Tatsuro Ouchi

Aggregation phenomenon of PEG-grafted chitosan in aqueous solution

Chitosan (deacetylated chitin), which is a naturally occurring polysaccharide having primary amino groups, is known to be insoluble in water because of its strong intermolecular hydrogen bonds. We prepared water-soluble poly(ethylene glycol) grafted chitosan (PEG-g-chitosan) through the chemical modification of chitosan and investigated its aggregation phenomenon in aqueous solutions. The solution properties of PEG-g-chitosans differ depending on the degree of introduction of PEG in aqueous solution, and were studied by measuring transmittance and light scattering. The PEG-g-chitosans could form aggregates spontaneously by means of their intermolecular hydrogen bonds in the aqueous solution. The PEG-g-chitosan aggregates could also uptake N-phenyl-1-naphthylamine (PNA) as a hydrophobic substance in neutral conditions and this PNA could be released from the aggregates in acidic conditions.

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.

Release behaviour of 5-fluorouracil from chitosan-gel microspheres immobilizing 5-fluorouracil derivative coated with polysaccharides and their cell specific recognition

In order to provide a device releasing drugs in a controlled manner and having targetability to specific organs or cells, chitosan-gel microspheres, CMS, crosslinked with glutaraldehyde, immobilizing 1-[N-(5-aminopentyl) carbamoyl]-5-fluorouracil, 1, coated with anionic polysaccharides, such as 6-O-carboxymethyl-N-acetyl-alpha-1,4-polygalactosamine (CM-NAPGA), 6-O-carboxymethyl-chitin, alginic acid and heparin, by polyelectrolyte complex membrane formation were prepared. When chitosan was crosslinked with glutaraldehyde, 1 was simultaneously immobilized into CMS by Schiffs base formation. Average diameter of CMS obtained was estimated to be about 0.5-1.0 micron by SEM observation. In physiological saline media, only free 5-FU was released from the CMS but 1 and any 5-FU derivative was not. Release rate of 5-FU from the CMS was reduced by coating with polyelectrolyte complex membrane of cationic chitosan and anionic polysaccharides. CMS coated with CM-NAPGA showed a lectin-mediated specific aggregation phenomenon by addition of Abrus precatorius agglutinin. Moreover, the CMS immobilizing 1 coated with CM-NAPGA showed higher growth-inhibitory effect against SK-Hep-1 (human hepatoma) cells in vitro than the CMS coated with other polysaccharides.

Release Behavior of 5-Fluorouracil from Chitosan-Gel Nanospheres Immobilizing 5-Fluorouracil Coated with Polysaccharides and Their Cell Specific Cytotoxicity

Abstract Small-sized chitosan-gel nanospheres, CNSs (average diameter 250 nm), containing 5-fluorouracil (5FU) or immobilizing 5FU derivatives (aminopentyl-carbamoyl-5FU or aminopentyl-ester-methylene-5FU) were prepared by the glutaraldehyde crosslinking technique and the emulsion method. When chitosan was crosslinked with glutaraldehyde, these 5FU derivatives were simultaneously immobilized to CNSs by means of Schiffs base formation. The CNSs were coated with anionic polysaccharides, such as 6-O-carboxymethyl-N-acetyl-α-l,4-polygalactosamine/Na (CM-NAPGA/Na), 6-O-carboxymethyl-chitin/Na (CM-chitin/Na), and sodium hyaluronate, through formation of a polyelectrolyte complex membrane to give CNS/polyanion, i.e., CNS/G, CNS/C, and CNS/H, respectively. The polyelectrolyte complex of polysaccharide was employed to achieve the controlled release and effective targeting of 5FU by the CNSs. The release rate of 5FU from the CNSs could be controlled by immobilization of 5FU, degree of deacetylation of chitosan use...

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.

Synthesis and enzymatic hydrolysis of lactic acid–depsipeptide copolymers with functionalized pendant groups

Since poly(lactic acid) is the biodegradable polyester having low immunogenicity and good biocompatibility, it is utilized as a medical material. However, poly(lactic acid) is a water-insoluble crystalline polymer having no reactive side-chain group. Thus, the use of poly(lactic acid) is limited. To modify the properties of poly(lactic acid) and to introduce the functionalized pendant groups to poly(lactic acid), we synthesized two kinds of lactic acid-depsipeptide copolymers with reactive pendant groups, namely poly[LA-(Glc-Lys)] and poly[LA-(Glc-Asp)]. This was done through ring-opening copolymerizations of L-lactide with the corresponding protected cyclodepsipeptides, cyclo[Glc-Lys(Z)] and cyclo[Glc-Asp(OBzl)], and subsequent deprotection of benzyloxycarbonyl and benzyl groups, respectively. By changing the mole fraction of the corresponding depsipeptide units, the solubility, thermal transition and degradation behavior of the modified poly(lactic acid) could be varied.

One‐Pot Synthesis of Novel Branched Polylactide Through the Copolymerization of Lactide with Mevalonolactone

Polylactide, which is a biodegradable and bio-absorbable polymer having low immunogenicity and good biocompatibility, has been mainly studied for biomedical applications. Branched polymers have different rheological and mechanical properties compared with their linear counterparts owing to their molecular architectures. We synthesized novel biodegradable polylactide having a branched structure composed of metabolically degradable and/or absorbable materials only. The branched polylactide was obtained from a one-pot copolymerization of L-lactide using metabolic intermediate dl-mevalonolactone as a bifunctional comonomer having both lactone ring and pendant hydroxy group. The glass transition point, melting point and crystallinity of the branched polylactide are lower than those of linear polylactide.

Preparation of poly(lactic acid)-grafted amylose through the trimethylsilyl protection method and its biodegradation

Biodegradable poly(lactic acid)-grafted amylose was synthesized using a trimethylsilyl (TMS) protection method. Tetrahydrofuran soluble, mostly trimethylsilyl protected amylose was prepared and reacted with potassium tert-butoxide to give the corresponding alkoxide. Poly(lactic acid)-grafted amyloses were obtained by ring-opening anionic polymerization of lactide using the polymeric alkoxides as initiators and subsequent removal of the TMS groups. The obtained graft copolymers show biodegradability and a micro-phase-separated morphology.

Synthesis and antitumor activity of conjugates of poly(α-malic acid) and 5-fluorouracils bound via ester, amide or carbamoyl bonds

Abstract Since poly(malic acid) is a biodegradable and bioabsorbable lactic acid-type polyester having pendent modifiable carboxylic acid groups, it is of interest as a polymeric carrier of drugs. In order to provide macromolecular pro-drugs of 5-fluorouracil (5FU), and so reduce the side-effects of 5FU, the covalent attachment of 5FUs to poly(α-malic acid) and poly(α-malic acid-co-lactic acid) through amide, ester or carbamoyl bonds was carried out. The antitumor activity of the resulting 5FU conjugates with poly(α-malic acid) was tested against P-388 lymphocytic leukemia in mice in vivo (i.p./i.p.). When the unreacted pendent carboxylic acid groups in the polyα-malic acid)-5FU conjugates were masked by methyl groups, conjugates were obtained which prolonged life and did not display acute toxicity at a dose of 200–800 mg/kg. These samples were therefore considered to be macromolecular pro-drugs of 5FU. Furthermore, for the purpose of elucidating the nature of 5FU release and main-chain cleavage in vivo, the hydrolyses of the pendent amide, ester or carbamoyl bonds and the main-chain ester bonds in poly(α-malic acid)-5FU conjugates and poly(α-malic acid-co-lactic acid)-5FU conjugates were studied in vitro at 37° C in aqueous solutions of various kinds.

Biodegradable microspheres having reactive groups prepared from L-lactic acid-depsipeptide copolymers

Poly(L-lactic acid)-based microspheres having chemically reactive groups on their surfaces, MS(LAGA) and MS(LAGL), were prepared from the respective biodegradable L-lactic acid (LA)-depsipeptide copolymers, poly[LA-(Glc-Asp)] and poly[LA-(Glc-Lys)], having low contents of depsipeptide units, by an oil-in-water (O/W) emulsion and solvent evaporation method. The average diameters of the dried microspheres obtained were estimated to be 390–450 nm by scanning electron microscopy (SEM). The amounts of functionalized groups located on their surfaces were found to increase with an increase in the content of depsipeptide units in the used copolymers as determined by a colloidal titration method. The ζ potential values of the MS(LAGA) and MS(LAGL) surfaces increased negatively and positively in proportion to the amount of carboxyl and amino groups, respectively. As one example of the chemical modification of the obtained microspheres, the immobilization of galactose residues onto the surface of MS(LAGA) was demonstrated. Moreover, the entrapment of 1-naphthalenesulfonic acid as a hydrophilic model drug into MS(LAGA) and MS(LAGL), and its release behavior were investigated.