Papers by Author: Kazuhiko Ishihara

Abstract: Photoreactive and cytocompatible polymer nanoparticles for immobilizing and photoinduced releasing proteins were prepared. A water-soluble and amphiphilic phospholipid polymer, poly (2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-4-(4-(1-methacryloyloxyethyl)-2-methoxy-5-nitrophenoxy) butyric acid (PL)) (PMB-PL) was synthesized. The PMB-PL underwent a cleavage reaction at the PL unit by photoirradiation at a wavelength of 365 nm. Additionally, the PMB-PL took polymer aggregate in aqueous medium and was used to modify the surface of biodegradable poly (L-lactic acid) (PLA) nanoparticle as an emulsifier. The morphology of the PMB-PL/PLA nanoparticle was spherical and approximately 130 nm in diameter. The carboxylic acid group in the PL unit could be used for immobilization of proteins by covalent bonding. The bound proteins were released by a photoinduced cleavage reaction. Within 60 sec, up to 90% of the immobilized proteins were released by photoirradiation and activity of the protein released in the medium was maintained as well as that the original proteins before immobilization. Octa-arginine (R8) could promote internalization of the protein/PLA/PMB-PL nanoparticles into cells when the R8 was co-immobilized on the nanoparticles. After that, photoirradiation induced protein release from the nanoparticles and proteins distributed more evenly inside cells. From these results, we concluded that PMB-PL/PLA nanoparticles have the potential to be used as smart carriers to deliver proteins to biological systems, such as the inside of living cells.

Abstract: Recently, much attention has been attracted to bio/blood compatible materials to suppress undesirable biological reactions that determine the fate of living organisms and materials. A phospholipid polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) unit, which is designed by inspiration of cell membrane surface structure, is the most promising polymer biomaterial with excellent bio/blood compatibility. Progress in living radical polymerization method initiated from the surface enables preparation of a dense polymer chains on the surface, which is called as a polymer brush. The polymer brush structure has narrow molecular weight distribution and controlled chain length. So, it is ideal surface to clarify the interactions between the biomolecules and biomaterial surface that has never done. In these regards, the poly(MPC) brush surfaces are expected to be a novel class of biomaterials, and have been extensively studied its unusual properties. In this review, surface-initiated living radical polymerization of MPC and the characteristics of the poly(MPC) brush surfaces are summarized from a viewpoint of biomaterials science.

Abstract: The phospholipid molecule is one of the typical components of the cell membrane. In particular, the phosphorylcholine polar group is an electrically neutral head group. Arrangement of phospholipid polar groups and construct the surface, we applied 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers bearing a phosphorylcholine group in side chain, which was designed with the cell membrane as an inspiration. Versatile polymers comprising MPC could be synthesized, and their specific biofunctions were evaluated. Establishing an ultimate interface between biological circumstances and artificial materials, so-called biointerfaces, with multiple functions is important from the viewpoint of biomaterials science. Nonspecific protein adsorption is essential for achieving versatile biomedical applications. Simultaneously, bioconjugation and retention of its biofunction are crucial for a high-performance interface. In this article, we would like to introduce effectiveness of interface with highly biological functions composed of the MPC polymers for constructing nanobiodevices and nanomedicine.

Abstract: Plastic substrates were modified with 2-methacryoyloxyethyl phosphorylcholine (MPC) copolymer brushes by surface-initiated atom transfer radical polymerization (ATRP) with a mean chain density of 2.44 chains/nm2 and a chain length around 50nm. Ellipsometry, X-ray photoelectron spectroscopy, gel permeation chromatography, nuclear magnetic resonance and atomic force microscopy were used to characterize the modified surfaces.
MPC was copolymerized with a methacrylate monomer including an active ester unit which can link covalently with antibodies. They latter were immobilized on these well-defined surfaces for characterization and a further use in enzyme-linked immunosorbent assay (ELISA).
All kinds of surfaces displayed a good density of immobilized antibodies mainly forming globular packs of around 500 units. A water-soluble MPC polymer-based stabilizer added to the antibody solution could further their individual immobilization.

Abstract: An anionic phospholipid copolymer PMBSSi was synthesized to construct a permanent coating with protein resistant units (2-methacryloyloxyethyl phosphorylcholine, MPC) as well as surface charges onto the silica based (quartz and glass) microchannel through a one-step modification. The coating showed a high property of minimizing the non-specific adsorption of both anionic and cationic proteins to a very low extent of less than 0.05 μg /cm2 on glass substrates. In addition, a significant cathodic EOF ((1.0±0.1) ×10-4 cm2/V·s) with approximately one-half of the EOF of the uncoated microchannel was achieved in coated microchannel at neutral pH. As a conclusion, PMBSSi coating is a simple but highly effective modification for reducing nonspecific protein adsorption, and promising to be applied in electrokinetic microfluidic systems.

Abstract: Non-biofouling surfaces with polymer-based substrate were prepared for manufacturing microfluidic devices. It was done by constructing biocompatible poly(2-methacryloyloxyethyl phosphorylcholine(MPC)) brushes using surface-initiated graft polymerization method based on dithiocarbamate as photoiniferter. The density and length of the polymer chains were varied by changing the composition of the photoiniferter moiety in the base polymer (macrophotoiniferter) and the photoirradiation time, respectively. The molecular weight and thickness of the poly(MPC)- grafted chains were 320 kDa and 95±14 nm, respectively. Characterizations of the poly(MPC) modified surfaces were conducted by water contact angle, X-ray photoelectron spectroscopy, atomic force microscope. Protein adsorption resistance of these modified surfaces was then investigated by contacting with human plasma protein dissolved in phosphate buffered saline. These poly(MPC)-modified surfaces effectively reduced protein adsorption.

Abstract: We investigated the bioconjugation of proteins on polymer nanoparticles covered with bio-inert phosphorylcholine groups. Poly[2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate-co-p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate] was used as an emulsifier and a surface modifier to prepare the poly (L-lactic acid) nanoparticles by a solvent evaporation technique. The diameter and surface potential of the nanoparticles were approximately 260 nm and –5 mV, respectively. We considered that the polymer chain that transformed the conformation by change in pH may be used as a method of controlling the bioreaction. The enzyme activity in a different pH under the coexistence of poly (glutamic acid) (PGA) and the enzyme was measured. Therefore, the enzyme activity increased in pH 6 in PGA/enzyme mixture system compared with that in pH 7 while the activity was constant in the enzyme single conjugation regardless of the pH change.

Abstract: We investigated phospholipid polymer hydrogels containing Fe3+ ions (PMA/PMB/Fe hydrogel) for their use as antiadhesive materials in the healing tissues. These hydrogels were prepared from the aqueous solutions of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)-comethacrylic acid) (PMA) and poly(MPC-co-n-butyl methacrylate) (PMB). The PMA/PMB hydrogel is formed by the intermolecular interactions between PMA and PMB, and it reversibly dissociates under physiological conditions. The addition of Fe3+ ions could control the gelation time and the dissociation time. Mechanical properties such as the gelation time and viscoelastic properties can be controlled by the FeCl3 concentration. With regard to biocompatibility, no evidence of inflammation was observed in vivo. Therefore, the PMA/PMB/Fe hydrogel has a potential to be used as an antiadhesive material.