Biomedical subjects

Mitsuru Akashi

Publications and source records attributed to Mitsuru Akashi.

At least 37 records · Page 2Linked to original sources

PubMed2005

Preparation and characterization of biodegradable nanoparticles based on poly(gamma-glutamic acid) with l-phenylalanine as a protein carrier.

The objective of the present study was to prepare nanoparticles composed of poly(gamma-glutamic acid) (gamma-PGA) and l-phenylalanine ethylester (l-PAE) in order to evaluate the possibility of using these nanoparticles as protein carriers. Novel amphiphilic graft copolymers composed of gamma-PGA as the hydrophilic backbone and l-PAE as the hydrophobic segment were successfully synthesized by grafting l-PAE to gamma-PGA using water-soluble carbodiimide (WSC). Due to their amphiphilic properties, the gamma-PGA-graft-l-PAE copolymers were able to form nanoparticles. The size of the gamma-PGA nanoparticles was measured by photon correlation spectroscopy (PCS) and showed a monodispersed size distribution with a mean diameter ranging from 150 to 200 nm. The solvents selected to prepare the gamma-PGA nanoparticles by a precipitation and dialysis method affected the particle size distribution. To evaluate the feasibility of vehicles for these proteins, we prepared protein-loaded gamma-PGA nanoparticles by surface immobilization and encapsulation methods. Ovalbumin (OVA) was used as a model protein and was immobilized onto the gamma-PGA nanoparticles or encapsulated into the inner core of these nanoparticles. Moreover, these OVA-encapsulated gamma-PGA nanoparticles could be preserved by freeze-drying process. The results of cytotoxicity tests showed that the gamma-PGA and gamma-PGA nanoparticles did not cause any relevant cell damage. It is expected that biodegradable gamma-PGA nanoparticles can immobilize proteins, peptides, plasmid DNA and drugs onto their surfaces and/or into the nanoparticles. These nanoparticles are potentially useful in pharmaceutical and biomedical applications.

Cell Proliferation↗

PubMed2005

Loading and release of charged dyes using ultrathin hydrogels.

The anionic dyes methyl orange (MO) and allura red (AR) were used as model drugs to assess the loading and release by layer-by-layer assembled ultrathin hydrogels prepared via the amide formation of poly(acrylic acid-co-N-isopropylacrylamide) with AAc contents of 5, 10, and 15 mol % plus poly(vinylamine hydrochloride). The amount of MO loaded was potentially controlled by changing the dye concentrations, film thickness, and AAc content of the copolymers. The release of AR was controlled by the NaCl concentration and pH. We conclude that the polymeric matrices of ultrathin hydrogels have great potential for the loading and release of charged drugs.

Acrylamides↗

PubMed2005

Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release.

Therapeutic agents based on DNA or RNA oligonucleotides (e.g., antisense DNA oligonucleotide, small interfering RNA) require a regulation of their kinetics in cytoplasm to maintain an optimal concentration during the treatment period. In this respect, delivery of functional nanoparticles containing these drugs into cytoplasm has been thought to have a potential for the cytosolic controlled gene release. In this study, we establish a protocol for the encapsulation of nanoparticles into liposome, which is further fused with ultra violet-inactivated Sendai virus to compose fusogenic liposomes. When nanoparticles were encapsulated in conventional liposomes, endocytosis-mediated uptake of nanoparticles was observed. In contrast, numerous amounts of nanoparticles were delivered into the cytoplasm without any cytotoxicity when the particles were encapsulated in fusogenic liposomes. Additionally, fusogenic liposome showed a high ability to deliver nanoparticles containing DNA oligonucleotides into cytoplasm. These results indicate that this combinatorial nanotechnology using fusogenic liposome and nanoparticle is a valuable system for regulating the intracellular pharmacokinetics of gene-based drugs.

Animals↗

PubMed2005

New fluorescence method for the determination of the critical micelle concentration by photosensitive monoazacryptand derivatives.

A novel fluorescence method for the determination of the critical micelle concentration (cmc) is reported. The cmc values of nonionic and anionic surfactants were evaluated utilizing a photosensitive monoazacryptand-Ba2+ complex, whose fluorescence intensity is sensitively changed by environmental conditions based on the photoinduced electron transfer (PET) mechanism as a fluorescent probe (PET method). Based on a comparison of the cmc values obtained by the PET method versus those obtained by conventional fluorescence-based methods as well as the values reported in the literature, one can conclude that the PET method is useful for the cmc determination. In particular, the PET method was more effective for the cmc determination of nonionic surfactants with very low cmc values (< 10(-5) M) than any other fluorescence-based method. In the cases of anionic surfactants, the PET method revealed the formation of the premicellar aggregates comprised of surfactant molecules and fluorescent probes below the cmc. Moreover, the hydrophobicity around the monoazacryptand-Ba2+ complex incorporated into various nonionic surfactant micelles was evaluated by this PET method.

Journal Article↗

PubMed2005

In vitro enzymatic degradation of nanoparticles prepared from hydrophobically-modified poly(gamma-glutamic acid).

Amphiphilic poly(gamma-glutamic acid) (gamma-PGA) was prepared by the introduction of L-phenylalanine ethylester (L-PAE) as a side chain. This gamma-PGA-graft-L-PAE formed monodispersed nanoparticles in water. The particle size of the gamma-PGA nanoparticles could be controlled by the degree of L-PAE grafting. The hydrolytic degradation and enzymatic degradation by gamma-glutamyl transpeptidase (gamma-GTP) of these gamma-PGA nanoparticles was studied by gel permeation chromatography (GPC) and scanning electron microscopy (SEM). The hydrolysis ratio of gamma-PGA was found to decrease upon increasing the hydrophilicity of the gamma-PGA. The degradation of the gamma-PGA backbone by gamma-GTP resulted in a dramatic change in nanoparticle morphology. With increasing time, the gamma-PGA nanoparticles reduced in size and finally disappeared completely.Time-course of the changes in the morphology of the gamma-PGA nanoparticles following incubation with gamma-glutamyl transpeptidase.

Biodegradation, Environmental↗

PubMed2005

Novel functional biodegradable polymer. III. The construction of poly(gamma-glutamic acid)-sulfonate hydrogel with fibroblast growth factor-2 activity.

Poly(gamma-glutamic acid)-sulfonate (gamma-PGA-S) hydrogel, with fibroblast growth factor (FGF)-2 activity was investigated as a novel, next-generation tissue-engineering material. gamma-PGA-net-gamma-PGA-S72 (S72-netgel) was prepared with gamma-PGA-S and gamma-PGA-S72 (72% sulfonated gamma-PGA) to provide the high mobility of gamma-PGA-S72 for FGF-2 activity. Cell adhesion and proliferation activities were evaluated on gamma-PGA and gamma-PGA-S hydrogels along with S72-netgels. Both cell adhesion and proliferation activities of gamma-PGA and gamma-PGA-S hydrogels were low. In contrast, S72-netgels had high cell adhesion and proliferation activities, because of their low swelling ratios and high sulfonic acid group concentrations. Furthermore, S72-netgels had high FGF-2 activity, because gamma-PGA-S72 retained FGF-2 activity when incorporated into S72-netgels. S72-netgels should be useful as next-generation tissue-engineering material containing FGF-2 activity.

Animals↗

PubMed2005

Fluorometric sensing of alkali metal and alkaline earth metal cations by novel photosensitive monoazacryptand derivatives in aqueous micellar solutions.

Novel monoazacryptand-type fluorescent chemosensors, (derived from an 18-crown-6) and (derived from a 15-crown-5) both with a pyrene ring as their photoresponsive moiety, were synthesized. Their fluorescence properties for alkali metal and alkaline earth metal cations in water were then examined. The detection of metal cations was accomplished by a change in the fluorescence intensity of the host compounds, based on a photoinduced electron transfer (PET) mechanism. In aqueous solution, showed little fluorescence upon the addition of Ba2+ because of the very weak complexation with Ba2+, but the presence of micelles of polyoxyethylene(10) isooctylphenyl ether (Triton X-100) enabled to show highly sensitive and selective Ba2+ detection among alkali metal and alkaline earth metal cations. With respect to the selective fluorescent detection of important metal cations (Na+, K+, Mg2+, Ca2+) relevant to living organisms, was found to detect K+ with high selectivity in aqueous micellar solutions of polyoxyethylene(20) sorbitan monostearate (Tween-60). The selectivity for metal cations was mainly dependent on the goodness of fit of the host cavity and the metal cation size. In the presence of anionic surfactants, detected alkaline earth metal cations more effectively than alkali metal cations.

Journal Article↗

PubMed2005

Short synthesis of skeleton-modified cyclodextrin derivatives with unique inclusion ability.

[reaction: see text] Skeleton-modified cyclodextrin (CD) derivatives, in which an alpha-(1,4)-glucosidic bond is converted into a beta-(1,4)-glucosidic bond, were conveniently synthesized by cleavage of a single glucosidic bond in permethylated and 2,6-di-O-methylated alpha- and beta-CDs and subsequent recyclization via the trichloroacetoimidate intermediates. The selective cleavage of an alpha-(1,4)-glucosidic bond of permethylated alpha- and beta-CDs was accomplished by stirring in 30% aq HClO(4) at 25 degrees C to give the corresponding maltohexaose and maltoheptaose derivatives, respectively. The cleavage of a glucosidic bond of hexakis(3-O-benzyl-2,6-di-O-methyl)-alpha-CD was successfully carried out in a mixed 60% aq HClO(4) and 1,4-dioxane solution (1:20). In the case of heptakis(3-O-benzyl-2,6-di-O-methyl)-beta-CD, the solvent-free reaction with p-toluenesulfonic acid was found to be effective for selective cleavage of one glucosidic bond. The permethylated beta-CD derivative with a beta-(1,4)-glucosidic bond (4b) exhibited higher inclusion ability toward sodium m-nitrobenzoate than the parent permethylated beta-CD, while these hosts showed the same inclusion ability toward sodium p-nitrobenzoate. On the other hand, the beta-(1,4)-type permethylated alpha-CD derivative 4a exhibited lower inclusion ability toward sodium p- and m-nitrobenzoates than the parent permethylated alpha-CD. Interestingly, host molecules 4a and 4b showed inclusion selectivity for sodium m-nitrobenzoate as compared with the corresponding para-isomer, in contrast to permethylated CDs which possessed para-isomer selectivity. On the other hand, host molecules 4a and 4b showed para-isomer selectivity toward sodium nitrophenoxide guests, indicating that the inclusion selectivity was remarkably influenced by the guest hydrophilic groups. (1)H NMR studies on complexes of those beta-(1,4)-type CD derivatives with p- and m-nitrobenzoates and p- and m-nitrophenolates were carried out to estimate their structures.

Cyclodextrins↗

PubMed2005

Controlled release based on the dissolution of a calcium carbonate layer deposited on hydrogels.

It is possible that inorganic materials conjugated to suitable organic materials may induce unique mechanical, optical and other functional properties. Therefore, artificial conjugation of organic and inorganic components is attractive for preparing novel functional materials. Recently, we developed an alternate soaking process for calcium salt formation on/in polymer materials. In this study, a poly(vinyl alcohol) (PVA) hydrogel-calcium carbonate (CaCO(3)) composite was prepared by the aforementioned process as a controlled release support. Brilliant blue FCF (Mw = 794), FITC labeled BSA (Mw = 6.6 x 10(4)), FITC labeled dextran-10 k (Mw = 9.5 x 10(3)) and FITC labeled dextran-40 k (Mw = 4.3 x 10(4)) were loaded into the composite as model drugs. CaCO(3) dissolution and model drug release rates increased with a decrease in buffer pH. In addition, model drug release rates increased with a decrease in model drug molecular weight. These results show that CaCO(3) layers on hydrogels behave as capping layers for model drug release; the release rate of model drugs can be controlled by the dissolution rate of CaCO(3) and the molecular weight of the drug.

Calcium Carbonate↗

PubMed2005

Apatite formed on/in agarose gel as a bone-grafting material in the treatment of periodontal infrabony defect.

The present study was designed to evaluate the effects of a hydroxyapatite/agarose (HA/agarose) composite gel formed by a novel alternate soaking process for the treatment of periodontal infrabony defects in three dogs. After creating two-wall infrabony periodontal defects on the medial aspect of the maxillary and mandibular second and forth premolars, the defects were implanted with temporary dental filling material (stopping) to induce inflammatory periodontal disease. Two months later, the mucoperiosteal flaps were raised, and after debridement, the infrabony defects were filled with one of the following three materials: (a) HA/agarose, (b) Bone ject (True-Bone Ceramic-collagen combined bone graft material, Koken, Japan), or (c) no material implantation (negative control). The animals were then randomly scheduled for sacrifice at 1, 2, and 6 months, and samples were taken for histological examination. In the HA/agarose gels, the 2-month postoperative cavities exhibited regeneration to new attachments with the apposition of a new cementum and well-oriented fibers. The neocementum was narrow and acellular, and the new bone apposition was limited. Six months postoperatively, newly formed bone was predominantly observed. The neocementum was wider and cellular. In the negative control, the 2-month postoperative cavities exhibited no regeneration of the cementum, nor any formation of periodontal pockets. The six-month postoperative cavities were nearly the same as the 2-month cavities. The Bone ject, 2-month postoperative cavities exhibited no regeneration of the periodontal tissue, nor any formation of periodontal pockets. Six months postoperatively, inflammatory granulation tissue was observed around the particles. The present study suggests that HA/agarose gels may play an important role in the regeneration of lost periodontal tissue.

Animals↗

PubMed2005

Induction of dendritic cell-mediated immune responses against HIV-1 by antigen-capturing nanospheres in mice.

Prophylactic vaccines, designed to elicit potent humoral and cellular immune responses to human immunodeficiency virus type 1 (HIV-1) antigens in mucosa, are the important approach to the protection of individuals against HIV-1 infection, since HIV-1 transmission is largely a result of sexual contact. In this study, a novel strategy has been developed to induce HIV-1-specific immune responses, which involves inactivated HIV-1-caputring concanavalin A (Con A)-immobilized nanospheres (HIV-NS) and their interaction with bone marrow (BM)-derived dendritic cells. HIV-NS were taken up by dendritic cells via cytoskeleton-dependent but mannose-binding site-independent phagocytosis. Serial stimulations to unprimed T-cells with HIV-1 gp120-capturing NS-pulsed dendritic cells could induce antigen-specific T-cell response. Intranasal administration of fluorescein isothiocyanate-labeled nanospheres (NS) in mice proved that the particles were taken up into pulmonary dendritic cells. Analysis of mice receiving intranasal immunizations with HIV-NS revealed that the mice efficiently induced the antibodies against HIV-1 in the genital tract and specific cytotoxic T-cells in the spleen. These results suggest that the use of HIV-1-NS may provide a novel and promising approach for the induction of humoral and cellular immune responses to HIV-1.

AIDS Vaccines↗

PubMed2005

Enhanced effects of lithocholic acid incorporation into liquid-crystalline biopolymer poly(coumaric acid) on structural ordering and cell adhesion.

A novel thermotropic liquid-crystalline biocopolymer, poly{trans-4-hydroxycinnamic acid (4HCA: trans-coumaric acid)-co-lithocholic acid (LCA)}, was synthesized by a thermal polycondensation of 4HCA and LCA. When the LCA composition of P(4HCA-co-LCA) was 0, 5, 7, 23, 27, and 45 mol%, the copolymers showed a nematic liquid-crystalline phase. The melting point of the copolymers was 120-250 degrees C depending on the LCA composition, and showed a maximum at 7 mol%. Wide angle X-ray diffraction (WAXD) analyses showed a typical diffraction pattern of a hexagonal arrangement for 0, 7, 23, and 27 mol% LCA composition, which were cooled from a nematic melt. The other polymers showed no distinct diffraction. In particular, the copolymer of 7 mol% LCA composition showed four distinct diffractions corresponding to spacings with a reciprocal ratio of 1, radical3, 2, and radical7, indicating the highest structural ordering of all copolymers prepared here. The results of the cell adhesion and static contact angle tests suggest that the 7 mol% LCA composition copolymer had the highest hydrophobicity and cell adhesion ability, which was easily controlled by altering in feed. We conclude that the structural ordering may have a significant correlation with cell adhesion activity.

Animals↗

PubMed2005

Nanosphere induced gene expression in human dendritic cells.

The molecular mechanisms of nanosphere-induced mucosal immunization are important to decipher, since this can form the basis for novel approaches in, e.g., nasal vaccination. In this study, we have investigated the effect of nanospheres as antigen carriers on immature human dendritic cells. The results clearly indicate that tetanus toxoid immobilized nanospheres have a direct effect on human monocyte derived dendritic cells and induce a specific transcriptional profile involving genes crucial for phagocytosis and a protective immune response.

B7-2 Antigen↗

PubMed2005

Fusogenic liposome can be used as an effective vaccine carrier for peptide vaccination to induce cytotoxic T lymphocyte (CTL) response.

We reported previously that fusogenic liposome (FL) introduced antigen protein encapsulated in the liposome directly into the cytoplasm of the antigen presenting cells, and that it induced immune responses. In the present study, we encapsulated TAX38-46, an HTLV-I derived protein and an antigen peptide model, into FL. The ability to induce effective cytotoxic T lymphocytes (CTL) responses in immunized mice was evaluated. Results showed FL could induce CTL response effectively and suggested that FL is a potential peptide vaccine carrier.

Animals↗

PubMed2005

[Spectra studies on the interaction of Mn2+ and poly N-isopropylacrylamide].

The interaction of Mn2+ and poly (N-isopropylacrylamide) (PNIPAAm) was studied by using UV-Vis, FTIR and fluorescence spectroscopic methods. The results indicated that Mn2+ could be bonded to oxygen atoms of carbonyl in PNIPAAm and form the complex of Mn2+ -PNIPAAm. It was found that there existed efficient Förster energy transfer from Mn2+ to PNIPAAm due to the emission spectra of Mn2+ overlapping the excitation spectra of PNIPAAm and that the emission peak of Mn2+ at 561 nm disappeared in Mn2+ -PNIPAAm complex. Therefore the fluorescence intensity at 307 nm was increased by 314%.

English Abstract↗

PubMed2004

Augmentation of antigen-specific immune responses using DNA-fusogenic liposome vaccine.

In an attempt to enhance the immunological efficacy of genetic immunization, we investigated a new biological means for delivering antigen gene directly to the cytoplasm via membrane fusion. In this context, we investigated fusogenic liposome (FL) encapsulating DNA as a possible genetic immunization vehicle. RT-PCR analysis indicated that a FL could introduce and express encapsulating OVA gene efficiently and rapidly in vitro. Consistent with this observation, an in vitro assay showed that FL-mediated antigen-gene delivery can induce potent presentation of antigen via the MHC class I-dependent pathway. Accordingly, immunization with FL containing the OVA-gene induced potent OVA-specific Th1 and Th2 cytokine production. Additionally, OVA-specific CTL responses and antibody production were also observed in systemic compartments including the spleen, upon immunization with the OVA-gene encapsulating FL. These findings suggest that FL is an effective genetic immunization carrier system for the stimulation of antigen-specific immune responses against its encoding antigen.

Animals↗

PubMed2004

Potent activation of antigen-specific T cells by antigen-loaded nanospheres.

Polystyrene nanospheres (NS) were found to be efficiently taken up by murine antigen-presenting cells (APC), especially bone marrow-derived dendritic cells (DC), in vitro and in vivo. The efficiency of NS uptake was not affected by the maturation state of DC. Both immature and mature DC had similar ability to take up NS in a dose- and time-dependent manner. Uptake and intracellular localization of NS was clearly demonstrated by confocal laser microscopy, using NS with fluorescence. DC could efficiently take up ovalbumin (OVA), when loaded on the surface of NS (OVA-NS). Consequently, OVA-NS-pulsed DC activated antigen-specific interferon (IFN)-gamma-producing T cells much more strongly than OVA-pulsed DC in vitro. These results suggest that NS can be used as an efficient antigen delivery system to DC for a variety of vaccines, such as an anti-human immunodeficiency virus type 1 vaccine.

Animals↗

PubMed2004

Sugar-amide surfactant micelles as effective reaction fields for enantioselective hydrolysis of alpha-amino acid esters.

The enantioselective hydrolysis of p-nitrophenyl esters of alpha-amino acids (Phe, Leu, Ala) was accomplished in micelles formed with the surfactants bearing one or two sugar-amide headgroups. The effect of structural variations in such sugar-amide surfactants on the rates and enantioselectivity (kD/kL) for hydrolysis of p-nitrophenyl esters of D- and L-phenylalanine hydrogen bromides (D- and L-PheONp) was studied. Both the hydrolysis rate of D-PheONp and the enantioselectivity increased with an increase in the alkyl chain length as well as an increase in the number of the alkyl chains in the maltobionamide-type surfactants. Enantioselectivity also increased with an increase in the sugar chain length from bisgluconamide to bismaltobionamide (by one glucose unit per each sugar chain) in the double-sugar-chain surfactants, but enantioselectivity was no longer influenced by a further increase to bismaltotrionamide. The stereochemistry of the linkage between the sugar units in the sugar chain remarkably affected the enantioselectivity: the maltobionamide-type surfactant, in which the two sugar units are connected by an alpha-1,4-glucosidic linkage, showed high enantioselectivity (kD/kL = 5.5), whereas the surfactant bearing cellobionamide headgroups (beta-1,4-glucosidic linkage) showed no enantioselectivity. Similar trends were observed when p-nitrophenyl esters of D- and L-leucine hydrogen bromides were used as substrates. On the other hand, the rates and enantioselectivity for hydrolysis of p-nitrophenyl esters of D- and L-alanine hydrogen bromides were not so largely affected by the structural variations in the sugar-amide surfactants. Additionally, the effects of the surfactant concentration and the reaction temperature on the rates and enantioselectivity for the hydrolysis of D- and L-PheONp were examined.

Amides↗