Jun-ichi Murata

Possibility of application of quaternary chitosan having pendant galactose residues as gene delivery tool

Since chitosan is a cationic natural polysaccharide having the formation ability of a polyelectrolyte complex with DNA, it is expected to be used as a carrier of DNA in gene delivery systems. So, in order to achieve an efficient gene delivery via receptor-mediated endocytosis, the synthesis of a novel polycationic polysaccharide derivative having recognizable saccharide residues, N,N,N-trimethyl(TM)-chitosan/galactose conjugate, was performed. The formation of a polyelectrolyte complex with DNA and the cellular recognition ability of TM-chitosan/galactose conjugate were tested, and then the possibility of its application as a gene delivery tool was investigated.

Design of quaternary chitosan conjugate having antennary galactose residues as a gene delivery tool

Abstract It is well-known that some kinds of saccharide play the important roles in biological recognition on cellular surface. So, they are expected to be applied for cellular recognition devices. Recently, it was reported that cluster glycosides were effective in the specific interaction between oligosaccharide chains and receptors. Since chitosan is a cationic natural polysaccharide, having formation ability of polyelectrolyte complex with DNA, it is expected to be used as a carrier of DNA in gene delivery systems. So, in order to achieve an efficient gene delivery via receptor-mediated endocytosis, the synthesis of novel polycationic polysaccharide derivative having recognizable branched saccharide residues, N,N,N-trimethyl(TM)-chitosan/tetragalactose antenna conjugate (TC-Gal4A20), was carried out. The cellular recognition ability of TC-Gal4A20 conjugate were tested, and then the possibility of its application as a gene delivery tool was investigated. TC-Gal4A20 conjugate showed high affinity to RCA120 lectin and its polycation-DNA complex had the ability of specific gene delivery to hepatocyte.

Immunological enhancement activity of muramyl dipeptide analogue/CM-curdlan conjugate

Abstract In order to provide a novel synthetic biological response modifier exhibiting high antitumor activity, GADP ( d -glucose analogue of muramyl dipeptide (MDP)) has been conjugated with carboxymethyl (CM)-curdlan (normal chain (1 → 3)-β- d -glucan) which has antitumor activity through stimulation of the host immuno-system. The immunological enhancement activities of the conjugate obtained were investigated against macrophage-like HL-60 (human promyelocytic leukemia) or U937 (human monoblast leukemia) cells to be enlarged by means of coupling of GADP with CM-curdlan. In this paper, in order to discuss the relationship between the immunological enhancement activities of the conjugate and its molecular structure, we synthesized GADP/CM-curdlan conjugates having various degrees of substitution of the carboxymethyl group (DCM) and degree of substitution of the GADP group (DGADP) per sugar unit. The GADP/CM-curdlan conjugate showed higher immunological enhancement activities than GADP derivative, CM-curdlan, or a mixture of GADP derivative and CM-curdlan in all the concentration ranges tested. The immunological enhancement activities of the GADP/CM-curdlan conjugate increased with increasing DGADP value. Although the immunological enhancement activities of CM-curdlan decreased with increasing in DCM value, those of GADP/CM-curdlan conjugate increased with increasing DCM value. We also synthesized the GADP/CM-dextran conjugate to investigate the effect of conjugation of GADP with immunologically active CM-curdlan. The immunological enhancement activities of GADP/CM-dextran conjugate was not higher than those of the mixture of GADP derivative and CM-dextran, or CM-dextran itself. These results suggested that the high immunological enhancement activities of GADP/CM-curdlan conjugates were due not only to giving polymeric character to GADP but also to the hybridization of GADP with immunologically active polysaccharide, curdlan. Moreover, from the results of the effect of DGADP and DCM values of the conjugate on activities and the results of the effect of the addition of calmodulin inhibitor to the macrophage-like cells activated by the conjugate on activities, the path of the activation of immunocompetent cells by the GADP/CM-curdlan conjugate was found to be different from that by CM-curdlan or free GADP derivative itself.

Preparation and biological properties of dicarboxy-glucomannan: enzymatic degradation and stimulating activity against cultured macrophages

Abstract Recently, it has been reported that some polysaccharides showed interesting biological or immunological activities. Glucomannan (GM) purified from Amorphophallus Konjac is a copolymer of 1,4-linked β- d -glucose and β- d -mannose, which has some mono-residual d -glucose or d -mannose branches at the 3-position of d -mannose units of the main chain. We prepared dicarboxy-gluco-mannan (DC-GM), a carboxylic acid derivative of glucomannan, by IO4−/ClO2− oxidation. The number-average molecular weight (Mn) of the DC-GM obtained was about 2·0 × 104. The chemical structure of DC-GM was investigated by gaschromatography. It showed high water-solubility and enzymatic degradation behavior by cellulase and β- d -glucosidase. The enzymatic degradation rate of the DC-GM was dependent on the degree of introduction of carboxylic acid group. The immunological enhancement activity of the DC-GM was evaluated in vitro by testing glucose consumption and β- d -glucuronidase activity against cultured macrophages, PMA (phorbol-12-myristate-13-acetate)-differentiated HL-60 (human promyelocytic leukemia) or U937 (human monoblast leukemia) cells. The DC-GM showed higher stimulating effects against such cultured macrophages than the other polysaccharide derivatives.

Design of macromolecular biological response modifier by immobilizing of D-glucose analogue of muramyl dipeptide on carboxymethyl-dextran having mannose branches

It is well known that muramyl dipeptide is a minimum required structure of bacterial peptidoglycan responsible for immunoadjuvant activity. Since mannose receptors exist on the surface of macrophages, polymers with branched mannose residues are expected to target moieties to macrophages. To achieve an efficient delivery of D-glucose analogue of muramyl dipeptide (GADP) via receptor-mediated endocytosis by mannose receptors on the surface of macrophages, GADP/carboxymethyl-dextran (CM-Dex)/Man conjugate was synthesized. Moreover, to study the effect of the introduction of mannose residues, we also synthesized GADP/CM-glucomannan (CM-GM) and GADP/CM-Dex conjugates. The immunological enhancement activities of their conjugates were evaluated by measurements of glucose consumption and beta-D-glucuronidase activity from macrophage-like cells. The GADP/CM-Dex/Man and GADP/CM-GM conjugates showed higher immunological enhancement activity than the GADP/CM-Dex conjugate. The immunological enhancement activity of GADP/CM-Dex/Man and GADP/CM-GM conjugates was decreased to the same level of immunological enhancement activity of GADP/CM-Dex conjugate under the presence of excess mannose. These results suggested that the introduction of mannose residues into GADP/CM-Dex conjugate could increase the affinity against macrophage and the immunological enhancement activity of GADP/CM-Dex conjugate itself.

Effect of dimerization of the D-glucose analogue of muramyl dipeptide on stimulation of macrophage-like cells.

N-Acetylmuramyl-L-alanyl-D-isoglutamine (MDP) is the minimum required structure responsible for the immunoadjuvant activity of the bacterial cell wall. The D-glucose analogue of MDP (GADP) was reported to show a higher immunoadjuvant activity than MDP itself. Although the mechanism of activation by MDP and the existence of receptor against MDP are not clear, the patch formation and cluster formation of receptors are important steps on the signal transduction by such bioactive molecules. It is expected that the cluster effect such as antennary oligosaccharides reported by Lee et al. increased the affinity of ligand against receptor and accelerated the patch formation and cluster formation of receptors. In order to discuss the effect of multivalent-ligand formation of GADP on the activation of immunocompetent cells in more detail, we have synthesized GADP dimers combined through various lengths of alkyl and poly(ethylene glycol) (PEG) spacer groups as the simple models of multivalent-ligand molecule of GADP and evaluated their immunological enhancement activities in vitro. The GADP dimers showed a higher level stimulatory activities against macrophage-like cells than free GADP and monomeric GADP derivatives.

Synthesis of muramyl dipeptide analogue—glucomannan conjugate and its stimulation activity against macrophage-like cells

Abstract Since the mannose receptors exist on the surface of macrophages, the branched mannose residues of glucomannan are expected to act as targeting moieties to macrophages. So, in order to achieve an efficient delivery of d -glucose analogue of muramyl dipeptide (GADP) via receptor-mediated endocytosis by mannose receptors on the surface of macrophages, the GADP/carboxymethyl(CM)-glucomannan conjugate was synthesized. Moreover, in order to study the relationship between the immunological enhancement activity of the conjugates and their mannose residues, we synthesized the GADP/CM-glucomannan conjugates having various degrees of substitution of carboxymethyl group in mol% per sugar unit (DCM) and GADP/CM-dextran conjugate through hybridization of GADP with dextran. The immunological enhancement activities of GADP/CM-glucomannan conjugates and GADP/CM-dextran conjugate were evaluated by measurements of the glucose consumption, the superoxide anion production and the β- d -glucuronidase activity from PMA (phorbol-12-myristate-13-acetate)-differentiated HL-60 ( human promyelocytic leukemia ) or U937 ( human monoblast leukemia ) cells as macrophage-like cells.

Synthesis of a MDP analogue/chitin conjugate that stimulates cultured macrophages

To provide a new synthetic biological response modifier which ex hibits a high immunopotentiation activity and antitumor activity, a hybrid conjugate of chitin with immobilized D-glucose analogue of muramyl dipeptide (MDP) (GADP) was synthesized. The stimulation activity of the conjugate against cultured macrophages was evaluated as an immunopotentiation activ ity in vitro by glucose consumption using PMA (phorbol-12-myristate-13- acetate)-differentiated HL-60 (human promyelocytic leukemia) cells and by superoxide anion (O2-) production from PMA-differentiated HL-60 cells. The stimulation activity of the GADP/chitin conjugate against cultured macrophages was greater than that of GADP derivative, carboxymethyl-chitin and a mixture of these two. The stimulation activity of GADP against cultured macrophages was increased by conjugation with chitin.