Akio Kishida

Polymyxin B binds to anandamide and inhibits its cytotoxic effect

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)‐immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.

Apatite coating on hydrophilic polymer-grafted poly(ethylene) films using an alternate soaking process

Previously, we developed a novel alternate soaking process and clarified that bone-like apatite was formed on/in organic polymer hydrogel matrices using this process. The present study focused on the apatite coating on hydrophilic polymer grafted poly(ethylene) (PE) films with various grafting densities and commonly used hydrophilic polymers, poly(acryl amide) (PAAm) and poly(acrylic acid) (PAAc) were employed. From X-ray diffraction analysis, hydroxyapatite was coated on PAAm- or PAAc- grafted PE films. The amount of apatite coated on PAAm-grafted PE (PAAm-g-PE) films increased with an increase in the reaction cycles and the grafting density of PAAm. Similar to PAAm-g-PE, the amount of apatite coated on PAAc-grafted PE (PAAc-g-PE) films increased linearly with an increase in the grafting density of the PAAc up to around 30 microg/cm2. While, no significant increase in the apatite coating on the PAAc-g-PE films was observed even after 50 reaction cycles when the grafting densities of PAAc were over 30 microg/cm2. Apatite coating was not observed on original PE films. Scanning electron microscopic images reveal that the aggregation of apatite crystals on all PAAm-g-PE films and PAAc-g-PE films with grafting density from 10 to 30 microg/cm2. On the other hand, a dense apatite layer with some cracks was coated when the grafting density of the PAAc chains was over 30 microg/cm2. These results indicated that it was possible to coat apatite on hydrophilic polymer grafted PE films by an alternate soaking process and that the apatite crystal morphology could be controlled as a function of polymer type and density.

Apatite formation on/in hydrogel matrices using an alternate soaking process: II. Effect of swelling ratios of poly(vinyl alcohol) hydrogel matrices on apatite formation

In our previous study, we reported a novel method of apatite formation on/in a three-dimensional hydrogel matrix. Using this method, bone-like apatite could be formed on/in the hydrogel matrix under normal conditions in vitro. A poly(vinyl alcohol) (PVA) gel was used as a model matrix. The method consists of two steps: first, water is transformed in a PVA gel with a CaCl2/Tris-HCl aqueous solution (pH 7.4) and second, the gel is soaked in a Na2HPO4 aqueous solution. In the present study, we report a detailed study of the effects of the swelling ratios of PVA gels on apatite formation. Cross-sectional observations and gravimetric measurements of PVA gels with various swelling ratios were done. The amount of apatite formed on/in PVA gels increased almost linearly with an increase in the reaction cycles. The rates of apatite formation on/in PVA gels largely depended on the swelling ratios, which were approximately 0.48, 0.61, 1.28, and 1.55 mg per cycle for swelling ratios of 4.1, 10.4, 16.8, and 30.1, respectively. The apatite content in PVA-apatite composites that was obtained by this method also increased with an increase of the reaction cycles. After six reaction cycles, a PVA gel with a high swelling ratio contains approximately 70 wt% of formed apatite in the composite. On the other hand, a gel with a low swelling ratio contains about 15 wt% of formed apatite in the composite. Cross-sectional views of the PVA gels after each cycle showed that apatite crystals were formed, not only on the surface of the gel but also within it after fifteen reaction cycles. The hydrogel-apatite composites that were obtained using an alternative soaking process will be useful as not only bone substitute materials but also as soft tissue adhesive materials.

Preparation and characterization of apatite deposited on silk fabric using an alternate soaking process

Apatite-deposited silk fabric composite materials were developed using a new alternate soaking process. The characteristics of deposited apatite were studied using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectrophotometry (FTIR), and X-ray photoelectron spectroscopy (XPS). Apatite weight increased with alternating soaking in a calcium solution [200 mM aqueous calcium chloride solution buffered with tris(hydroxymethyl) aminomethane and HCl (pH 7.4)] and a phosphate solution (120 mM aqueous disodium hydrogenphosphate) changed every hour. SEM showed that apatite deposited after 21 or more repeated soakings was over 20 microm thick. XRD showed that with alternate soakings, the apatite crystals deposited on silk fabric elongated along the c axis. FTIR and XPS indicated the existence of carbonate, HPO(4)(2-), and Na(+) ions in addition to constituent ions of hydroxyapatite. A loss of HPO(4)(2-) and Na(+) ions in the deposit upon further soaking might be associated with an increasing apatite crystallinity. Apatite deposited on silk by the alternate soaking process was a deficient apatite containing carbonate, HPO(4)(2-), and Na(+) ions as in a natural bone tissue. Thus, this apatite-silk composite material might be potentially bioactive.

Graft copolymers having hydrophobic backbone and hydrophilic branches. XXIII. Particle size control of poly(ethylene glycol)‐coated polystyrene nanoparticles prepared by macromonomer method

Monodisperse polymeric nanospheres, which consist of polystyrene cores and poly(ethylene glycol) (PEG) branches on their surfaces, were prepared by the dispersion copolymerization of styrene (St) with PEG macromonomers that had a methacryloyl (MMA-PEG) or p-vinylbenzyl (St-PEG) end group in various organic solvent/water media. Electron spectroscopy for chemical analysis (ESCA) of the nanosphere surfaces indicated that PEG macromonomer chains were favorably located on their surfaces. The morphologies of the nanospheres were observed via a scanning electron micrograph (SEM), and particle sizes were estimated by a submicron particle analyzer. When both the concentration of macromonomers and molecular weight were higher, small nanospheres in diameter were obtained. Larger nanospheres in diameter were obtained using macromonomers with low molecular weight at lower concentration. The functions that correlate the diameter (Dn) on different concentration units were Dn = K[St]0.64[MMA-PEG]−0.53±0.01[I]−0.49 and Dn = K[St]0.80[St-PEG]−0.69±0.01[I]−0.22, where [I], [St], [MMA-PEG], and [St-PEG] are initiator, styrene, MMA-PEG, and St-PEG macromonomer concentration in feed, respectively. When the reaction parameters such as the molecular weight of the macromonomers were properly chosen, the particle size could be controlled in a range from ca. 80 to 3100 nm.

Graft copolymers having hydrophobic backbone and hydrophilic branches. XI. Preparation and thermosensitive properties of polystyrene microspheres having poly (N-isopropylacrylamide) branches on their surfaces†

Thermosensitive microspheres with 0.4–1.2 μm diameter consisting of a polystyrene core and poly(N-isopropylacrylamide) (polyNIPAAm) branches on their surfaces were prepared by the free radical polymerization of a polyNIPAAm macromonomer and styrene in ethanol. Electron spectroscopy for chemical analysis (ESCA) of the microsphere surface suggested that polyNIPAAm chains were favorably located on the surface of the microspheres. The morphology of the microspheres was observed by transmission electron micrograph (TEM) and the particle size of was estimated by submicron particle analyzer. The molecular weight of the polyNIPAAm macromonomer, the ratio of the macromonomer and styrene, and the polymerization temperature affected the particle size. Thermosensitive properties of polyNIPAAm-coated polystyrene microspheres were evaluated by the turbidity of their dispersion solutions and the hydrodynamic size of the miocrospheres. The transmittance in dispersion solutions changed clearly, similar to oligoNIPAAm and polyNIPAAm macromonomers. In addition, the particle size of microspheres decreased with rising temperature. These results were explained by the thermosensitivity of polyNIPAAm branches on the microsphere surface.

Oral peptide delivery using nanoparticles composed of novel graft copolymers having hydrophobic backbone and hydrophilic branches

Nanoparticles composed of new graft copolymers having a hydrophobic backbone and hydrophilic branches were prepared by the dispersion copolymerization of hydrophilic polyvinyl macromonomers with styrene in a polar solvent. The potential of these nanoparticles as carriers for oral peptide delivery, was investigated using salmon calcitonin (sCT) in rats. The rate of sCT incorporated in nanoparticles was high and was affected by the macromonomer structure. Anionic nanoparticles having poly(methacrylic acid) macromonomer chains on their surfaces showed the highest incorporating activity. When the mixture of sCT and nanoparticles was administered orally, the decrease in the blood ionized calcium concentration was greater than that after oral administration of sCT aqueous solution. This hypocalcemic effect was also affected by the macromonomer structure, and the absorption of sCT was enhanced most strongly by nanoparticles having poly(N-isopropylacrylamide) macromonomer chains. However, the calcium concentration changed less when the nanoparticle concentration was low. On the other hand, the hypocalcemic effect was independent of the nanoparticle size and molecular weight of the macromonomers. The absorption enhancement of sCT by the nanoparticles probably results from both bioadhesion to the gastrointestinal (GI) mucosa and the increase of the stability of sCT in the GI tract. These nanoparticles were demonstrated to be useful carriers for incorporating highly water-soluble peptides and for enhancing peptide absorption via the GI tract.

Synthesis and functionalities of poly(N-vinylalkylamide). V. Control of a lower critical solution temperature of poly(N-vinylalkylamide)

N-vinyl-n-butyramide (NVBA), N-vinylisovaleramide (NVIVA), and N-vinyl-n-valeramide (NVVA), which are N-vinylalkylamides with different alkyl groups were synthesized and their solution behavior in a polymeric form was examined. Copolymers of N-vinylisobutyramide (NVIBA) with N-vinylacetamide (NVA), NVIBA with NVVA, and NVVA with NVA were prepared by the solution polymerization to control the LCSTs. The resultant polyNVBA showed a lower critical solution temperature (LCST) sharply at 32°C, but polyN-vinylisovaleramide (polyNVIVA) and polyN-vinyl-n-valeramide(polyNVVA) that have n-butyl and isobutyl groups, respectively, on their side chains were insoluble even in cold water. The water solubility of the resulting polymers was found to vary, depending on the molecular shapes as well as the side chain length of the alkyl groups in question. The copolymers consisting of NVVA, NVIBA, and NVA in water showed LCSTs sharply between 10 and 90°C, depending on changes in their comonomer content. It was found that the changes in LCST that are caused by the incorporation of comonomers are due to changes in the overall hydrophilicity of the polymer.

Apatite formation on/in hydrogel matrices using an alternate soaking process (III) : Effect of physico-chemical factors on apatite formation on/in poly(vinyl alcohol) hydrogel matrices

The aim of this study is to clarify the physico-chemical factors which influence apatite formation on/in a hydrogel during a novel alternate soaking process. A poly(vinyl alcohol) (PVA) gel was used as a model matrix. The amount of apatite formed on/in PVA gels decreased with an increase in the reaction temperature during the same reaction cycles. This suggested that the equilibrium swelling ratios decreased with increasing reaction temperatures; that is, the diffusion of calcium and phosphate ions reduced at high reaction temperature. However, the crystallinity of apatite formed on/in PVA gels was greater at higher reaction temperatures. The amount of apatite formed on/in PVA gels increased with an increase in the calcium and phosphate solution concentrations, and increased by shaking at the first three reaction cycles. A few influences could be observed when the solution volume was changed, however, the soaking order was not effective in this study. These results indicate that the amount of apatite formation on/in PVA gels can be controlled by changing the reaction temperature and the Ca- and P-solution concentrations, and that the crystallinity of apatite can be also changed by controlling the reaction temperatures.

Optimized chemical structure of nanoparticles as carriers for oral delivery of salmon calcitonin

Nanoparticles having two kinds of surface hydrophilic polymeric chains were prepared by the free radical copolymerization between styrene and hydrophilic macromonomers terminating in vinylbenzyl groups. Their potential as carriers for oral peptide delivery was investigated using salmon calcitonin (sCT) in rats. After oral administration of mixtures of sCT and nanoparticles, the ionized calcium concentration in blood was measured. The absorption of sCT was significantly enhanced by nanoparticles having poly-N-isopropylacrylamide (PNIPAAm) chains on their surfaces. This enhancement effect was considerably increased by introducing cationic poly-vinylamine (PVAm) groups to the surface of PNIPAAm nanoparticles. The absorption enhancement depended on the ratio of NIPAAm and VAm macromonomers to styrene in the nanoparticle preparation. In contrast, the introduction of nonionic poly-vinylacetamide (PNVA) groups eliminated completely the absorption-enhancing function of PNIPAAm nanoparticles. It was suggested that this disappearance was due to the shielding of PNIPAAm groups by PNVA groups. The enhancement effect of sCT absorption by nanoparticles was greatly dominated by their chemical structure that was closely related to surface characteristics. Optimization of the chemical structure on the basis of the mechanism of the absorption enhancement resulted in the further improvement of sCT absorption.