Immunoactivity of polymer microspheres with their hydrophilic/hydrophobic heterogeneous surface sensitized with an antibody

Styrene/2-hydroxyethyl methacrylate polymer microspheres consisting of various polymer compositions were produced by emulsifier-free seeded emulsion polymerization technique. Using these microspheres, which should have hydrophilic/hydrophobic heterogeneous surface, the effects of surface hydrophilicity on the main, fundamental requirements for an immunomicrosphere — high colloidal stability, sensitive immunologic agglutinability and insensitive non-specific agglutinability — were studied in detail. There was a region of the surface hydrophilicity that satisfied the three requirements simultaneously.Part CII of the series Studies on Suspension and Emulsion.     

Participation of electrolyte cations in albumin adsorption onto negatively charged polymer latices

The participation of electrolyte cations in the adsorption of bovine serum albumin (BSA) onto polymer latices was investigated. The latices used were hydrophobic polystyrene (PS), and hydrophilic copolymers, i.e., styrene (St)/2-hydroxyethyl methacrylate(HEMA) copolymer [P(St/HEMA)] and styrene/acrylamide (AAm) copolymer [P(St/AAm)]. Three kinds of electrolyte cations (Na⁺, Ca²⁺, Mg²⁺) were used as the chloride. The amount of BSA adsorbed in every cation medium showed a maximum near the isoelectric point (iep, pH about 5) of the protein. The amounts of BSA adsorbed onto copolymer latices (except in the acidic pH region lower than the iep) were considerably smaller than that onto PS latex because of the steric repulsion and the decrease in the hydrophobic interaction between BSA and copolymer latices. In the acidic pH region, there was little difference in the amount of BSA adsorbed in every cation medium. However, in the pH region higher than the iep, the amounts of BSA adsorbed (particularly onto PS latex) in divalent cations (Ca²⁺ and Mg²⁺) media were relatively greater compared with that in a monovalent (Na⁺) one. This result was interpreted on the basis of the differences in such effects of electrolyte cations as dehydration power, suppression of the electrostatic repulsion, and binding affinity to BSA molecule. Ion Chromatographic estimation of the amounts of electrolyte cations captured upon BSA adsorption (in pH > 5) revealed that divalent cations were incorporated into the contact interface between the latex and BSA molecule so as to prevent the accumulation of anion charge and facilitate the protein adsorption.     

Binding of naproxen and 1-anilino-8-naphtalenesulphonic acid to albumin: Characterization of their binding sites in bovine serum albumin

The binding of naproxen (NP) and 1-anilino-8-naphtalenesulphonic acid (ANS) to bovine serum albumin (BSA) has been studied by equilibrium dialysis and spectrophotofluorometry, respectively. The drug protein (D/P) ratio is found to determine the nature of binding sites for NP but not for ANS. At low D/P ratio, NP possesses a specific site in BSA and one of the tryptophan residues is part of it. Fluorometric investigation reveals non-displacement of ANS from its sites by the binding of NP to its specific site. At high D/P ratio, NP instead causes localized conformational changes in BSA. Furthermore, it is possible to infer the location of NP's specific site as loop 4 of BSA. NP and ANS binding sites have also been examined by employing Bromocresol Green (BCG) as a spectrophotometric probe. Competition studies are also able to establish distinct binding pattern for the two ligands in BSA. The displacement pattern points out the presence of independent sites for NP (specific site) and ANS (initially occupied site) in BSA in spite of their amphipathically similar nature.     

Preparation of micron-size monodisperse polymer microspheres having cationic groups

Micron-size monodisperse crosslinked polymer microspheres having chloromethyl groups were prepared by seeded copolymerization of styrene, divinylbenzene, and chloromethylstyrene in the presence of 2.1- monodisperse polystyrene seed particles produced by dispersion polymerization. The modification reaction of chloromethyl groups on the surfaces with polyamines such as triethylenetetramine and ethylenediamine was carried out. From the measurements of potential and the amount of chloride ion released, the introduction of a large number of cationic groups at the surfaces was confirmed.Part CXXXI of the series Studies on suspension and Emulsion.     

Competitive adsorption of fibrinogen and albumin onto polymer microspheres having hydrophilic/hydrophobic heterogeneous surface structures

The competitive adsorption of bovine fibrinogen (BFb) and bovine serum albumin onto polymer microspheres from the mixture solution was examined under various protein-to-microsphere ratios using various homopolymer microspheres and poly(2-hydroxyethyl methacrylate)/polystyrene composite microspheres having heterogeneous surface structures consisting of both hydrophilic and hydrophobic parts. They were produced by emulsifier-free (seeded) emulsion polymerizations. The selective adsorption of BFb was not observed for the homopolymer microspheres, but observed for the composite polymer microspheres having optimum compositions.Part CXXXVIII on the series Studies on Suspension and Emulsion     

Production of anomalous polymer microspheres having uneven surfaces by “stepwise” heterocoagulation technique

Anomalous polymer microspheres having uneven surfaces were produced by stepwise heterocoagulation technique of small polymer particles (SPs) onto large polymer particles (LPs).SPs andLPs have surface charges opposite to each other in the emulsion states.SPs were produced by emulsion copolymerization of styrene and methacryloyloxyethyltrimethylammonium chloride, andLPs by emulsion terpolymerization of styrene, butyl acrylate and methacrylic acid, with nonionic emulsifier being used in both cases. Maximum covering ofLP bySPs was obtained under the conditions that both emulsions were blended without the coagulation at pH 3 at room temperature and then left stand to coagulate with each other at 70 °C for 4 h at pH 9.Part CXI of the series Studies on Suspension and Emulsion.     

XPS analysis (ESCA) of the surface composition of poly(styrene/2-hydroxyethyl methacrylate) microspheres produced by emulsifier-free emulsion polymerization

Polymer microspheres composed of various compositions of styrene and 2-hydroxyethyl methacrylate (HEMA) were produced by batch emulsifier-free emulsion polymerization. The HEMA content at the surface, [HEMA] _s , of the microspheres powdered by freeze-drying was determined by both quantitativeC ^1s /O ^1s analysis andC ^1s peak shape analysis of the x-ray photoelectron spectroscopic spectra. When the HEMA content in the microsphere, [HEMA] _p , was less than about 5 mole%, the [HEMA] _s values determined by the two different methods showed good agreement. At [HEMA]_p above 5 mole %, [HEMA]_s values determined by the first method were about 15 mole % greater than those determined by the second. They both showed a similar tendency with the [HEMA] _s being higher than the [HEMA] _p , e.g., when [HEMA] _p was 1 mole %, [HEMA] _s was 11 mole %. The intensity of the satellite peak due to the ^* transition of the benzene ring of the styrene component decreased with an increase in [HEMA] _p , to zero at 5 mole % of [HEMA] _p . These results indicate that the HEMA component is localized at the surface.Part CVIII of the series Studies on Suspension and Emulsion.     

Development of new hydrophilic polymer supports based on dimethylacrylamide

A survey of the development of hydrophilic resins based on dimethylacrylamide (DMA) is presented. It describes the preparation of DMA-based resins by suspension copolymerization of DMA and acrylamide-based crosslinking and functional monomers. Swellability data for a number of DMA-based resins are tabulated, demonstrating that the resins swell to approximately the same extent in dichloromethane as they do in water, methanol, dimethylformamide or dimethylsulphoxide, but they swell only slightly or not at all in tetrahydrofuran. Typical chemical reactions employed for quantitative estimation or derivatization of resin-bound functional groups, including those involving intra-resin crosslinking, are discussed. The development of composite polymer supports, in which the DMA-based polymer is supported on kieselguhr particles or an amphiphilic ion exchange polymer is supported on DMA-based resin beads, is also covered briefly.     

Polymer coatings onto anodized aluminum plate by anodic electrodeposition from alkali-swollen carboxylated acrylic emulsion

Polymer coating of an anodized aluminum plate was carried out by anodic electrodeposition from alkali-swollen ethyl acrylate-methyl methacrylate-methacrylic acid (45/45/10, mole ratio) terpolymer emulsion with dimethyl aminoethanol. The alkali-swollen state was controlled by heating time at 60°C prior to the elctrodeposition. The similar complicated behaviors were observed in the relationships between the alkali-swollen state and the primary current density, the current density after 2 min, the quantity of electricity, and the amount of polymer deposited. The appearance of coatings changed correspondingly with such behaviors. These results indicate that the properties of the coatings are complicatedly affected by the degree of alkali-swollen state.Part CXII of the series Studies on Suspension and Emulsion.     

Estimation of distribution of vinyl groups in micron-sized monodisperse polymer microspheres by bromine titration method

The distribution of vinyl groups in polystyrene/poly(styrene-divinylbenzene) (PS/P(S-DVB)) composite microspheres produced by seeded copolymerization of S and DVB in the presence of 2.1-m monodisperse PS seed particles was estimated from the amount detected with bromine titration method as a function of the reaction time under various conditions.Part CXXIII of the series Studies on Suspension and Emulsion.     

Chemical deposition coatings from emulsifier-free anionic polymer emulsion onto anodized aluminum plate under stirring conditions

The chemical deposition from an anionic acrylic copolymer emulsion onto an anodized aluminum plate was carried out under various stirring conditions. The amount of polymer deposited was affected by the stirring rate. At higher stirring rates, the surface flatness of the obtained coating decreased, but the transparency (which was affected by the traces of bubbles, due to evolution of hydrogen gas from the surface of the plate in the deposition process) was improved. Under optimum stirring conditions an excellent coating having a flat surface and good transparency was obtained.Part CXVIII of the series Studies on Suspension and Emulsion.     

Preparation of micron-size monodisperse polymer particles having highly crosslinked structures and vinyl groups by seeded polymerization of divinylbenzene using the dynamic swelling method

Micron-size monodisperse polystyrene/polydivinylbenzene (PS/PDVB) composite particles having highly crosslinked structures and vinyl groups were prepared as follows. First, 1.9 m-size monodisperse PS seed particles produced by dispersion polymerization were dispersed in ethanol/water (70/30, w/w) solution which dissolved divinylbenzene (DVB) monomer, benzoyl peroxide as an initiator and poly(vinyl alcohol) as a stabilizer. The PS seed particles were swollen with a large amount of DVB monomers to 4.3 m in diameter while keeping good monodispersity by the dynamic swelling method, where water was slowly added, continuously, with a micro feeder into the dispersion. And then, the seeded polymerization of the absorbed DVB was carried out.Part CXXXV of the series Studies on Suspension and Emulsion     

Preparation of asymmetric polymer film by emulsion blend technique

Two kinds of anionic polymer emulsions, of which particle sizes were greatly different, were blended and then cast on a release-paper at 30 °C. One was poly(butyl acrylate) emulsion, 0.46n in diameter, produced by emulsifier-free emulsion polymerization, and the other was ethyl acrylate-methyl methacrylate (1/1, mole ratio) copolymer emulsion, 0.02m in diameter, produced by emulsifier-containing emulsion polymerization. The film produced had asymmetric surface properties: the air-side surface was nontacky and the bottom-side surface was tacky. The forming mechanism is discussed.     

Effect of surface charge on adsorption of bovine serum albumin 2. Interaction of protein molecules with an anionic monolayer,as studied by ellipsometry,radiotracer and surface tension measurements

The adsorption of bovine serum albumin (BSA) onto an anionic monolayer of sodium docosylsulfate (SDocS) spread at the air/water interface was studied by ellipsometry. The adsorption behavior of BSA was estimated from the observed changes in phase differences and in the ratio of reflection coefficients. The dynamic process of BSA adsorption was measured after the injection of BSA solution into the aqueous substrate of SDocS monolayer. The gentle stirring of the substrate solution for 10 min was found to be enough to make the solution homogeneous without damaging the monolayer. The adsorption characteristics of BSA onto a negatively charged surface was compared with that onto a positively charged surface previously reported.The amount of adsorption depended on time and showed a maximum with an initial rapid rise, followed by gradual decrease toward the ultimate equilibrium value. The amount and time of the maximum adsorption depended on the concentration of BSA added to the aqueous substrate.Separate radiotracer measurement, using³⁵S-labeled SDocS monolayer, which is insoluble by itself, revealed that SDocS is solubilized into the bulk solution when BSA is added to the aqueous substrate.     

Simulation of a monolayer adsorbed on a heterogeneous surface: effect of the size and shape of the adsorbate on the structure of the interface and the distribution of the adsorption energies

At the low temperature limit, the effect on the size and shape factors of the adsorbate molecule is shown in a simulation of a monomolecular phase adsorbed on a heterogeneous surface. This factor is reflected both in the theoretical distribution of the adsorption energies and the packing of the adsorbed phase.     

Light scattering study of solution properties of bovine serum albumin,insulin, and polystyrene under moderate pressure

Brillouin scattering experiments have been performed on very dilute solutions of bovine serum albumin (BSA) in pH=4.7,I=0.1 M NaCl; insulin in pH=7.5,I = 0.1 M NaCl and polystyrene latex suspensions in aqueous medium at room temperatureT = 20₁ °C. The samples were first characterized by quasi-elastic light scattering (QELS) to determine the translational diffusion coefficient (D _T), hydrodynamic radius (R _H), and polydispersity of each of the samples at normal pressure. Subsequently, the samples were subjected to Brillouin scattering studies where the spectrum of the scattered light was analyzed by using a piezo-electric scannend five-pass Fabry-Perot interferometer. The hydrostatic pressure (P) acting on the samples was varied in the range from 1B to 825 B in steps of 100 B each. The pressure dependence of the measured sound velocityV _s (P) could be least square fitted tov _s (P)=A₀ +A ₁ P +A ₂ P ² with experimental error (± 1 %). This data has been interpreted consistent with an earlier work within the framework of modified Tait's equation of state and presumption of linear pressure dependence of the adiabatic bulk modulus in the moderate pressure regime.     

Protein immobilization on surface modified latices bearing aldehyde groups

A new method for providing polymeric microspheres with aldehyde functional groups capable of covalent reaction with proteins is described. Benzyl halide groups on small molecules, polymeric resins, and the surfaces of surfactant-free poly(styrene-co-chloromethylstyrene) were converted to benzaldehyde groups by oxidation with 2-nitropropane in aqueous sodium methoxide. The modified microspheres containing aldehyde groups can be reacted with protein under milder conditions than the parent microspheres. The activity of a monoclonal antibody, Phe 1.9, was higher when it was immobilized on the aldehyde-containing beads than following immobilization on the parent beads.     

Molecular properties of acetone and some of its chloroderivatives adsorbed at the surface of water

The relations between the electric surface potential (V) and the surface tension () of aqueous solutions of acetone, chloroacetone, 1,3-dichloroacetone, and their concentration were investigated. The vertical components of dipole moments of the above mentioned compounds were determined using the Helmholtz equation. The calculations were carried out on the basis of surface excess values, which were obtained from surface tension measurements and surface potential changes. Once the vertical component of dipole moments were found and the orientation of adsorbed molecules was assumed, the local dielectric permittivities of the surface monolayer were estimated.     

Polymer adsorption on fine particles; the effects of particle size and its stability

The effects of particle size on polyacrylamide (PAAm,M _w =59×10⁴, 500×10⁴) adsorption were investigated using a series of well-characterized hematite (-Fe₂O₃) dispersions. The -Fe₂O₃ particles with highly monodisperse and nearly spherical shape ranged in radius from 23 nm to 300 nm. the maximum amount of PAAm adsorption (M _m ) in each system, showed a steady increase with decreasing particle radius and was influenced strongly by particle concentrations in the medium. Furthermore, it was realized that the diameter of -Fe₂O₃ particles after treatment with PAAm under different particle concentrations decreased with increasing particle concentration. The relation between particle concentration in the medium and particle size after treatment was also influenced by the medium pH, i.e., at the medium pH close to the isoelectric point of -Fe₂O₃ particles (pH^o=9.2), the particle size after treatment increased with increasing particle concentration. All these results suggest that in the system of ultra-fine particles, the mixing process between particle-particle and polymerparticle will play an important role on the conformation of adsorbed polymer layer.     

Interface stabilization and micelle formation in blends with a block copolymer

The phase separation behavior of ternary blends of two homopolymers, PMMA and PS, and a block copolymer of styrene and methylmethacrylate, P(S-b-MMA), was studied. The homopolymers were of equal chain length and were kept at equal amounts. Two copolymers were used with blocks of equal length, which exceeded or equaled that of the homopolymer chains. Varied was the copolymer contentf. Films were cast from toluene, which is a nonselective solvent. The morphologies of the cast films were compared with the structure of the critical fluctuations in solution, which were calculated in mean field approximation. The axis of blend compositionsf can be divided into parts of dominating macrophase and microphase separation. Above a transition concentrationf _o, all copolymer chains are found in phase interfaces. Belowf _o, part of them form micelles within the homopolymer phases.