Tsunehiko Fukuda

Disulfide crosslinked stearoyl carrier peptides containing arginine and histidine enhance siRNA uptake and gene silencing

The siRNA has been expected to apply for several diseases such as cancer since siRNA specifically silences the disease-associated genes. However, effective gene carriers should be developed to overcome the low siRNA stability in vivo, form stable complexes and facilitate intracellular uptake of siRNA. In this study, to develop a safe and efficient siRNA carrier, stearoyl (STR) peptides with Cys (C), Arg (R), and His (H) residues that can form disulfide cross linkages via Cys (C) were synthesized, and their suitability as siRNA carriers was evaluated. The particle size of STR-CH(2)R(4)H(2)C/siRNA complexes was about 100 nm. The cellular uptake ability after transfection with FAM-siRNA with STR-CH(2)R(4)H(2)C, CH(2)R(4)H(2)C, or STR-GH(2)R(4)H(2)G was significantly higher than that with FAM-siRNA only. STR-CH(2)R(4)H(2)C showed the highest cellular uptake ability when compared with CH(2)R(4)H(2)C and STR-GH(2)R(4)H(2)G. STR-CH(2)R(4)H(2)C did not induce substantial cytotoxicity. The intratumor injection of STR-CH(2)R(4)H(2)C/vascular endothelial growth factor (VEGF) siRNA (siVEGF) complexes achieved a high anti-tumor effect in tumor bearing mice. These results suggest STR-CH(2)R(4)H(2)C has potential of effective siRNA carrier possible to exercise silencing effect in vitro and in vivo.

Development of cell-penetrating peptide-modified MPEG-PCL diblock copolymeric nanoparticles for systemic gene delivery.

To develop a safe and efficient systemic non-viral gene vector, methoxy poly(ethylene glycol) (MPEG)/poly(epsilon-caprolactone) (PCL) diblock copolymers conjugated with a Tat analog through the ester or disulfide linkage were synthesized and their suitability as a systemic non-viral gene carrier evaluated. The physicochemical properties of the MPEG-PCL diblock copolymers were determined by GPC, (1)H NMR and FT-IR spectroscopy. The particle sizes and in vitro (COS7 and S-180 cells) transfection efficiencies and cytotoxicity were evaluated. Furthermore, the luciferase activity was then determined in various tissues after intravenous injection of MPEG-PCL-SS-Tat/pCMV-Luc complex into mice bearing S-180 cells. The particle sizes of the MPEG-PCL-Tat copolymers with or without pDNA were about 40 and 60nm, respectively. The luciferase activity in COS7 cells transfected with pCMV-Luc with MPEG-PCL-ester-Tat or MPEG-PCL-SS-Tat was higher than that with pDNA only. MPEG-PCL-SS-Tat greatly increased the transfection efficiency compared to MPEG-PCL-ester-Tat in COS7 and S-180 cells. In an in vitro cytotoxicity test MPEG-PCL-SS-Tat did not induce any remarkable cytotoxicity. In an in vivo experiment, the synthesized MPEG-PCL-SS-Tat copolymers promoted the delivery and expression of pDNA into tumor tissue in tumor-bearing mice. In conclusion, this vector might be applicable as a tumor-targeting non-viral systemic gene carrier in the clinical setting.

Muramyl Dipeptide Derivatives with Multiprenylacetyl Group. Synthesis and Immunological Activities

Several linear and branched all-trans-multiprenylacetic acids were synthesised, and introduced to the 6 position of the sugar moiety of muramyl dipeptide and its analog, via an amino acid as a linking unit. Compared with the saturated stearoyl derivative, all the compounds having a multiprenylacetyl group exhibited more potent adjuvant activity on the induction of delayed-type hypersensitivity to N-acetyl-3-(4-arsonophenylazo)-L-tyrosine. The derivatives with larger branched side chains tended to have increased activity.

p-Tolylmethylsulphonyl: a new amino-protecting group in peptide synthesis

The p-tolylmethylsulphonyl group for the protection of the Iµ-amino group of lysine, which is readily removed with anhydrous hydrogen fluoride but is strongly resistant to trifluoroacetic acid or dilute hydrogen chloride, can be applied to both solid-phase and solution synthesis of peptides.

Versatile Nuclear Localization Signal-Based Oligopeptide as a Gene Vector

To develop a versatile nuclear-targeted gene vector, nuclear localization signal (NLS) oligopeptides combining cysteine (C), histidine (H), and stearic acid (STR) were investigated in this study. The original SV40 sequence (SV40: Pro-Lys-Lys-Lys-Arg-Lys-Val) was selected as the NLS sequence, and physical characterizations of various NLS-based oligopeptides (CSV40C, STR-CSV40C, and STR-CH2SV40H2C), including mean diameter, zeta-potential, complex condensation, and decondensation, were evaluated. In addition, cellular and nuclear uptake of plasmid DNA (pDNA) and gene expression in COS7 and dendritic cells (JAWS II) were determined. As a result, C and STR enhanced formation of a smaller and more stable nano-complex with pDNA based on ionic interactions, the disulfide linkage and hydrophobic interactions. STR-CSV40C and STR-CH2SV40H2C had significantly higher cellular uptake ability and transfection efficiency than SV40 and CSV40C. In particular, STR-CH2SV40H2C had higher nuclear uptake and gene expression efficiency than STR-CSV40C. Furthermore, STR-CH2SV40H2C could deliver pDNA to the nuclei and had high gene expression efficiency in dendritic cells. Our results indicate that STR-CH2SV40H2C is a promising gene delivery system in non- or slow-dividing cells.