Masami Mizu

Schizophyllans carrying oligosaccharide appendages as potential candidates for cell-targeted antisense carrier.

Schizophyllans carrying beta-lactoside and alpha-mannoside appendages were prepared from native schizophyllan through NaIO4 oxidation followed by reductive amination using aminoethyl-beta-lactoside and alpha-mannoside, respectively. The resulting schizophyllans form stable macromolecular complexes with polynucleotides, such as poly(C) and poly(dA). Specific affinity between these macromolecular complexes and saccharide-binding proteins was demonstrated by surface plasmon resonance and agarose gel staining assays. beta-lactoside-appended schizophyllan enhanced an antisense activity in hepatocytes which express lactoside-binding proteins on their cell surfaces.

Lactose-appended schizophyllan is a potential candidate as a hepatocyte-targeted antisense carrier.

A schizophyllan (beta-1,3-glucan) derivative carrying lactose-appendages prepared by reductive amination can form stable macromolecular complexes with polynucleotides, shows excellent affinity with a lactose-binding lectin, and effectively mediates gene transfection into hepatocytes.

Polysaccharide-polynucleotide complexes. Part 7. Hydrogen-ion and salt concentration dependence of complexation between schizophyllan and single-stranded homo RNAs

Schizophyllan belongs to a beta-1,3-D-glucan family, which exists as a random coil in dimethyl sulfoxide (DMSO) and as a triple helix in water, respectively. The schizophyllan single chain forms a complex with single-stranded homo RNAs in water/DMSO mixed solvents. Using circular dichroism, we studied the complexation and its stability as a function of apparent pH (pH(*)) in a mixed solvent system and as a function of the salt concentration. The complex is formed in the pH(*) range 6.5-10, and dissociated in the pH(*) range 4-6. Both poly(A) and poly(C) adopt a double strand in the pH(*) range 4-6 and a single strand in the pH(*) range 6.5-10. Therefore, the conformational change of each polynucleotide is responsible for dissociation/association of the complex, i.e., the single strand of the polynucleotides can form complexes, whereas the double one cannot. This result indicates that hydrogen bonding and similarity of the helix parameters are essential for the complex formation. The melting temperature of the complex reaches the maximum around 0.05 M of NaCl and KCl, and the value of the maximum temperature depends on the cation species.

Thermally induced conformational-transition of polydeoxyadenosine in the complex with schizophyllan and the base-length dependence of its stability

A single chain of schizophyllan, one of the β-1,3-glucan family, can form a stoichiometric complex with poly(dA) and the poly(dA)’s conformation and the complex stability strongly depends on the base length.

Polysaccharide–polynucleotide complexes. Part 12. Enhanced affinity for various polynucleotide chains by site-specific chemical modification of schizophyllan

Recently, we found that schizophyllan (SPG), which is a natural and neutral polysaccharide, can form macromolecular complexes with certain polynucleotides. In order to increase the stability and the affinity of the complexes, we synthesized a 2-aminoethanol-appended SPG (N-s-SPG) by utilizing periodate oxidation and reductive amination. Although the modification level was only 2.4 ± 0.3%, the melting temperature of the poly(C)–N-s-SPG complex was increased by 8 °C comparing with the poly(C)–s-SPG complex. In addition, the conformation of the complex was scarcely perturbed by the introduction of amino groups. When we formed complexes of N-s-SPG and other polynucleotides, the melting temperature of their complexes increased without exception. Moreover, the amino modification induced complexation with poly(U), which does not form a complex with SPG. The affinity for the short chain nucleotide was also enhanced. These results indicate that the present modification method is quite useful to improve complex stability and we believe, therefore, that this strategy will make it possible to apply SPG to a novel gene carrier.