning microscopy demonstrated that proteins were located in the central part of the spherical particle and also between bilayers. The study of biological (antitrypsin and antichymotrypsin) activity demonstrated partial spatial shielding of active sites of proteins entrapped in liposomes. The effect of an ionic detergent on the activity of the encapsulated BBI and BPTI is consistent with this hypothesis and suggests that this shielding is reversible. Stability of liposomes was examined using three various media modeling gastrointestinal fluids (gastric and intestinal juices and fluids). Data obtained indicate that the prepared liposomes seem to be promising formulations for BBI and BPTI delivery.

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.

A novel approach of colorimetric quantification of chitosan based on the derivatization reaction of its primary amino groups with o-phthalaldehyde and a thiol – N-acetyl-l-cysteine has been developed. The reaction of equal volumes of sample solution and the reagent solution was allowed to proceed for 1h, and then the absorbance values were measured at 340nm against the reference solution. The procedure conditions have been optimized for chitosan assay in the presence of polyanionic electrolyte dextran sulphate (pH 8.9, the reagent solution: 4.0mM o-phthalaldehyde, 2.6mM N-acetyl-l-cysteine, 0.25M NaCl). The method has proven to be convenient and reliable for quantitative determination of either the concentrations of chitosans of various molecular weights or their degree of deacetylation. The different reactivity of chitosans and proteins can be used in order to determine chitosan in presence of the protein. This approach ensured accurate assay within the chitosan concentrations ranging from 0.01 to 0.15mg/ml and could be applied for quantitative analysis of chitosan in protein-loaded microparticles.

Interaction between duodenase (a granase family member) from bovine duodenal mucosa and recombinant antichymotrypsin (rACT) and its P1 variants has been studied. Association rate constants (k a) were 11, 6.8, and 17 mM−1·sec−1 for rACT, ACT L358M, and ACT L358R, respectively. Natural antitrypsin (AT) compared to ACT was a 20 times more effective duodenase inhibitor (in terms of k a). Duodenase interacted with P1 variants of ACT via a suicide mechanism with stoichiometry of the process SI = 1.2. The nature of the P1 residue of the inhibitor did not influence the interaction if other residues did not meet conformational requirements of the duodenase substrate-binding pocket. Also, interaction of duodenase with ACT variants containing residues from AT reaction center loop (rACT P2-P3′, rACT P3-P4′, rACT P4-P3′, and rACT P6-P4′) was studied. The inhibition type ([E]0 = 1·10−7 M, 25°C) was revealed to be reversible-like, and efficacy of inhibition decreased with increase in the substituted part of the reactive center loop. Constants of inhibition (K i) were measured. Efficacy of interaction between the enzyme (duodenase) and inhibitor depends on topochemical correspondence between a substrate-binding pocket of the enzyme and substrate structure.

Methods for preparing colloidal delivery systems for drugs of different chemical structures have been developed and optimized. Proteins were encapsulated in bioadhesive biodegradable starch microparticles and liposomes from negatively charged and zwitter-ionic soybean phospholipids. Proteins and a poorly watersoluble anticancer drug-tamoxifen-were encapsulated in nanoparticles based on the amphiphilic graft block copolymer dextran-poly(ɛ-caprolactone). In vitro release studies showed sustained release of proteins and tamoxifen in different media.

The review considers commercial insulin formulations. Special attention is paid to difficulties and strategies of the development of alternative hormone delivery systems (buccal, transdermal, intranasal, pulmonary and oral). At the moment there is only one approved formulation of the noninvasive insulin in the world.

New nanoscaled polymeric carriers have been prepared on the basis of different amphiphilic water-soluble derivatives of poly-N-vinylpyrrolidone (PVP). The polymer self-assembly and interaction with model proteins (Bowman–Birk soybean proteinase inhibitor (BBI) and its hydrophobized derivatives) were studied in aqueous media. The possibility of inclusion of both BBI and hydrophobized oleic acid derivatives of BBI in amphiphilic PVP aggregates was investigated. It was ascertained that polymeric particles of size 50–80 nm were formed in certain concentrations of amphiphilic PVP and poorly soluble dioleic acid derivatives of BBI. Such polymeric aggregates are capable of solubilization of dioleoyl BBI with a concomitant prevention of its inactivation at low pH values.

Graft block copolymers of poly(ɛ-caprolactone) containing various amounts of dextran [dextranpoly(ɛ-caprolactone) copolymers] have been prepared and characterized. The methods of obtaining nanoparticles based on the dextran-poly(ɛ-caprolactone) graft block copolymers have been elaborated and optimized. It has been demonstrated that the electrokinetic potential of nanoparticles may be varied relative to the composition of the polymer, the structure of nanoparticles, and the ionic strength of solution. Under physiological conditions, the nanoparticles behave as stable colloids. On the basis of the experimental evidence, conclusions concerning the structure of the nanoparticles have been made.