Norio Nishi

Immunological activity of chitin and its derivatives

The effect of chitin and its derivatives on the activation of peritoneal macrophages in vivo, on the suppression of tumour growth in syngeneic mice and on the protection of the host against bacterial infection was examined. Thirty percent deacetylated chitin (30% DA-chitin), 70% DA-chitin and carboxymethyl-chitin (CM-chitin) induced cytotoxic macrophages most effectively. Chitosan, hydroxyethyl-chitin, dihydroxypropyl-chitin (DHP-chitin) and DHP-chitosan had moderate activities. Phosphorylated-, sulphonated- or acetyl-chitin, however, were less effective. Both 70% DA-chitin and DHP-chitosan were most active on the suppression of Meth-A tumour growth in BALB/c mice, and 30% DA-chitin had a moderate effect. For the stimulation of non-specific host resistance against Escherichia coli infection, 30% and 70% DA-chitin were effective.

Adjuvant activity of chitin derivatives in mice and guinea-pigs

Adjuvant activity of chitin derivatives was examined in guinea-pigs and mice. Among derivatives of chitin tested, 30 and 70% deacetylated chitin (DAC-30 and DAC-70), were active as adjuvants for the circulating-antibody formation to bacterial alpha-amylase and for the induction of delayed-type hypersensitivity to azobenzenearsonate-N-acetyl-L-tyrosine. DAC-70 enhanced the helper T cell function, the generation of alloreactive cytotoxic T lymphocytes, and the activity of natural killer cells in mice, however, it was inactive as mitogen. Other derivatives of chitin showed weaker or no adjuvant activity compared with DAC-70. Carboxymethyl-, hydroxyethyl and dihydroxypropyl-chitin showed weak mitogenic activities on normal spleen cells.

Bioactive chitin derivatives. Activation of mouse-peritoneal macrophages by O-(carboxymethyl)chitins.

The effect of O-(carboxymethyl)chitins (CM-chitins) on the activation of mouse-peritoneal macrophages in vivo and their mitogenic activity on mouse spleen-cells were investigated. The induction of cytotoxic macrophages is enhanced by an increase of negative charge at O-6 and decreased by further modification at O-3 of the GlcNAc residue. CM-Chitins had a minor effect on mitogenic activity that was independent of the site of modification; partially N-deacetylated chitins had little activity. Although there was remarkable enhancement of accessibility to lysozyme upon modification at O-6 of the GlcNAc residue, the accessibility was decreased by further substitution at O-3.

Facile preparation and inclusion ability of a chitosan derivative bearing carboxymethyl-β-cyclodextrin

Abstract Coupling carboxymethylated β-cyclodextrin and partially deacetylated chitin (Mw = 7300) afforded a new type of functional chitosan derivative having an ability to form an inclusion complex. The property to form an inclusion complex was studied using a fluorescent dye, 6-(p-toluidino)-2-naphthalene-6-sulfonate (TNS), as the guest molecule. It was found that the presence of the cyclodextrin bonded to chitosan enhanced the relative intensity of TNS fluorescence significantly (3.6 times as much as that of β-cyclodextrin). Fluorometric titration revealed that the 1:1 stoichiometrical complex was formed and that the equilibrium constant was 1.13-1.68 × 103 M−1. Ionic interaction between the sulfonic acid moiety of TNS and the amino group of chitosan was suggested from experiments which involved changing the pH and ionic strength of the buffer solution as well as adding a surface-active agent.

Blood compatible aspects of DNA-modified polysulfone membrane—protein adsorption and platelet adhesion

DNA was used as a biomaterial to modify the polysulfone (PSf) membrane by blending it with PSf. The blood compatibility of the membranes was then investigated. The water contact angle decreased, and the hydrophilicity increased when a single strand DNA was blended with PSf. Because of the hydrophilic surface, the DNA-blended PSf membranes had a lower protein adsorption than the PSf membrane, but it was not significantly decreased due to the interaction between the DNA and proteins. Circular dichroism (CD) spectroscopy was used to examine the changes in the secondary structure of the proteins after adsorption onto the polymer surface and desorption from the polymer surface into the SDS solution. The conformation of the proteins adsorbed onto the PSf membrane and desorbed from the PSf membrane significantly changed, but that of the proteins for the DNA-blended PSf membranes differed only slightly from the native one. The number of platelets that adhered on the surface of the DNA-blended PSf membranes was reduced compared to that on the PSf membrane. This suggested that DNA can be regarded as a biopolymer to modify PSf, and contributes to the hydrophilic and hemocompatible wipers on the surface of the hydrophobic PSf membranes.

Mesoscopic pattern formation of nanostructured polymer assemblies

Abstract Hierarchical mesoscopic structures of the nanoscopic supramolecular assemblies, which consist of polyelectrolytes and bilayer-forming amphiphiles, are prepared by a simple and new solvent-casting method. Submicron scale 2-D structures, e.g. regular dots, stripes, and networks, are formed when highly diluted organic solutions of polymer assemblies are cast on solid surfaces. Dynamic mesoscopic regular structures, the so-called ‘dissipative structures’, formed in the non-equilibrium processes of solvent-casting are fixed as hierarchically structured polymer assemblies.

Highly phosphorylated derivatives of chitin, partially deacetylated chitin and chitosan as new functional polymers: preparation and characterization

New functional chitin derivatives, i.e. variously phosphorylated derivatives of chitin and deacetylated chitins (DA-chitins), were prepared by the reaction of chitin or DA-chitins with phosphorus pentoxide in methanesulphonic acid. Degree of substitution (DS) of the products estimated from i.r. spectra, 13C n.m.r. spectra and elemental analyses increases with increase in the amount of phosphorus pentoxide employed, and products become nearly fully substituted when 4.0 mol. equiv. of phosphorus pentoxide to the residue is employed. 13C n.m.r. study suggested that the hydroxyl groups of C-3 and C-6 positions are equally substituted with the phosphate group. Chitin phosphates (P-chitins) are easily soluble in water independent of DS. However DA-chitin phosphates (P-DA-chitins) of high DS are insoluble in water owing to the amphoteric property, while those of low DS are water soluble. The water-soluble products behave as typical polyelectrolyte viscometrically. Two pKa values of the introduced phosphate groups were estimated to be around 3.7 and 8.2–8.5 by potentiometric titration. Preliminary experiment for adsorption of Cu2+ ion onto P-chitin (DS 1.0) by gel filtration showed that one copper ion was chelated to two phosphated N-acetyl-d-glucosamine residues, suggesting its high metal-binding ability.

Laminin-1 peptide-conjugated chitosan membranes as a novel approach for cell engineering

Laminin, a major component of the basement membrane, has diverse biological activities. Recently, we identified various biologically active sequences on laminin‐1 by using a large set of synthetic peptides. Chitosan, a polysaccharide, is biodegradable and has been used as a biomaterial. Here, we conjugated several biologically active laminin peptides onto chitosan membranes and measured the cell attachment activity of peptide‐conjugated chitosan membranes with various cell types. The active laminin peptide‐conjugated chitosan membranes promoted cell attachment with cell type specificity. A99 (AGTFALRGDNPQG)‐chitosan membrane promoted cell attachment with well‐organized actin stress fibers. This adhesion was inhibited by EDTA but not by heparin. AG73 (RKRLQVQLSIRT)‐chitosan membrane promoted cell attachment with filopodia formation, and this adhesion was inhibited by heparin but not by EDTA. These data suggest that the A99‐chitosan membrane interacted with an integrin cellular receptor and that the AG73‐chitosan membrane promoted proteoglycan‐mediated cell attachment, as previously reported. Furthermore, both AG73‐chitosan and A99‐chitosan membranes effectively promoted neurite outgrowth with PC12 rat pheochromocytoma cells. We conclude that conjugation on a chitosan membrane is applicable for testing quantitatively the biological activity of synthetic peptides and that these constructs have a potential ability to serve as bioadhesive materials for tissue regeneration and engineering.

Surface modification of nonporous glass beads with chitosan and their adsorption property for transition metal ions

Abstract A new hybrid material that adsorbs transition metal ions was prepared by immobilizing chitosan on the surface of nonporous glass beads. The glass beads, prepared by etching in aqueous NaOH at 100°C, were first reacted with γ-aminopropyltriethoxysilane (APES) to introduce amino groups on the surface. Subsequently, the resulting aminated beads were treated with glutaraldehyde at 25°C to change the amino groups into aldehyde groups. Finally, chitosan of average molecular weight 40,000 was introduced via the aldehyde groups through a Schiffs reaction. After complete acid-hydrolysis of the immobilized chitosan, the Svennerholm method for glucosamine analysis showed that 0.3% (w/w) chitosan had been successfully introduced on the glass beads. Atomic absorption spectroscopic analysis of eluants of a column of the chitosan-modified glass beads showed that metal ions such as Cu 2+ , Ag + , Pb 2+ , Fe 3+ , and Cd 2+ were more than 90% entrapped on a column of beads prepared in this manner.

Preparation and characterization of DNA films induced by UV irradiation.

Large amounts of DNA-enriched materials, such as salmon milts and shellfish gonads, are discarded as industrial waste. We have been able to convert the discarded DNA to a useful material by preparing novel DNA films by UV irradiation. When DNA films were irradiated with UV light, the molecular weight of DNA was greatly increased. The reaction was inhibited by addition of the radical scavenger galvinoxyl suggesting that the DNA polymerization with UV irradiation proceeded by a radical reaction. Although this UV-irradiated DNA film was water-insoluble and resistant to hydrolysis by nuclease, the structure of the DNA film in water was similar to non-irradiated DNA and maintained B-form structure. In addition, the UV-irradiated DNA film could effectively accumulate and condense harmful DNA-intercalating compounds, such as ethidium bromide and acridine orange, from diluted aqueous solutions. The binding constant and exclusion number of ethidium bromide for UV-irradiated DNA were determined to be 6.8 +/- 0.3 x 10(4) M(-1) and 1.6 +/- 0.2, respectively; these values are consisted with reported results for non-irradiated DNA. The UV-irradiated DNA films have potential uses as a biomaterial filter for the removal of harmful DNA intercalating compounds.