Haruma Kawaguchi

Functional polymer microspheres

Functional particles are prepared directly by heterogeneous polymerization, e.g. emulsion polymerization and dispersion polymerization. Modification of existing particles is another method to prepare functional particles. The features of nano- or microparticles such as large specific surface area, high mobility, easy recovery from the dispersion and reversible dispersibility, etc., are utilized for the exhibition of functions. Medical and biochemical applications of particles, including absorbents, latex diagnostics, affinity bioseparators and drug and enzyme carriers, are the most practical ones at present. Optical and opto-electrical functions of particles are attracting attention. Some particles exhibit unique rheological behavior under special conditions and will expand their applications. In this review article, examples of the above-mentioned functional particles are reviewed and discussed.

High-performance affinity beads for identifying drug receptors

We have developed a method using novel latex beads for rapid identification of drug receptors using affinity purification. Composed of a glycidylmethacrylate (GMA) and styrene copolymer core with a GMA polymer surface, the beads minimize nonspecific protein binding and maximize purification efficiency. We demonstrated their performance by efficiently purifying FK506-binding protein using FK506-conjugated beads, and found that the amount of material needed was significantly reduced compared with previous methods. Using the latex beads, we identified a redox-related factor, Ref-1, as a target protein of an anti-NF-κB drug, E3330, demonstrating the existence of a new class of receptors of anti-NF-κB drugs. Our results suggest that the latex beads could provide a tool for the identification and analysis of drug receptors and should therefore be useful in drug development.

Hydrogel microspheres III. Temperature-dependent adsorption of proteins on poly-N-isopropylacrylamide hydrogel microspheres

Precipitation polymerization of N-isopropylacrylamide (NIPAM) with methylenebisacrylamide (MBAAm) in water at 70°C gave thermosensitive hydrogel microspheres. The adsorbability of proteins on the poly-NIPAM microspheres was found to depend on temperature. Below the lower critical solution temperature (LCST) of poly-NIPAM in an aqueous medium, that is, around 32°C, the microspheres hold a large amount of water inside and their surface is hydrophilic enough to suppress the adsorption of proteins. On the contrary, above 32°C, the micropheres deswell and their surface becomes hydrophobic and, consequently, susceptible to adsorption of a large amount of proteins. Proteins once adsorbed on the microspheres at a high temperature could be desorbed more or less by lowering the temperature to below 32°C. The extent of desorption at low temperatures was found to depend on the incubation time for adsorption at high temperatures.

Thermosensitive pickering emulsion stabilized by poly(N-isopropylacrylamide)-carrying particles.

Poly(N-isopropylacrylamide) (PNIPAM)-carrying particles were characterized as thermosensitive Pickering emulsifiers. Emulsions were prepared from various oils, such as heptane, hexadecane, trichloroethylene, and toluene, with PNIPAM-carrying particles. PNIPAM-carrying particles preferentially formed oil-in-water (O/W)-type emulsions with a variety of oils. All the emulsions stabilized by PNIPAM-carrying particles were stable for more than 3 months as long as they were stored at room temperature. However, when the emulsions were heated from room temperature to 40 degrees C, at which point the PNIPAM layer caused a coil-to-globule transition, phase separation occurred. Thus, by using thermosensitive PNIPAM-carrying particles as emulsifiers, the stability of the Pickering emulsions could be controlled by a slight change in temperature.

Phagocytosis of latex particles by leucocytes. I. Dependence of phagocytosis on the size and surface potential of particles.

A series of latex particles, having different sizes and surface structures, were prepared and the dependence of phagocytosis of latex particles by leucocytes on the particle size and surface potential was investigated by measuring the oxygen consumption of leucocytes. Most of the phagocytic behaviour in the initial stages can be explained by susceptibility of particles to heterocoagulation i.e. attachment of small particles (latex particles) onto large particles (leucocytes) by the colloidal attractive force between the two kinds of particles. Specific behaviour for fine particles seems to be attributed to the contribution of steric stabilization by the hydrated layer on the particle surface and to the inability for the leucocytes to recognize very fine particles as foreign materials.

Smart latexes for bioseparation

Monodisperse, thermosensitive microspheres with sub-micron diameters are used for separation and collection of proteins and other biomolecules. The thermosensitivity gives the microspheres two valuable features. One is the controllability of affinity between microsphere and protein with temperature. The quality and quantity of proteins to be adsorbed on the microspheres can be controlled with temperature. The other feature is reversible control of dispersion stability. Microspheres which have adsorbed target proteins in the dispersion can be easily recovered by changing temperature.

Development of novel magnetic nano-carriers for high-performance affinity purification

We developed novel magnetic nano-carriers around 180 nm in diameter for affinity purification. Prepared magnetic nano-carriers possessed uniform core/shell/shell nano-structure composed of 40 nm magnetite particles/poly(styrene-co-glycidyl methacrylate (GMA))/polyGMA, which was constructed by admicellar polymerization. By utilizing relatively large 40 nm magnetite particles with large magnetization, the magnetic nano-carriers could show good response to permanent magnet. Thanks to uniform polymer shell with high physical/chemical stability, the magnetic nano-carriers could disperse in a wide range of organic solvent without disruption of core/shell structure and could immobilize various kinds of drugs. We examined affinity purification using our prepared magnetic nano-carriers with anti-cancer agent methotrexate (MTX) as ligand. Our magnetic nano-carriers showed higher performance compared to commercially available magnetic beads in terms of purification efficiency of target including extent of non-specific binding protein.

Enzyme immobilization on thermosensitive hydrogel microspheres

Precipitation polymerization of N-isopropylacrylamide, acrylamide, and methylenebisacrylamide in water at 70°C resulted in thermosensitive hydrogel microspheres. Carboxyl groups on the microspheres were introduced by hydrolysis, and amino groups by the Hofmann reaction of amide units on the microspheres. Trypsin was immobilized on the carboxylated microspheres using carbodiimide. Phase transitions were detected using a hydrophobic fluorescence probe. The temperatures at which a phase transition occurred were increased by immobilizing enzymes. The enzymatic activity of the immobilized enzymes decreased above the transition temperature. This was attributed to (i) a decrease in the diffusion of substrate; and (ii) entrapment of enzyme in the surface layer of the microspheres. In an attempt to overcome the entrapment, enzymes were immobilized via a hydrophilic spacer (α-(carboxymethyl)-ω-(carboxymethoxy)-poly(oxy-1,2-ethanediyl), PEO acid) to the microspheres. These enzyme-carrying hydrogel microspheres were found to show an enzymatic activity independent of temperature, even though these conjugates show a phase transition at the lower critical solution temperature.

Preparation of DNA-carrying affinity latex and purification of transcription factors with the latex.

We have developed DNA-carrying latex particles for the separation and purification of transcription factors. These particles consist of styrene (St), glycidyl methacrylate (GMA) and divinylbenzene (DVB). It was confirmed that the ethanolamine-treated surface of these particles suffered no nonspecific adsorption of proteins. To the latex particles sequence-specific DNA oligomers were immobilized via covalent coupling. A transcription factor, E4TF3, was efficiently purified to homogeneity using the latext particles. In contrast, the purification using DNA-carrying Sepharose gel yielded poor results. Compared to DNA-carrying Sepharose gel, the latex particles exhibited several times higher efficiency in the purification of E4TF3 from the crude nuclear extract.

Direct purification of multiple ATF/E4TF3 polypeptides from HeLa cell crude nuclear extracts using DNA affinity latex particles

We developed a method using affinity latex particles to rapidly and efficiently purify DNA-binding proteins directly from crude cell extracts. The particles are composed of a styrene core and a polyglycidyl methacrylate surface, to which DNA oligomers were immobilized by means of epoxy groups. Multiple polypeptides were copurified, which bound to the ATF/E4TF3-binding site from crude nuclear extracts of HeLa cells, within a few hours. Affinity-purified polypeptides stimulated transcription in vitro from a promoter in which ATF/E4TF3-binding sites were present. At least eight polypeptides with molecular masses of 116, 80, 65, 60, 55, 47, 45, and 43 kDa were copurified. About 2 micrograms of the 43-kDa protein was purified directly from 8 mg of crude nuclear extracts. All the polypeptides directly bound to the same DNA sequence and were thought to form a family. The results indicated that the particles are useful for quickly purifying various DNA-binding proteins directly from crude cell extracts.