[Polyelectrolyte complexes of lactoferrin and pH-sensitive microparticles on their basis].

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.

[Immune reactivity of human lens structures in norm, age-related cortical and secondary opacification].

Using immunohistochemical methods, the immune reactivity of human lens epithelium and fibers to NSE, S-100 protein, Vim, alpha-SMA and EMA was studied in 10 normal persons and in 25 patients with its age-related cortical and secondary cataract. It was demonstrated that in age-related cortical and secondary cataract lens epithelium and fibers became more reactive to antibodies against NSE, S-100 protein and Vim, but showed no immunopositivity to alpha-SMA and EMA. Thus, the data obtained suggest some common pathogenetic mechanisms of age-related cortical and secondary cataract development with the formation of Adamyuk-Elschnig pearls.

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, 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.

Characteristics of binding of zwitterionic liposomes to water-soluble proteins.

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).

Effect of reactive center loop structure of antichymotrypsin on inhibition of duodenase activity.

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 degrees 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.

[Liposomal formulations of protein proteinase inhibitors: preparation and specific activity].

Here we investigated encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)). The influence of the PC/PE w/w ratio on the incorporation efficiency 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 between the bilayers. The biological activity (antitrypsin and antichymotrypsin) assay of the protein entrapped in liposomes showed its active sites were spatially shielded. The effect of an ionic detergent on the activity of the encapsulated BBI and BPTI confirmed this hypothesis and suggested that this shielding is reversible. The liposomes stability in three various media-artificial gastric juice and intestinal fluids was also examined. The liposomes prepared seem to be promising formulations for BBI and BPTI delivery.

[Noninvasive insulin delivery systems].

The review considers commercial insulin formulations. Special attention is paid to the difficulties and strategies of 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.

Interaction between human alpha2-macroglobulin and duodenase, a serine proteinase with dual specificity.

Interaction between a serine proteinase from bovine duodenum and human serum alpha(2)-macroglobulin (alpha(2)-MG) was studied. alpha(2)-MG is established to be one of the most effective duodenase inhibitors. The enzyme is completely inhibited in less than 30 sec at equimolar ratio of the inhibitor and enzyme (concentration 2 x 10(-8) M). Under identical conditions, the rate of duodenase association with alpha(2)-MG is at least 2.5-fold higher than the rate of chymotrypsin association with this inhibitor. The interaction with duodenase results in proteolysis of the inhibitor subunit in the "bait region". Similarly to other proteases, duodenase in the complex with alpha(2)-MG retains the intact catalytic apparatus and ability to hydrolyze some small substrates. But the duodenase-inhibitor complex is fully inactive to proteins (bovine serum albumin). The stoichiometry of the enzyme interaction with the inhibitor is 2 : 1 (mol/mol). Based on the association rate constant and the termination time of the duodenase and alpha(2)-MG in vivo association, alpha(2)-MG is suggested to be a physiological regulator of the enzyme.

Interaction of native Bowman-Birk soybean protease inhibitor and its hydrophobized derivative with multilamellar vesicles of soybean phospholipids.

The interaction of native Bowman-Birk soybean protease inhibitor (BBI) and its hydrophobized derivative with multilamellar vesicles of various soybean phospholipids was investigated. Decrease in pH and introduction of negatively charged components to the lipid mixture increased BBI content in the protein-lipid complex. This suggests a contribution of electrostatic forces in the protein-lipid interaction. Protein hydrophobization insignificantly influenced BBI binding to lipids. In the complex with lipids, both proteins (BBI and its hydrophobized derivative) retained high anti-chymotrypsin activity (75-100%), which was not influenced by the presence of the ionic detergent sodium deoxycholate.

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 micro m), or 2) salting out of insulin from solution by sodium chloride (aggregate size of 5-13 micro m). 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.

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 10(8)-10(9) 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.

Interaction of polymer aggregates based on stearoyl-poly-N-vinylpyrrolidone with blood components.

Stearoyl-poly-N-vinylpyrrolidone (PVP-stear) of various molecular weights (M(n) = 1500-5500) self-assemble in aqueous medium. Particles prepared from PVP-stear were characterized in terms of shape and size distribution, and the mechanical stability of the particles was studied. The interaction of PVP-stear and its aggregates with blood components was investigated. Aggregates formed by the polymers with M(n) = 1500-3500 in the presence of human serum are stable. The direct lytic action of PVP-stear preparations was studied using sheep and human erythrocytes. The influence of PVP-stear aggregates on the activation of complement system both on classical and alternative pathways was examined. The aggregates prepared from PVP-stear of various molecular weights had no effect on the activation of the complement system.

Cationic inhibitors of serine proteinases from buckwheat seeds: study of their interaction with exogenous proteinases.

The inhibition of exogenous serine proteinases of different origin by cationic protease inhibitors BWI-1c, -2c, -3c, and -4c from buckwheat (Fagopyrum esculentum Moench) seeds has been studied. High efficiency of the inhibitors in binding bovine trypsin and chymotrypsin as well as their broad antiprotease effect, including inhibition of proteinases secreted by fungi and bacteria, has been demonstrated. According to the data obtained, it is proposed that cationic inhibitors from buckwheat seeds may participate in the defense of plants against fungal and bacterial infection.

Self-assembling systems based on amphiphilic poly-N-vinylpyrrolidone and their interaction with model proteins.

Polymeric particles formed by stearoyl-poly-N-vinylpyrrolidone (PVP-stear) of M(n) = 2600 were obtained in aqueous solution, and their shape and size distribution were characterized. The size of the particles was shown to decrease with an increase in the ionic strength of the solution. Interaction of PVP-stear and its aggregates with model proteins (Bowman-Birk soybean proteinase inhibitor (BBI) and its hydrophobized derivatives) was studied. The possibility of inclusion of both native BBI and oleoylic derivative of BBI in the PVP-stear polymeric aggregates was investigated. It was established that polymeric particles with a diameter of 30 nm formed under certain concentration ratios between PVP-stear and poorly soluble dioleoyl BBI are capable of solubilization of dioleoyl BBI as well as prevention of its inactivation at low pH values.

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 alpha-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 micro m 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.

Effect of membranotropic and mucoadhesive formulations of protein proteinase inhibitors on bovine herpes virus-1 reproduction.

The lipidized derivatives of Bowman-Birk soybean protease inhibitor (BBI) containing one to three oleoyl groups were synthesized and characterized. The (ole)(1)- and (ole)(2)BBI were demonstrated to have 200- and 100-fold higher uptake into Caco-2 cell monolayers compared to native BBI. The acylated BBI had increased affinity to elastase-like proteases. Aprotinin-loaded starch/bovine serum albumin microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride and characterized for their morphology, size and release of the inhibitor. Various formulations of protein proteinase inhibitors were tested for their influence on BHV-1 reproduction in cell cultures. Native aprotinin possessed palpable dose-dependent effect inhibiting the virus reproduction up to 4.0 lg (10,000-fold). The bioadhesive, biodegradable aprotinin-loaded microcapsules were the most effective decreasing virus infectious titer up to 4.0 lg and delaying the cytopathic effect up to 144 h in lesser doses of aprotinin. The lipophilic derivative (ole)(1)BBI was shown to exhibit effective inhibition (>100-fold) of BHV-1 reproduction unlike native BBI.

[Interaction of duodenase with human blood serpins]. / Vzaimodeistvie duodenzay s serpinami krovi cheloveka.

The interaction of duodenase, a new serine protease from a small group of Janus-faced proteases, with serpins, alpha 1-protease inhibitor (alpha 1-PI) and antichymotrypsin (ACT) from human blood serum, was studied. The stoichiometry of the inhibition process was found to be 1.2 and 1.3 mol/mol for alpha 1-PI and ACT, respectively. The presence of a stable enzyme-inhibitory complex duodenase-alpha 1-PI was confirmed by SDS-PAGE. No formation of the duodenase-ACT complex was demonstrated; instead, the band of the cleaved inhibitor was indicated upon the ACT hydrolysis. The suicide mechanism of the duodenase interaction with the human blood serpins was proved. The association rate constants (Ka, M-1 s-1) were 2.4 +/- 0.3 x 10(5) for alpha 1-PI and 3.0 +/- 0.4 x 10(5) for ACT. These results indicate the possibility of the regulation of duodenase activity by endogenous serpins. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

Study of antiproteinase activity of acylated derivatives of Bowman-Birk soybean proteinase inhibitor.

The effect of acylation of Bowman-Birk soybean proteinase inhibitor (BBI) by derivatives of various unsaturated fatty acids on inhibition of trypsin, alpha-chymotrypsin, and human leukocyte elastase was investigated. Inhibition (K(i)) and kinetic (k(ass), k(diss)) constants of interaction between proteases and acylated BBI derivatives were determined. For mono-, di-, and triacylated BBI derivatives, insertion of two oleic residues into the BBI molecule was demonstrated to be more potent for exhibiting antiproteinase activity.

Encapsulation of proteins by layer-by-layer adsorption of polyelectrolytes onto protein aggregates: factors regulating the protein release.

A novel method of protein encapsulation is proposed. Preformed protein aggregates are covered with polyelectrolyte layers by means of layer-by-layer adsorption. The polyelectrolyte membrane prevents protein leakage out of the capsule. Using chymotrypsin as a model enzyme the capsule wall selective permeability was demonstrated for substrates and inhibitors of different molecular weight and solubility.

Potential of block copolymer- and immuno-conjugates for tumor-targeted delivery of Bowman-Birk soybean proteinase inhibitor.

The present work reports the effect of conjugation of the anticarcinogenic and antitumor soybean Bowman-Birk protease inhibitor (BBI) with amphiphilic block copolymer of ethylene oxide and propylene oxide (PEO-PPO) as well as with monoclonal antibody via clinical dextran (D) on tumor-targeted delivery of BBI.