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Hypercrosslinked polystyrene as a novel type of high-performance liquid chromatography column packing material. Mechanisms of retention.

An experimental material, Chromalite 5HGN (Purolite, UK), that represents hypercrosslinked polystyrene as a new type of neutral stationary phase for HPLC was examined. The material contains no functional groups, but is compatible with any kind of nonpolar and highly polar mobile phase, and even with water. It is chemically resistant and thermally stable. When using aqueous organic mobile phases, Chromalite 5HGN works similar to standard C18 reversed-phase packings, but is characterized by much greater hydrophobicity and, sometimes, unusual selectivity. When using nonpolar mobile phases, i.e. under "quasi normal-phase" conditions, the retention is mostly governed by the interactions between pi-electronic systems of the adsorbent and adsorbate. Adding highly polar, even hydrophilic solvents into the mobile phase, leads to a shift of retention times toward the "reversed-phase" kind of chromatography, which gives an additional possibility in fine tuning the column selectivity.

Chromatography, High Pressure Liquid↗

Adsorption thin-layer chromatography and viscometry of polystyrenes in solvent mixtures.

The effect of the thermodynamic quality of solvent mixtures on the adsorption behavior of macromolecules under dynamic conditions was investigated. The chromatographic behavior of polystyrenes (PSs) in various mixed eluents was studied under conditions of adsorption and size-exclusion thin-layer chromatography as well as at the exclusion-adsorption transition point with silica gel KSKG adsorbent. The thermodynamic quality of the solvent mixtures used in the chromatographic experiments was determined viscometrically. The dependences of the intrinsic viscosity of PSs on solvent composition for solvent-non-solvent, two solvents, and theta-solvent-solvent mixtures were obtained. A correlation was found between Snyder's polarity indices for the solvent mixtures used in polymer chromatography under "critical conditions" and the intrinsic viscosity values of PSs in the same mixtures.

Chromatography, Thin Layer↗

Liquid chromatography and differential scanning calorimetry studies on the states of water in polystyrene-divinylbenzene copolymer gels.

The thermal phase transition behavior of water incorporated in crosslinked polystyrene-divinylbenzene copolymer (PS-DVB) gel packings for liquid chromatography was investigated by differential scanning calorimetry. Free or bulk water, freezable bound or intermediate water, and nonfreezing water were observed for TSKgel Styrene-250 samples, while only free water and nonfreezing water were observed for TSKgel Styrene-60 samples. Freezable bound water and nonfreezing water in these gel samples were considered to be water confined in pores of the polymer gels. A liquid chromatographic method for determination of the amounts of stationary phase water was applied to the characterization of water in the PS-DVB beads in the columns and it was found out that any water in the PS-DVB gels did not function as the stationary phase contrary to the water sorbed in hydrophilic polymer gels; not only freezable bound water but also nonfreezing water in hydrophobic PS-DVB gels are similar to bulk water with respect to the affinity to the solute compounds.

Calorimetry, Differential Scanning↗

Polystyrene nanoparticles based on poly(butyl methacrylate-g-methoxypoly(ethylene glycol)) and poly(methyl methacrylate-g-methoxypoly(ethylene glycol)) graft copolymers.

The solubilization of styrene by poly(butyl methacrylate-g-methoxypoly(ethylene glycol)) and poly(methyl methacrylate-g-methoxypoly(ethylene glycol)) graft copolymers has been examined. From turbidity measurements the solubility limit of the monomer in the micelles was obtained and the distribution coefficients were evaluated. Dynamic light scattering revealed that below the solubility limit, solubilization leads to a slight increase in micelle size, while above the solubility limit, there is a dramatic increase in particle size and turbidity as oil-in-water emulsions are formed through coalescence of monomer-swollen micelles. Polymerizations carried out below the solubility limit using the graft copolymer micelles as templates resembled microemulsion polymerizations in nature and led to very fine sterically stabilized polystyrene latex particles. Through careful control of the monomer concentration and the polymerization temperature it was possible to obtain spherical nanosize latex particles with similar size to those of the micelle precursors (10 nm) up to 11% monomer by weight. Polymerizations above the solubility limit, on the other hand, showed similarities with emulsion polymerizations and resulted in larger particles with higher polydispersity.

Emulsions↗

Covalent immobilization of proteins onto photoactivated polystyrene microtiter plates for enzyme-linked immunosorbent assay procedures.

Enhancement of the speed and sensitivity of an ELISA technique was achieved by doing it on a polystyrene microtiter plate preactivated by a simple photochemical reaction. Immobilization of Epicoccum nigrum antigen (allergenic antigen) or goat anti-rabbit IgG onto the photoactivated plates was found to occur in only 45 min with higher binding than that obtained through adsorption during the same period onto the untreated surface. Nearly 1.5-2-folds higher readings were obtained when the ELISA was carried out with the solid phase prepared on the photoactivated surface rather than on the untreated surface. Moreover, solid phases prepared on the activated surface could detect IgE (E. nigrum antibody) even at 1/50 (v/v) dilutions, whereas a solid phase prepared on the untreated surface failed to do so. Around three times higher ELISA values were obtained in the activated plate than the untreated plate when IgE was diluted to 1/5 (v/v). Such photoactivated surface could be of great importance in diagnostic tests involving the ELISA technique particularly to confirm false negative cases and for other immunoassays such as radioimmunoassay procedures.

Animals↗

Adsorption studies of tritium-labeled peptides on polystyrene surfaces.

In this study, three presentation formats of an epitope peptide (hepta-peptide), derived from the human chorionic gonadotropin amino acid sequence, were compared for adsorption to the polystyrene wells of a microELISA plate. The peptides had either a free N-terminus, an Ata-group or a linear (Lys)7-extension at the N-terminal. In order to measure the adsorption properties, all peptides were tritiated by synthesizing an additional 3H-labeled glycyl residue to the N-terminus of their peptide sequence. Over a broad range of peptide concentrations used as coat solution, extension of the peptide by an Ata-group consistently increased adsorption by a factor of 1.5 to 3 compared to the free parent peptide. Of the three peptides studied, the Ata-peptide showed the highest surface coverage of 0.6 mg/m2 when 1.0 mmol/l was offered as the concentration of peptide in the coating solution. The highest surface coverage observed for the parent peptide was 0.4 mg/m2 (at 1.5 mmol/l). The lysyl (K7) peptide showed a maximum plateau value of 0.2 mg/m2, and therefore the lysyl (K7) extension reduced the peptide surface coverage at relatively high coat concentrations (above 0.1 mmol/l) compared to the parent peptide. At lower input concentrations (below 0.1 micromol/l), however, the packing density of the lysyl (K7) peptide was up to 25 times higher when compared to the other two peptide analogs. We conclude that better adsorption as well as improved antibody binding activity and (functional) affinity could explain the higher reactivity observed in ELISA procedures when peptides are N-terminally extended by an Ata-group or lysyl (K7) extension.

Adsorption↗

Determination of polybrominated diphenyl ethers and PBDD/Fs during the recycling of high impact polystyrene containing decabromodiphenyl ether and antimony oxide.

A high impact polystyrene (HIPS) plastic, flame retarded with decabromodiphenyl ether (DecaBDE) and antimony trioxide (Sb2O3) has been repeatedly processed and the amount of debromination of the flame retardant and possible formation of PBDD/F was monitored. No indications of debromination were found. The reprocessed (recycled) plastic was found to comply with the German Chemicals Banning Ordinance. The concentrations of relevant PBDD/F congeners were at least one order of magnitude below the regulated limit values for PBDD/F (1 ppb for the sum of four congeners, 5 ppb for the sum of all eight regulated congeners).

Bromine↗

Anomalous colloidal stability of protein coated polystyrene latex beads studied by small angle light scattering.

Results of small angle laser light scattering experiments carried out on polystyrene latex beads coated with lysate of Plasmodium falciparum (Pf) antigen (Ag) and human seropositive sera (10:1 ratio) are reported for various NaCl concentrations (0-300 mM). The protein coated beads showed time-dependent coagulation. The normalised intensity of scattered light I(s)(t)/I(0) showed I(s)(t)/I(0)=1+(Gammat)(delta) behaviour with the coagulation rate, Gamma and exponent, delta showing anomalous dependence on NaCl concentration. The coagulation rate exhibited strong increase up to NaCl concentration of 50 mM, above this and up to 300 mM the coagulation rate was found to remain independent of NaCl concentration yielding non-DLVO behaviour. The same was true for delta which increased from 1.04+/-0.06 to 6.94+/-0.07 as NaCl concentration was raised from 0 to 50 mM. Above 50 mM it remained constant with delta=6.94+/-0.07. Results are discussed through Smoluchowski aggregation kinetics and theoretical construction of interparticle interaction potentials relevant to our problem.

Animals↗

Adhesion, apoptosis and cytokine release of human mononuclear cells cultured on degradable poly(urethane urea), polystyrene and titanium in vitro.

Early interactions between materials and mononuclear cells may influence the viability and secretory response of the cells. Such effects may in turn influence the subsequent inflammatory and repair phases around the materials. In the present study, it was examined if mononuclear cells cultured in vitro either unstimulated or stimulated with lipopolysaccharide (LPS) (10ng/ml) revealed differences regarding cell viability and apoptosis. A major interest was to study the influence of different material properties on the parameters of the inflammatory response upon cell adhesion to materials with widely different surface chemical properties but similar surface topography: degradable poly(urethane urea) (PUUR), cell culture treated polystyrene (PS) surfaces, and commercially pure (c.p.) titanium (Ti). Finally, the secretion of the proinflammatory tumor necrosis factor-a (TNF-alpha) and the downregulating interleukin-10 (IL-10) cytokines was examined in the supernatants from 24h mononuclear cell cultures. No differences in cell viability as measured by lactate dehydrogenas (LDH) were observed between the three materials. The number of material-surface adherent cells was higher on PUUR than the more hydrophilic PS and Ti as judged by quantification of material surface-associated DNA, light microscopic morphological examination of DAPI-stained cells and SEM. LPS increased the number of adherent cells, irrespective of the type of material. The lowest number of apoptotic (annexin-V) and necrotic (propidium iodide) mononuclear cells was detected on PUUR. LPS decreased the number of both apoptotic and necrotic cells, irrespective of material. Low TNF-alpha levels were detected in unstimulated conditions, irrespective of material types. A significantly lower amount of TNF-alpha was found with unstimulated cells on PUUR than on Ti. A significantly higher IL-10 level was detected in unstimulated Ti cultures compared with PUUR and PS. Secretion of IL-10 was predominantly stimulated by LPS on PUUR and Ti. The data indicate that material-related differences are expressed in differences in cell adherence, apoptosis and cytokine secretion. Further, degradable PUUR has equal or less cell-activating properties than Ti and PS under in vitro conditions.

Absorbable Implants↗

Adsorption of immunoglobulin G and anti-factor VIII inhibitory antibody from haemophiliac plasma to derivatized polystyrenes.

Modified polystyrene resins containing sulphonate groups and tyrosyl sulphamide or tyrosyl methyl ester sulphamide groups have been investigated with respect to their potential for selective binding of anti-Factor VIII inhibitory antibodies from plasma. Adsorption of total immunoglobulin G and of a monoclonal antibody to Factor VIII was measured following addition of the radioiodinated proteins to normal plasma, plasma depleted of Factor VIII by adsorption on a resin coupled to anti-Factor VIII antibody, and haemophiliac plasma containing various levels of inhibitory anti-Factor VIII antibody. Depletion of anti-Factor VIII antibody from the haemophiliac plasmas by incubation with the resins was also measured by Bethesda assay. The modified resins and their corresponding unmodified "controls' showed similar binding of total immunoglobulin G. However, only resins containing either sulphonate or a combination of sulphonate and tyrosyl sulphamide groups showed evidence of selective adsorption of anti-Factor VIII antibody from plasma.

Adsorption↗

Growth behavior of fibroblasts on microgrooved polystyrene.

We investigated the contact guidance phenomenon of rat dermal fibroblasts (RDF) on microgrooved polystyrene substrates. Grooves were 1 microm deep, and between 1 and 10 microm wide. Light microscopy and digital image analysis (DIA) showed that RDF were oriented on all microgrooved substrates. Scanning electron microscopy showed that RDF cultured on 1 or 2 microm wide grooves were positioned on top of the ridges. On the wider 5 and 10 microm grooves, the cells were able to descend into the grooves. In confocal laser scanning microscopy, focal adhesions were lying in the same direction as the actin filament where they attached to. DIA confirmed an orientational behavior of focal adhesions and actin filaments on microgrooves. There were no differences in the measured orientation between the different grooves. Besides, no obvious preference was found for focal adhesions to lie along edges of the surface ridges. Transmission electron microscopy showed that focal adhesions were able to bend along the edges of ridges. On the basis of our observations, we suggest that the breakdown and formation of fibrous cellular components, especially in the filopodium, is influenced by the microgrooves. The microgrooves create a pattern of mechanical stress, which influences cell spreading and cause the cell to be aligned with surface microgrooves.

Animals↗

Preparation of poly(ethylene glycol)-polystyrene block copolymers using photochemistry of dithiocarbamate as a reduced cell-adhesive coating material.

This article reports a novel preparation method of poly(ethylene glycol) (PEG)-polystyrene (PST) amphiphilic block copolymers with well-defined block lengths by using photopolymerization of an iniferter, benzyl N,N-diethyldithiocarbamate. PEG macroiniferters, which were prepared by end-capping of PEG monomethyl ethers with benzyl N,N-diethyldithiocarbamate group at one end, were irradiated with UV light in the presence of styrene (ST). NMR analyses showed that the PST block was chain-extended from the PEG block, resulting in the preparation of PEG-PST block copolymers. The number-average molecular weights of the copolymers increased almost linearly with irradiation time, light intensity, and concentration of ST. The polydispersities of the copolymers remained relatively small throughout the reaction (Mw/Mn approximately 1.3). The composition of two PEG-PST block copolymers thus obtained was as follows: PEG (Mn; 1.9 x 10(3) gmol(-1))-PST (3.0 x 10(3) gmol(-1)) and PEG (4.9 x 10(3) gmol(-1))-PST (2.6 x 10(3) gmol(-1)). These copolymers were coated onto a poly(ethylene terephthalate) film surface. X-ray photoelectron spectroscopy analyses and water wettability measurements showed that the PST block was enriched at the outermost layer as cast in air, whereas upon immersion into water, the PEG block was oriented toward water. Enhanced wettability was observed for the diblock copolymer with a higher PEG content. Significantly reduced cell adhesion was observed on both the coated surfaces. Thus, the PEG-PST block copolymer may function as a cell adhesion-resistant coating which reduced cell-substrate interaction.

Animals↗

Attachment of hyaluronic acid to polypropylene, polystyrene, and polytetrafluoroethylene.

Surfaces of polypropylene (PP), polystyrene (PS) and polytetrafluoroethylene (PTFE) were activated with radio frequency plasmas Ar and NH3 to aminate the polymer surface and were subsequently reacted with hyaluronic acid (HA) in one of the three different attachment schemes. Results show that ammonia plasma treated polymers were more reactive toward HA attachment. The three chemistry schemes consisted of two distinct approaches: (1) direct attachment of the HA to the aminated surface, and (2) extending the reactive group away from the surface with succinic anhydride and then reacting the newly formed carboxylic acid group with an adipic dihydrazide modified HA (HA-ADH). The latter scheme proved to be more effective, suggesting that steric effects were involved with the reactivity of the HA with surface groups. These HA-coated polymers are a candidate for cell attachment and growth.

Ammonia↗

Orientation of cell adhesion and growth on patterned heterogeneous polystyrene surface.

Studies of neurite outgrowth or cell migration, two important processes in neuronal networks formation, are facilitated by cell culture models capable of orientating cellular growth and of designing a well-defined cellular pattern. Heterogeneous polystyrene surfaces composed of oxygen plasma-treated stripes (PSox) with a low hydrophobicity separated by non-treated areas (PS) have these properties. In this study, to guide cell growth, we developed a cell culture model using these supports and we identified the molecular factors involved in cellular orientation. When the heterogeneous supports were not coated, proteins from a serum culture medium were required for cells to line up on PSox. On the other hand, cell orientation on coated surfaces was clearly influenced by competitive adsorption of adhesive proteins such as fibronectin or collagen and anti-adhesive molecules as pluronic F68 or albumin. Attachment factors were adsorbed on PSox stripes while adsorption of anti-adhesive molecules on the most hydrophobic PS areas prevented cell adhesion or growth. Thus, we describe the preparation of a cell culture substrate that succeeded in orientating cell growth and that led to a line of cells on adhesive PSox stripes ranging from 2 to 100 microns width.

Animals↗

Immuno affinity purification of foot and mouth disease virus type specific antibodies using recombinant protein adsorbed to polystyrene wells.

The specificity of foot and mouth disease virus (FMDV) serological tests depends largely on the quality and purity of the antibodies used. Such type specific antibodies can be generated by hybridoma technology. Alternatively, the specific antibodies can be selected from polyclonal serum by immunoaffinity chromatography using recombinant protein/peptide bound affinity matrices. Based on this approach, we purified selectively antibodies against the major epitopes of VP 1 of FMDV serotype Asia 1 using recombinant protein adsorbed to polystyrene wells. Optimum buffer conditions were standardised for efficient elution. Buffer consisting of 4 M MgCl2 with 75 mM HEPES pH 6.5 was found to be optimum with respect to elution efficiency of bound antibodies and integrity of antigen. The specific reactivity of eluted antibodies was confirmed by dot-enzyme linked immunosorbent assay (dot-ELISA) and antigen capture reverse transcription polymerase chain reaction (Ag/RT-PCR). The effect of temperature and repeated elution on the stability of coated protein were studied.

Animals↗

Monitoring for adsorption of human serum albumin and bovine serum albumin onto bare and polystyrene-modified silver electrodes by quartz crystal impedance analysis.

The adsorption of human serum albumin (HSA) and bovine serum albumin (BSA) from PBS (pH 7.4) onto bare and polystyrene (PS)-modified silver electrodes was in situ monitored using quartz crystal impedance analysis. The adsorption characteristics of HSA and BSA were discussed by analyzing piezoelectric parameter simultaneous responses. Experimental results indicated that for both HSA and BSA, the amount adsorbed on bare silver was more than that on PS-modified surface. The BSA amount adsorbed on the two surfaces was more than that of adsorbed HSA. A kinetic model was developed to describe the adsorption process and fitted to the experimental data of frequency shift. It was shown that HSA adsorption could be described by a kinetic equation involving two consecutive reactions. At lower concentration, BSA adsorption only involved the first reaction. At higher concentration, BSA adsorption on PS-modified surface involved two consecutive reactions. All fitted results were well in agreement with the corresponding experimental results. The regression values of reaction rate constants for the HSA and BSA adsorption were obtained. These data exhibited difference in adsorption kinetics under different conditions.

Adsorption↗

In vitro uptake of polystyrene microspheres: effect of particle size, cell line and cell density.

Uptake of polycation-DNA particles is the first step in achieving gene delivery with non-viral vehicles. One of the important characteristics determining uptake of DNA particles is their size. Here we have characterized the ability of several cell lines to internalise labelled polystyrene microspheres of different sizes. All the cell lines tested ingested 20-nm microspheres avidly. With larger microspheres (93, 220, 560 and 1010 nm) cell type as well as growth related differences were observed. Whereas some cell lines (HUVEC, ECV 304 and HNX 14C) took up microspheres up to 1010 nm even when the cells were confluent, others did not take up many microspheres larger than 93 nm (Hepa 1-6 and HepG2). In one cell line (KLN 205), uptake of 93-, 220- and 560-nm microspheres was avid in growing cells, but not detectable when they were confluent. In KLN 205 cells, a good correlation was found between the uptake of 560-nm microspheres and the uptake of a peptide-DNA polyplex formulation, when it was prepared under conditions leading to small particle sizes. Little correlation was found when the polyplex formulation was allowed to aggregate.

Amino Acid Sequence↗

Receptor-mediated delivery of all trans-retinoic acid to hepatocyte using poly(L-lactic acid) nanoparticles coated with galactose-carrying polystyrene.

All trans-retinoic acid (RA)-loaded poly(L-lactic acid) (PLA) nanoparticles coated with galactose-carrying polymer, as hepatocyte-specific targeting material using galactose ligands as recognition signals to asialoglycoprotein receptors were prepared by the diafiltration method. Effects of released RA from its loaded nanoparticles on morphology and DNA synthesis of hepatocytes were studied. Receptor-mediated endocytosis of the nanoparticles was checked by fluorescence and confocal laser microscopy. It was found that the shapes of most hepatocytes attached onto polystyrene dish precoated with collagen solution were flat and spreading at low concentration of RA for the RA-loaded nanoparticles, whereas their shapes were round at even low concentration of RA when RA was mixed with the nanoparticles. From the fluorescence and confocal laser microscopic studies, it was suggested that the nanoparticles coated with galactose-carrying polymers were internalized by the hepatocytes through the receptor-mediated mechanism. The RA-loaded nanoparticles were more potent stimulators of hepatocyte DNA synthesis than the free RA system in the presence of epidermal growth factor (EGF) owing to the controlled release of RA from the RA-loaded nanoparticles.

Animals↗