Tatsuya Okuda

Time-programmed dual release formulation by multilayered drug-loaded nanofiber meshes

To develop a drug carrier that enables time-programmed dual release in a single formulation, multilayered drug-loaded biodegradable nanofiber meshes were designed using sequential electrospinning with the following construction: (i) first drug-loaded mesh (top), (ii) barrier mesh, (iii) second drug-loaded mesh, and (iv) basement mesh (bottom). The drug release speed and duration were controlled by designing morphological features of the electrospun meshes such as the fiber diameter and mesh thickness. Control of the timed release of the second drug-the retardation period-was accomplished by appropriate design of the barrier mesh thickness. An in vitro release experiment demonstrated that the tetra-layered construction described above with appropriate morphological features of each component mesh can provide timed dual release of the respective drugs. The time-programmed dual release system using the multilayered electrospun nanofiber meshes was demonstrated as a useful formulation for advanced multidrug combination therapy requiring regiospecific administration of different drugs at different times. The potential use of the present multilayered formulation is discussed for application to biochemical modulation as one administrative strategy for use in sequential chemotherapy employing multiple anti-tumor drugs.

Characters of dendritic poly(L-lysine) analogues with the terminal lysines replaced with arginines and histidines as gene carriers in vitro.

The development of a non-viral gene delivery system into cells is an important key to realize the safe delivery of therapeutic genes without the side effects often pointed out for viral vectors. We have shown that dendritic poly(L-lysine) of the 6th generation (KG6) shows high transfection efficiency into several cultivated cells with low cytotoxicity. Here, to investigate the effect of substituting terminal cationic groups on the gene delivery into cells, we synthesized KGR6 and KGH6, in which terminal amino acids were replaced by arginines and histidines, respectively. DNA-binding analysis showed that KGR6 could bind to the plasmid DNA as strongly as KG6, whereas KGH6 showed decreased binding ability. KGR6 showed 3- to 12-fold higher transfection efficiency into several cultivated cells than KG6. In contrast, KGH6 showed no transfection efficiency. However, once KGH6 was mixed with the DNA under acidic conditions (pH 5.0), DNA-complexes were formed and they showed high transfection efficiency compared to that in KG6-mediated transfection. DNA-complexes of KGH6 formed under acidic conditions were 1-2 microm and spherical, and relatively stable under neutral conditions. The size and spherical shape of the complexes were the same as those of KG6. The unique character of KGH6 will be one of the basic and valuable tools which will enable us to construct a functional gene transfection system in vitro and in vivo.

Time-dependent complex formation of dendritic poly(L-lysine) with plasmid DNA and correlation with in vitro transfection efficiencies

Dendritic poly(L-lysine) of the 6th generation shows high transfection efficiency into several cultivated cells with low cytotoxicity. In order to understand the mechanism of complex formation with plasmid DNA, the complex was observed using atomic force microscopy. After mixing for 15 min, 1-2 microns assemblies of complexes composed of several small particles (50-200 nm) were observed. At the same time, individual small complexes of 50 to 500 nm were observed on a mica surface. After incubation for 2 h, only the large complexes were found on the mica surface. As a result of further dynamic light scattering analysis and measurement of the transfection efficiency at different time points, the transfection efficiency of KG6 was found to increase with increasing size of the DNA-complexes. This result indicates that large complexes of more than 1 micron are major species that contribute to transfection in vitro.

Gangliosides Act as Co-receptors for Salmonella enteritidis FliC and Promote FliC Induction of Human β-Defensin-2 Expression in Caco-2 Cells

Antimicrobial peptides such as defensins are crucial for host defense at mucosal surfaces. We reported previously that Salmonella enteritidis flagellin (FliC) induced human β-defensin-2 (hBD-2) mRNA expression in Caco-2 cells via NF-κB activation (Ogushi, K., Wada, A., Niidome, T., Mori, N., Oishi, K., Nagatake, T., Takahashi, A., Asakura, H., Makino, S., Hojo, H., Nakahara, Y., Ohsaki, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Moss, J., and Hirayama, T. (2001) J. Biol. Chem. 276, 30521–30526). In this study, we examined the role of ganglioside as co-receptors with Toll-like receptor 5 (TLR5) on FliC induction of hBD-2 expression in Caco-2 cells. Exogenous gangliosides suppressed FliC induction of hBD-2 promoter activity and binding of FliC to Caco-2 cells. Incorporation of exogenous ganglioside GD1a into Caco-2 cell membranes increased the effect of FliC on hBD-2 promoter activity. In support of a role for endogenous gangliosides, incubation of Caco-2 cells with dl-threo-2-hexadecanoylamino-3-morpholino-1-phenylpropanol, a glucosylceramide synthase inhibitor, reduced FliC induction of hBD-2 promoter activity. GD1a-loaded CHO-K1-expressing TLR5 cells had a higher potential for hBD-2 induction following FliC stimulation than GD1a-loaded CHO-K1 cells not expressing TLR5. FliC increased phosphorylation of mitogen-activated protein kinase, p38, and ERK1/2. Exogenous gangliosides GD1a, GD1b, and GT1b each suppressed FliC induction of p38 and ERK1/2 phosphorylation. Furthermore, FliC did not enhance luciferase activity in Caco-2 cells transfected with a plasmid containing a mutated activator protein 1-binding site. These results suggest that gangliosides act as co-receptors with TLR5 for FliC and promote hBD-2 expression via mitogen-activated protein kinase.

Novel poly(ethylene glycol) derivatives with carboxylic acid pendant groups: Synthesis and their protection and enhancing effect on non-viral gene transfection systems

Novel carboxylic acid pendant-containing poly(ethylene glycol) (PEG) derivatives (PEGCs) were synthesized by the chemical modification of the carbon-carbon double-bond side chain of copoly(allyl glycidyl ether/ethylene oxide) (copoly(AGE/EO)). PEG-C showed a protecting ability for the DNA/polycation complex from the albumin-induced aggregation. It also expressed the enhanced efficiency on the polycation-mediated gene transfection on the cultured cells (up to 3-4-times higher), probably due to its disperse-stabilizing property and also the proton-sponge effect.

Development of PEGylated cysteine-modified lysine dendrimers with multiple reduced thiols to prevent hepatic ischemia/reperfusion injury

To inhibit hepatic ischemia/reperfusion injury, we developed polyethylene glycol (PEG) conjugated (PEGylated) cysteine-modified lysine dendrimers with multiple reduced thiols, which function as scavengers of reactive oxygen species (ROS). Second, third, and fourth generation (K2, K3, and K4) highly branched amino acid spherical lysine dendrimers were synthesized, and cysteine (C) was conjugated to the outer layer of these lysine dendrimers to obtain K2C, K3C, and K4C dendrimers. Subsequently, PEG was reacted with the C residues of the dendrimers to obtain PEGylated dendrimers with multiple reduced thiols (K2C-PEG, K3C-PEG, and K4C-PEG). Radiolabeled K4C-PEG ((111)In-K4C-PEG) exhibited prolonged retention in the plasma, whereas (111)In-K2C-PEG and (111)In-K3C-PEG rapidly disappeared from the plasma. K4C-PEG significantly prevented the elevation of plasma alanine aminotransferase (ALT) activity, an index of hepatocyte injury, in a mouse model of hepatic ischemia/reperfusion injury. In contrast, K2C-PEG, K3C-PEG, l-cysteine, and glutathione, the latter two of which are classical reduced thiols, hardly affected the plasma ALT activity. These findings indicate that K4C-PEG with prolonged circulation time is a promising compound to inhibit hepatic ischemia/reperfusion injury.

S/O-nanodispersion electrospun fiber mesh effective for sustained release of healthy plasmid DNA with the structural and functional integrity

Localized and sustained delivery of the therapeutic genes using a solid carrier matrix is a potential approach to develop highly curative treatments. Electrospun nanofiber mesh of biodegradable polymer has been applied extensively as a carrier for localized and sustained delivery of drugs, proteins, and DNA, but it remains difficult to release sufficient amounts of DNA while maintaining structural and functional integrity. To realize the stable sustained release of the healthy plasmid DNA (pDNA) from electrospun fiber mesh, a novel method was examined for loading pDNA into the fibers based on solid-in-oil (S/O) nanodispersion of pDNA in organic solvent for electrospinning polymer solution: S/O nanodispersion electrospinning. A prepared pDNA-loaded fiber mesh made of biodegradable polymer showed sustained release of pDNA without burst release. From luciferase activity-based in vitro transcription–translation assay, pDNA released from meshes of the S/O nanodispersion retained about 60% luciferase activity of control pDNA, whereas pDNA released from the meshes of simple mixing showed only about 5% activity, indicating that S/O nanodispersion electrospinning is effective for loading pDNA into electrospun fiber meshes while maintaining their healthy functions. Effectiveness of S/O nanodispersion electrospinning was verified for fabricating a sustained release carrier matrix for high molecular weight bioactives, including therapeutic genes.