Shoichiro Asayama

Synthesis of Novel Polyampholyte Comb-Type Copolymers Consisting of a Poly(l-lysine) Backbone and Hyaluronic Acid Side Chains for a DNA Carrier

The polyampholyte comb-type copolymers consisting of a poly(L-lysine) (PLL) main chain, a DNA binding site, and hyaluronic acid (HA) side chains, cell-specific ligands, have been prepared as the DNA carrier targeting sinusoidal endothelial cells of liver. The reducing end of HA and epsilon-amino groups of PLL were covalently coupled by reductive amination to obtain the resulting comb-type copolymers (PLL-graft-HA). The chain length of HA was controlled by the enzymatic hydrolysis of high-molecular weight HA. Since HA formed polyion complexes with PLL, the coupling reaction was carried out with high-ionic strength media to suppress polyion complex formation. The reaction proceeded in a homogeneous system, leading to a high efficiency of coupling (>70%) of HA onto the PLL backbone. By using the enzymatic hydrolysis of HA and the reductive amination reaction between HA and PLL with high-ionic strength media, it is possible to prepare the various comb-type copolymers with a defined density and a defined length of HA side chains. Furthermore, we also find that these polyampholyte comb-type copolymers vary their assembling structure in water in response to two kinds of environmental factors, i.e., ionic strength and pH. Finally, a 1H NMR study reveals that the PLL backbone efficiently interacts with DNA molecules despite the presence of HA side chains having negative charges.

Targeted gene delivery to sinusoidal endothelial cells: DNA nanoassociate bearing hyaluronan-glycocalyx

Liver sinusoidal endothelial cells (SECs) possess unique receptors that recognize and internalize hyaluronic acid (HA). To develop a system for targeting foreign DNA to SECs, comb‐type polycations having HA side chains were prepared by coupling HA to poly(L‐lysine) (PLL). The HA‐grafted‐PLL copolymer (PLL‐g‐HA) thus formed was mixed with DNA in 154 mM NaCl to form soluble nanoassociates bearing hydrated hyaluronate shells. Agarose gel retardation assays revealed selective interaction of the PLL backbone with DNA despite the presence of polyanionic HA side chains. To determine whether the PLL‐g‐HA/DNA complexes were recognized by SEC HA receptors in vivo, we injected Wistar rats i.v. via the tail vein with PLL‐ g‐HA complexed to a β‐galactosidase expression plasmid (pSV β‐Gal) labeled with 32P. One hour postinjection, >90% of the injected radioactivity remained in the liver. Administration of the PLL‐g‐HA complexed to an FITC‐labeled DNA revealed that the carrier‐DNA complex was distributed exclusively in SECs. A large number of SECs expressing β‐galactosidase was detected along the sinusoidal lining after transfection with PLL‐g‐HA/pSV β‐Gal. Moreover, PLL‐g‐HA effectively stabilized DNA triplex formation. In conclusion, the new PLL‐g‐ HA/DNA carrier system permits targeted transfer of exogenous genes selectively to the SECs.

Bi-phasic polycation for the DNA carrier responding to endosomal pH

Abstract The bi-phasic polycation consisting of a poly(1-vinylimidazole) (PVIm) backbone and ‘lactosylated’ poly( l -lysine) (PLL) side-chains has been prepared as a DNA carrier with a proton-buffering effect at endosomal pHs (=5–6). The comb-type copolymer PVIm- graft -PLL was prepared by using the macromonomer method. First, a poly( N e -carbobenzoxy- l -lysine) macromonomer was radically copolymerized with VIm. Then, the e-amino groups of the PLL- grafts and the reducing end of lactose were covalently coupled by reductive amination. The resulting lactosylated PVIm- graft -PLL comb-type copolymer (PVIm- graft -PLL · Lac) exhibited a dual ionic character owing to the two cationic segments in the copolymer, as determined by acid–base titration. Agarose gel retardation assays proved that the stronger basic segment PLL · Lac- grafts worked as the anchor segment for DNA/PVIm- graft -PLL · Lac complex formation. Turbidity measurements showed that the DNA/PVIm- graft -PLL · Lac complex exhibited a slight turbidity at physiological pH (=7.4) and significant turbidity at endosomal pH, owing to the basicity (protonation–deprotonation) of the imidazole groups. It is worth noting that the solubility of the DNA/polycation complex in water decreased in spite of the increase of the net positive charges of DNA/polycation mixture. Due to the lactose groups, the interaction of DNA with the PLL · Lac- graft was different from that with unmodified PLL- graft . The difference of the interaction caused the solubility change of the DNA/polycation complex at endosomal pHs.

COMB-TYPE COPOLYMERS FOR CONTROLLED DNA DELIVERY

Various comb-type copolymer containing a polycation as a main chain was design to construct delivery systems of DNAs. The comb-type copolymers having cell-specific polysaccharides were proved to be useful to deliver DNA to the target cells in vivo. Of interest, the copolymers with abundant side chains of hydrophilic polymers are capable of stabilizing DNA triplex. Further, injectable nanoparticles for controlled releases of DNAs were fabricated from the copolymer and a biodegradable polymer.