Biological Activity of Hybrid Vaterite-Pectin Microparticles Towards Bacteria E. coli and Human Neutrophils.

Interaction of microbiota with hybrid vaterite-pectin microparticles as an attractive multifunctional vehicle for mucosal delivery should not provoke inflammation. Our purpose was to study the reaction of bacteria E. coli strain Mg1655 and isolate SharL from a patient with Crohn disease on the cultivation with hybrid microparticles and vaterite, and the subsequent activation of neutrophils. Vaterite-pectin microparticles enhanced leakage of ATP from bacteria. For E. coli Mg1655, the concentration of DNA decreased, while intracellular ATP increased. For E. coli SharL, the intracellular ATP decreased with simultaneous growth of DNA. Bacteria and microparticles together did not enhance activation of neutrophils in comparison with the particles per se in the medium without serum and in comparison with bacteria in the medium supplemented with serum; microparticles did not reduce functional activity of neutrophils.

Analysis of Cytokines and ATP in Plucked Hair Follicles.

The concentrations of ATP, IL-6, and IL-10 were measured in extracts of plucked hair follicles from healthy volunteers (normal values) and patients with androgenetic alopecia and then, ATP, IL-6, and IL-10 content was calculated for each follicle. The resulting values were directly proportional to hair follicle length, except for IL-6. The concentration of extracted ATP correlated with lactate dehydrogenase activity indicating cell damage. In patients with androgenetic alopecia, IL-10 content exceeded the normal values in follicles with a length <1 mm and ATP content surpassed the normal in follicles >2 mm long. The content of IL-6 and IL-10 measured by ELISA was comparable with results of mRNA expression assayed by RT-PCR, which attested to moderate level of gene expression. The content of ATP and IL- 10, but not IL-6 depended on the length of plucked hair follicle and on pathogenetic factors affecting hair growth.

The mechanism of catalase loading into porous vaterite CaCO3 crystals by co-synthesis.

Porous vaterite CaCO3 crystals are nowadays extensively used as high-capacity bio-friendly sacrificial templates for the fabrication of such protein-containing nano- and micro-particles as capsules and beads. The first step in the protein encapsulation is performed through loading of the protein molecules into the crystals. Co-synthesis is one of the most useful and simple methods proven to effectively load crystals with proteins; however, the loading mechanism is still unknown. To understand the mechanism, in this study, we focus on the loading of a model protein catalase into the crystals by means of adsorption into pre-formed crystals (ADS) and co-synthesis (COS). Analysis of the physico-chemical characteristics of the protein in solution and during the loading and simulation of the protein packing into the crystals are performed. COS provides more effective loading than ADS giving protein contents in the crystals of 20.3 and 3.5 w/w%, respectively. Extremely high loading for COS providing a local protein concentration of about 550 mg mL-1 is explained by intermolecular protein interactions, i.e. formation of protein aggregates induced by CaCl2 during the co-synthesis. This is supported by a lower equilibrium constant obtained for COS (5 × 105 M-1) than for ADS (23 × 105 M-1), indicating a higher affinity of single protein molecules rather than aggregates to the crystal surface. Fitting the adsorption isotherms by classical adsorption models has shown that the Langmuir and BET models describe the adsorption phenomenon better than the Freundlich model, proving the aggregation in solution followed by adsorption of the aggregates into the crystals. We believe that this study will be useful for protein encapsulation through CaCO3 crystals using the COS method.

Binding of Mucin by E. coli from Human Gut.

Cells of E. coli isolates from the gut of healthy volunteers (N=5) and patients with Crohn's disease (N=5) and laboratory E. coli strain DH5α bound mucin in vitro in similar amounts ranging from 0.02 to 0.12 mg/mg of bacterial dry weight. Binding was evaluated by the decrease in optical absorption of mucin solution at 214 nm after incubation with bacteria. Detailed analysis of mucin binding by one of isolates showed that during incubation of 0.09 mg/ml bacteria in 0.15 M NaCl containing 0.1 mg/ml mucin at 25oC, maximum binding was reached in 30 min, while in the presence of 14 mM α-methyl mannoside, mucin binding decreased by 46% (p<0.05). Confocal microscopy revealed intensive binding of FITC-labeled mucin to the surface of a small number of bacterial cells. Mucin binding did not significantly affect zeta potential of bacteria and their energetic status assessed by ATP content; at the same time, ATP content in the extracellular environment slightly increased.

Protein loading into porous CaCO3 microspheres: adsorption equilibrium and bioactivity retention.

Formulation of proteins into particulate form is a principal strategy to achieve controlled and targeted delivery, as well as to protect fragile protein molecules. Control over size, mechanical properties, and surface area (porosity) of particulate proteins has been successfully achieved by hard templating under mild conditions using porous CaCO3 microspheres. A crucial step in this approach, which determines protein content, is the loading of proteins into the CaCO3 microspheres. In this study, the adsorption of different proteins into the microspheres has been investigated. Proteins differing in characteristics such as molecular weight and charge have been employed: catalase (Cat), insulin (Ins), aprotinin (Apr), and protamine (Pro). Thermodynamics of adsorption equilibria have been studied, together with quantitative and qualitative analysis of protein loading and distribution in the microspheres. Protein interaction with the CaCO3 microspheres is not limited by the diffusion of protein molecules (protein dimensions are significantly smaller than microsphere pores) but is determined by the protein affinity for the microsphere surface. Cat and Ins bind much more strongly to the microspheres than Apr and Pro, which can be explained by electrostatic attractive forces. Protein binding/release and protein biological activity have been investigated as a function of pH. It is shown that pH variation during the adsorption process plays a principal role and defines not only the amount of protein adsorbed/released but also protein biological activity. Protein adsorption and microsphere elimination (by EDTA) do not affect protein bioactivity. In addition to applications for protein particle/capsule formulations, the findings of this study might help in understanding protein interactions with carbonate minerals such as calcium carbonate, which is used as a natural material for multiple applications.

[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.

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.

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.

[Modification of basic pancreatic trypsin inhibitor by fatty acid derivatives]. / Modifikatsiia osnovnogo pankreaticheskogo ingibitora proteinaz proizvodnymi zhirnykh kislot.

A procedure was developed for the modification of basic pancreatic trypsin inhibitor (BPTI) by N-hydroxysuccinimide esters of oleic and stearic acids in a DMSO-DMF-dioxane-pyridine mixture with a temporary citraconyl protection of the amino group belonging to its active site. The BPTI derivatives containing from one to three acylated amino groups were obtained. It was shown that the hydrophobized BPTI with one amino group modified retained practically the full activity of the native inhibitor. An increase up to three acylated groups resulted in a decrease in the specific activity of the inhibitor to 28% of the initial activity. An increase in hydrophobicity of the modified inhibitors was demonstrated by the spectral method and by the distribution in a water-triton X-114 system.

[Natural proteinase inhibitors as a basis for creating new drugs]. / Prirodnye ingibitory proteinaz kaka osnova dlia sozdaniia novykh lekarstvennykh sredstv.

Isolation of the proteinases inhibitors, available for medicinal purposes, was described, where the inhibitor of the Kunitz type was obtained from bovine pancreas and the inhibitor of the Bowman-Birk type from soybeans. Screening of the immobilization procedures was carried out, which enabled the authors to produce the polymeric conjugates of the proteinase inhibitors exhibiting the maximal rate of activity against pancreatic proteinases and granulocyte elastases. Pharmacokinetics of the proteinase inhibitors obtained was studied. High molecular derivatives of the inhibitors from the bovine pancreas circulated in rat blood in larger quantities and longer, their total clearance was 5 times than native inhibitor preparations. The preparations containing these inhibitors from bovine pancreas exhibited a high therapeutic efficiency in treatment of rats with hemorrhagic pancreatitis and acute liver failure in rabbits.

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.

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.

Isolation and characterization of soybean Bowman-Birk inhibitor from different sources.

A chromatographic procedure for isolation of different isoforms of Bowman--Birk soybean trypsin inhibitors was developed. The number of isoforms was shown to depend on soybean cultivar. The amount of the classical Bowman--Birk inhibitor (BBI) in different soybean cultivars, commercial flour, and processing products was analyzed. BBI reaches its highest concentration in freshly milled seeds. Storage conditions optimum for preservation of maximum inhibitory activity in soybean raw material were developed. The use of indirect enzyme immunoassay for BBI detection during its isolation from different sources was demonstrated.