Design of polymer particle dispersions (latexes) in the course of radical heterophase polymerization for biomedical applications.

Dispersions of polymer particle (DPPs) are increasingly being exploited both as biomolecule carriers, and as markers in various DPP biomedical applications related to cell and molecular biology, enzymology, immunology, diagnostics, in vitro and in vivo visualization, bioseparation, etc. Their potential to reduce reaction scales, lower costs, improve the rate, sensitivity, selectivity, stability and reproducibility of assays governs the diversity of their bioapplications. This review focuses on the design of DPPs with innovative special properties in the course of free radical heterophase polymerization that provides careful control of both macromolecular and colloidal properties. We demonstrate approaches that, according to the polymerization technique, regulate the particle size, shape, particle size distribution, morphology, surface chemistry and functionality, as well as the formation of organic-inorganic hybrid DPPs. The production of bioreagents based on DPPs and their use in bioassay are also reviewed.

Submicron polyacrolein particles in situ embedded with upconversion nanoparticles for bioassay.

We report a new surface modification approach of upconversion nanoparticles (UCNPs) structured as inorganic hosts NaYF4 codoped with Yb(3+) and Er(3+) based on their encapsulation in a two-stage process of precipitation polymerization of acrolein under alkaline conditions in the presence of UCNPs. The use of tetramethylammonium hydroxide both as an initiator of acrolein polymerization and as an agent for UCNP hydrophilization made it possible to increase the polyacrolein yield up to 90%. This approach enabled the facile, lossless embedment of UCNPs into the polymer particles suitable for bioassay. These particles are readily dispersible in aqueous and physiological buffers, exhibiting excellent photoluminescence properties, chemical stability, and also allow the control of particle diameters. The feasibility of the as-produced photoluminescent polymer particles mean-sized 260 nm for in vivo optical whole-animal imaging was also demonstrated using a home-built epi-luminescence imaging system.

High-throughput Method of One-Step DNA Isolation for PCR Diagnostics of Mycobacterium tuberculosis.

The efficiency of one-step and multi-step protocols of DNA isolation from lysed sputum samples containing the Mycobacterium tuberculosis complex has been compared. DNA was isolated using spin-cartridges containing a special silica-based sorbent modified with fluoroplast and polyaniline, or using an automated isolation system. One-step isolation using the obtained sorbent has been shown to ensure a significantly lower DNA loss and higher sensitivity in the PCR detection of Mycobacterium tuberculosis as compared to a system based on sorption and desorption of nucleic acids during the isolation.

[Isolation and determination of activity of IgA1 protease from Neisseria meningitidis].

A method of the isolation and purification of IgAl protease from a culture of Neisseria meningitidis serogroup A has been developed. Three inactivated intermediates of the production of the meningococcal vaccine, a culture liquid, as well as a supernatant and sediment obtained by the precipitation of bacterial cells by cetavlon, served as a starting material. The purity of IgA1 protease was determined by SDS-PAGE. An immunoenzyme assay for determining the IgA1 protease activity has been devised. The yield of the enzyme with a specific activity of 0.5 to 4 million units/mg from 103 g of the cetavlon precipitate (40 l of culture liquid) was about 600 mug. It was shown that IgAl protease isolated from serogroup A meningococcus is capable of protecting experimental animals (mice) infected with meningococcus of serogroup B.

[Biodegradable microparticles with immobilized peptide for wound healing].

Thrombin receptor agonist peptide (TRAP-6) may effectively replace thrombin for stimulation of damaged tissue regeneration. (Thrombin employment is limited by its high cost, instability and proinflammatory effect at high concentrations.) Immobilization of TRAP-6 into a poly(D,L)-lactide-co-glycolide (PLGA)-based matrix can protect peptides from a destruction by peptidases located in a wound area, and can also provide controlled release of the peptide. PLGA microparticles with immobilized peptide were produced by double emulsion/evaporation technique. An observation of microparticle morphology by scanning electron microscopy highlighted that peptide immobilization resulted in the increase of the microparticle porosity. TRAP-6 release kinetics was characterized by burst increase of TRAP-6 concentration in HEPES buffer solution (pH 7.5) for first 2 hours from the beginning of the experiment, and TRAP-6 complete release occurred for 20 hours. An investigation of TRAP-6 destruction by scanning electron microscopy revealed that the increase of microparticle size and surface porosity were observed already after 1 day of incubation in the buffer solution, and an aggregation of destructing microparticles was obvious by the 7th day of the incubation. Thus, peptide immobilization into PLGA microparticles can allow to develop a novel controlled release drug delivery system.

[A composite polyaniline-containing silica sorbent for DNA isolation]. / Kompozitsionnyi polianilinsoderzhashchii kremnezemnyi sorbent dlia vydeleniia DNK.

A composite sorbent based on porous glass beads modified with thin polyaniline coating was prepared by precipitating aniline polymerization in the presence of carrier particles. It was shown that the modification ensures the uniform coating of the inner surface of the carrier pores with the polymer layer approximately 70 A thick. It was shown that the resulting material retains the initial porosity of the carrier and is selective in the separation of nucleic acids and proteins. The polyaniline-coated sorbents were shown to be efficient for both the preparative DNA isolation from bacterial lysates and for analytical purposes, in particular, for studying DNA fragmentation during apoptosis proceeding under UV irradiation of cell lysates of colon carcinoma. The morphological and chromatographic characteristics of the new sorbent were demonstrated to be similar to those of the polyfluorobutadiene sorbent.

[Coprecipitation of the Pseudomonas fluorescens lipase with hydrophobic compounds as an approach to its immobilization for catalysis in nonaqueous media]. / Soosazhdenie lipazy Pseudomonas fluorescens s gidrofobnymi soedineniiami kak podkhod k ee immobilizatsii pri katalize v nevodnykh sredakh.

The precipitation of N-cetylamine, N-cetylacetamide, hexan-1,2-diol, cetyl alcohol, and poly(butyl metacrylate) in acetone-water media in the presence of the lipase from Pseudomonas fluorescens was found to be accompanied by the coprecipitation of the enzyme. Within the lyophilized coprecipitates, the lipase exhibits a high catalytic activity and enantioselectivity in the reaction of (1RS)-phenylethanol acetylation with vinyl acetate in t-butyl methyl ether. In order of increasing lipase activity, the coprecipitates can be arranged in the series: cetyl alcohol, poly(butyl metacrylate), hexadecane-1,2-diol, N-cetylamine, and N-cetylacetamide, with the activity 2.5- to 19-fold exceeding the activity of the native enzyme. The immobilization of the lipase on solid supports, such as Celite 545 (physical sorption) and Eupergit C250L (covalent binding), in the presence of hexadecane-1,2-diol was found to increase the esterifying activity of the enzyme. The English version of the paper.

[Polymer coatings with immobilized thrombin and peptides: preparation and use for wound healing]. / Polimernye pokrytiia s immobilizovannymi v nikh trombinom ili peptidami: poluchenie i primenenie dlia ranozazhivleniia.

Polymer dressings with encapsulated thrombin or synthetic peptides which can mimic thrombin action are employed for wound healing. Paper describes the method for preparation of these hydrogel composites of PVCL-CaAlg [poly(N-vinyl caprolactam-calcium alginate). The effect of encapsulated thrombin/peptides on tissue repair process have beet investigatat in vivo experiments using a mouse model of wound healing. The developed dressings accelerated wound healing: thascan be used as a basis for creation of novel formulations with controlled drug release for wound therapy.

A study of moss (Marchantia polymorpha) thylakoid membrane lipids in monolayers.

Monolayers of seven fractions of natural lipids (phosphatidyl inositol, sulfoquinovosyl diacylglycerol, phosphatidylcholine, digalactosyl diacylglycerol, phosphatidyl glycerol, phosphatidylethanolamine, monogalactosyl diacylglycerol), isolated from the photoautotrophic cell culture of the moss Marchantia polymorpha grown under normal and light-stress conditions, have been prepared for the first time. We have shown that the high-intensity light affects the area occupied by the lipid molecule. In the case of digalactosyl diacylglycerol and phosphatidyl glycerol fractions, after the light stress the area significantly increased from 0.50 to 0.80 nm2 and from 0.47 to 0.63 nm2, respectively, and in the case of the sulfoquinovosyl diacylglycerol fraction, the area decreased from 0.40 to 0.32 nm2. These results are in agreement with our previous data on the redistribution of the double bonds in the aliphatic chains of these lipids and can be used to characterize the state of the lipid bilayer of the thylakoid membranes.

Immobilized thrombin receptor agonist peptide accelerates wound healing in mice.

To accelerate the healing processes in wound repair, attempts have been repeatedly made to use growth factors including thrombin and its peptide fragments. Unfortunately, the employment of thrombin is limited because of its high liability and pro-inflammatory actions at high concentrations. Some cellular effects of thrombin in wound healing are mediated by the activation of protease activated receptor-1 (PAR-1). The thrombin receptor agonist peptide (TRAP:SFLLRN) activates this receptor and mimics the effects of thrombin, but TRAP is a relatively weak agonist. We speculated that the encapsulated peptide may be more effective for PAR-1 activation than nonimmobilized peptide and developed a novel method for TRAP encapsulation in hydrogel films based on natural and synthetic polymers. The effects of an encapsulated TRAP in composite poly(N-vinyl caprolactam)-calcium alginate (PVCL) hydrogel films were investigated in a mouse model of wound healing. On day 7 the wound sizes decreased by about 60% under TRAP-chitosan-containing PVCL films, as compared with control films without TRAP. In the case of TRAP-polylysine-containing films no significant decrease in wound sizes was found. The fibroblast/macrophage ratio increased under TRAP-containing films on day 3 and on day 7. The number of proliferating fibroblasts increased to 150% under TRAP-chitosan films on day 7 as compared with control films. The number of [3H]-thymidine labeled endothelial and epithelial cells in granulation tissues was also enhanced. Thus, the immobilized TRAP to PVCL-chitosan hydrogel films were found to promote wound healing following the stimulation of fibroblast and epithelial cell proliferation and neovascularization. Furthermore, TRAP was shown to inhibit the secretion of the inflammatory mediator PAF from stimulated rat peritoneal mast cells due to augmentation of NO release from the mast cells. The encapsulated TRAP is suggested to accelerate wound healing due to the anti-inflammatory effects and earlier development of the proliferative phase of wound healing.

Cellular localization of the galactose-binding lectin from human serum.

An SL2 lectin was isolated from human serum and characterized previously; cellular localization of the lectin was studied using polyclonal rabbit antibodies. According to cytofluorimetry, anti-SL2 antibodies bound only to lymphocytes and monocytes but not to other blood cells. Antibodies bound to Jurkat T cell lymphoma but did not interact with IM-9 cells of B cell origin. Moreover, the Jurkat cells bound oligosaccharides having the highest affinity to SL2 (GalNAcalpha_and Fucalpha1-2Gal), and this interaction was inhibited by anti-SL2 antibodies. Lysis of the Jurkat cells with subsequent electrophoresis and Western blotting indicates that anti-SL2 antibodies recognized a 14-kD protein.

[Synthesis of amphiphilic photochromic benzo-15(18)-crown-5(6)-ethers and their properties in monolayers]. / Sintez amfifil'nykh fotokhromnykh benzo-15(18)-kraun-5(6)-éfirov i ikh issledovaniie v monosloiakh.

New amphiphilic photochromic benzo-15(18)-crown-5(6) ethers (APC) differing in the position of the octadecyl substituent and the size of the crown cavity were synthesized. The compounds form stable monolayers in the air/water and air/alkaline metal salt solution interfaces. The results of the pressure isotherm measurements, atomic force microscopy (AFM), and electronic spectroscopy show that the structure of the monolayers formed depends on the structure of the parent APC and the nature of the cation in salt solutions. The area per molecule of APC in the monolayer (specific area) is the smallest on the water surface and increases by 20-40% on the aqueous subphase surface with an increasing concentration of salts therein to indicate the formation of APC complexes with the metal cations. When the hydrophobic aliphatic substituent is displaced from position 3 to position 5 of the benzothiazole ring, the specific area on the surface of water and subphases decreases twofold, which indicates the compactization of the monolayer on this modification. A reversible E-Z-photoisomerization of APC was found in the monolayers formed in the salt solution/air interface. The features of the reaction are defined by the specific organization of the amphiphilic molecules in the monolayer and by the nature of the cation.

[Turbidimetric study of latex agglutination in 2,4-dichlorophenoxyacetic acid analysis]. / Turbidimetricheskie issledovaniia reaktsii lateksnoi aggliutinatsii pri opredelenii 2,4-dikhlorofenoksiuksusnoi kisloty.

By the example of 2,4-dichlorophenoxyacetic acid (2,4-D) assay by the inhibition of latex agglutination, new synthetic polymer microspheres for the conjugation with antibodies to 2,4-D and agglutinators based on ovalbumin and polyacrylamide were developed and characterized. The effect of various parameters (the concentration of reagents, the type and the degree of modification of the microsphere surface, and the nature of the carrier in the composition of the agglutinator) on the rate of agglutination and the maximal optical absorption observed during the reaction were studied by turbidimetry. The optimal parameters were found for the assay of 2,4-D by the inhibition of latex agglutination with turbidimetric registration of the results.

Features of the behaviour of double-chained cationic surfactant monomer in a monolayer at the water-gas interface.

We studied the formation of a double-chained cationic monomer, N,N-diallyl-N-cetyl-N-dodecyloxycarbonyl methylammonium bromide, in a monolayer at the water-gas interface. An increase in surface pressure induced the transition from the liquid-expanded to liquid-condensed state with tail-packing and then head-packing of the monomer, which was accompanied by a decrease in the mobility of monomer molecules. An increase in the ionic strength of the subphase led to the compression of the layer, and addition of ethanol or detergents caused its dilution. Polyethylene glycol and polyacrylic acid induced the formation of a complex between the monomer and polymer.

[Molecular design of polymeric materials for biotechnology and medicine]. / Molekuliarnoe konstruirovanie polimernykh materialov dlia biotekhnologii i meditsiny.

This review describes new polymer materials for biomedical applications developed in the Polymers for Biology Laboratory of the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences. These include composite rigid sorbents for biochromatography, polymer dispersions for immunoassay, polymer hydrogels for immobilization of enzymes and cells, and polymer ultra thin films as biomembrane models and materials for biosensors. Some general and specific properties of these new materials and models as well as examples of their applications are discussed.

[Effect of surfactants on peroxidase activity. I. Effect of anionic surfactants]. / Vliianie poverkhnostno-aktivnykh veshchestv na aktivnost' peroxidazy. I. Vliianie anionnykh PAV.

The effect of various anionic surfactants on the initial rate of 5-aminosalicylic acid peroxidation with horseradish peroxidase was studied. With increasing surfactant concentration, this rate first decreased, then increased, and again decreased. We conclude that these changes were due to an inhibition of the enzyme, a subsequent change in the enzyme conformation accompanied by the enhanced accessibility of its active site to the substrate, and the final protein denaturation, respectively.

[Thrombin--a regulator of reparative processes in wound healing]. / Trombin--reguliator reparativnikh protsessov pri zazhivlenii ran.

Thrombin, binding to receptors of the protease activated receptor (PAR) family, is involved in wound healing by inducing the reparation processes and regulating the activity of mast cells, which secrete mediators of inflammation. Using thrombin receptor agonist peptide (TRAP-6) for the activation of rat mast cells, effect of several receptors, including PAR-1, on mast cells was demonstrated. It was shown that TRAP increases the concentration of Ca2+ in the cytoplasm of mast cells and regulates cell degranulation, while releasing nitrogen oxide. Thrombin encapsulated in poly(N-vinyl caprolactam)-calcium alginate (PVCL-Ca-Alg) hydrogel films promotes wound healing in rats as demonstrated by the acceleration of fibroblast proliferation and neovascularization.

Immunoreagents based on polymer dispersions for immunochemical assays.

Immunoreagents based on polymer dispersions consisting of unimodal polyacrolein (PAL) microspheres with diameters in the range 0.3-2.0 microns have been prepared and evaluated by various immunoassay techniques such as immunoradiometric assay of ferritin and microtitre particle agglutination and immunofiltration dot assay of group-specific polysaccharide of S. pyogenes (A-PS) in comparison with conventional carriers and methods. The antibodies were covalently or indirectly bound to the PAL. The coupled antibodies to ferritin retained a high average affinity (Ka = 4.5 x 10(9) M-1). In comparison with microcrystalline cellulose-based immunosorbent, more than an order-of-magnitude lower amount of PAL-IgG was necessary for the analysis of ferritin. Use of PAL-IgG gave a higher sensitivity of assay with a detection limit of 0.7 x 10(-13) M l-1 and a wider concentration range of antigen detection (about four orders of magnitude) without manifestation of the high-dose hook effect. Particle agglutination assay of A-PS in microtitre plate was shown to be a simple, demonstrative and highly sensitive one-step analytical method with a detection limit of 0.05 ng A-PS/ml or 10(4) cells/ml. The sensitivity of immunofiltration assay using both enzyme and latex markers was shown to be approximately the same (50 ng A-PS/ml) and the duration of the assay was 3-5 min. No cross-reaction of latex conjugates with non-A Streptococcus cell lysates were observed.

[Composite fluorine polymer-containing sorbents for isolation and purification of biopolymers]. / Kompozitsionnye ftorpolimersoderzhashchie sorbenty dlia vydeleniia i ochistki biopolimerov.

Composite fluoropolymer-containing sorbents based on porous silicas were synthesized for the isolation and purification of biopolymers under nondenaturing conditions. Examples of the application of these sorbents in the separation of various mixtures of peptides and proteins and purification of nucleic acids from various sources (plasmid DNA and DNA from nucleated human blood cells) using the cartridge, column, and batch (sorption in a stirred volume) methods are presented. It was shown that the sorbents can be used in laboratory practice because they are selective to nucleic acids (DNA and RNA) and proteins. These materials combine the mechanical properties of the inorganic matrix with the specific sorption properties of the polymer phase and exhibit enhanced stability to alkaline hydrolysis. Alternative methods of preparing sorbents containing polytetrafluoroethylene, polytrifluorostyrene, and polyfluorobutadiene are described. By the example of polyfluorobutadiene-containing sorbents, a completely new method for obtaining fluorinated polymer phases was developed: the polymer phase was preliminarily formed on the surface of porous disperse carriers and was fluorinated with xenon difluoride.