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Biomedical subjects

Nobuhiro Nishiyama

Publications and source records attributed to Nobuhiro Nishiyama.

At least 19 recordsLinked to original sources
PubMed2006

PEGylated gene nanocarriers based on block catiomers bearing ethylenediamine repeating units directed to remarkable enhancement of photochemical transfection.

The therapeutic usefulness of macromolecular drugs such as plasmid DNA is often limited by the inefficient transfer of macromolecules to the cytosol. Photochemical internalization (PCI) technology, in which the endosomal escape of DNA or its complex is assisted by co-incubated photosensitizers that photodamage endosome membrane, offers a solution for this problem. A series of poly(ethylene glycol) (PEG)-based block polycatiomers with increasing number of ethylenediamine repeating unit at side chain of polycatiomers were complexed with pDNA to form the PEGylated polyplexes as a biocompatible gene carrier. Dendrimeric phthalocyanine (DPc)-incorporated micelle was used to assist the gene transfer of these polyplexes in a light-inducible manner. As a result, the light-inducible transfection activity was significantly enhanced as the number of amino group at the side chain of PEG-b-polycatiomer increased. The polyplex from PEG-b-polycatiomer having the longest ethylenediamine structure achieved approximately 1000-fold enhancement of transfection upon photoirradiation. This result supports the underlying hypothesis that photochemical transfection and proton sponge effect of polycations can work synergistically to enhance the transfection efficiency. With careful balance between photochemical transfection enhancement and cytotoxicity, PEG-b-polycatiomers used in this study might be a potential candidate for in vivo PCI-mediated gene transfer.

Algorithms↗
PubMed2006

Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery.

Polymeric micelles, self-assemblies of block copolymers, are promising nanocarrier systems for drug and gene delivery. Until now, several micellar formulations of antitumor drugs have been intensively studied in preclinical and clinical trials, and their utility has been demonstrated. Even compared with long-circulating liposomes, polymeric micelles might have several advantages, such as controlled drug release, tissue-penetrating ability and reduced toxicity such as hand-foot syndrome and hypersensitivity reaction. Importantly, critical features of the polymeric micelles as drug carriers, including particle size, stability, and loading capacity and release kinetics of drugs, can be modulated by the structures and physicochemical properties of the constituent block copolymers. Also, nano-engineering of block copolymers might allow the preparation of polymeric micelles with integrated smart functions, such as specific-tissue targetability, as well as chemical or physical stimuli-sensitivity. Thus, polymeric micelles are nanotechnology-based carrier systems that might exert the activity of potent bioactive compounds in a site-directed manner, ensuring their effectiveness and safety in the clinical use.

Animals↗
PubMed2006

Polyion complex micelles for photodynamic therapy: incorporation of dendritic photosensitizer excitable at long wavelength relevant to improved tissue-penetrating property.

A polymeric micelle (DPcZn/m) system, which is formed via an electrostatic interaction of anionic dendrimer phthalocyanine (DPcZn) and poly(ethylene glycol)-poly(l-lysine) block copolymers (PEG-b-PLL), was prepared for use as an effective photosensitizer for photodynamic therapy. DPcZn/m exhibited strong Q band absorption around 650 nm, a useful wavelength for high tissue penetration. Dynamic light scattering studies indicated that the DPcZn/m system has a relevant size of 50 nm for intravenous administration. Under light irradiation, either DPcZn or DPcZn/m exhibited efficient consumption of dissolved oxygen in a medium to generate reactive oxygen species and an irradiation-time-dependent increase in photocytotoxicity. The photodynamic efficacy of the DPcZn was drastically improved by the incorporation into the polymeric micelles, typically exhibiting more than two orders of magnitude higher photocytotoxicity compared with the free DPcZn at 60-min photoirradiation.

Cell Culture Techniques↗
PubMed2006

Pulsatile cardiac tissue grafts using a novel three-dimensional cell sheet manipulation technique functionally integrates with the host heart, in vivo.

We devised a method of fabricating easily transplantable scaffoldless 3D heart tissue, made with a novel cell-sheet (CS) technology from cultured cardiomyocytes using a fibrin polymer coated dish. In the present study, we tested in vivo electrical communication which is essential for improving heart function between the host heart and the grafted CS. The epicardial surface of the ventricle of an anesthetized open-chest nude rat was ablated by applying a heated metal. Bilayered CS was obtained from neonatal rat primary culture. CS was transplanted onto the injured myocardial surface (sMI) (sMI+sheet group). The rats were allowed to recover for 1 to 4 weeks, to stabilize the grafts. Action potentials (APs) from the excised perfused heart were monitored by the fluorescence signal of di-4ANEPPS with a high speed charge-coupled device camera. The APs were observed under epicardial pacing of the host heart or the CS grafts. The pacing threshold of the current output was measured in the sMI+sheet group and in the nongrafted sMI group at the center of the sMI and in the normal zone (Nz). Bidirectional AP propagation between the sMI and Nz was observed in the sMI+sheet group (n=14), but was blocked at the marginal area of the sMI in the sMI group (n=9). The ratio of the pacing threshold (sMI/Nz) was significantly lower in the sMI+sheet than in the sMI group (3.0+/-0.7, 19.0+/-6.1 respectively P<0.05). There were neither spontaneous nor pacing-induced arrhythmias in these two groups. Bidirectional smooth AP propagation between the host heart and the grafted CS was observed. This finding suggested functional integration of this CS graft with the host heart without serious arrhythmia.

Action Potentials↗
PubMed2006

A PEG-based biocompatible block catiomer with high buffering capacity for the construction of polyplex micelles showing efficient gene transfer toward primary cells.

Nonviral gene vectors from synthetic catiomers (polyplexes) are a promising alternative to viral vectors. In particular, many recent efforts have been devoted to the construction of biocompatible polyplexes for in vivo nonviral gene therapy. A promising approach in this regard is the use of poly(ethylene glycol) (PEG)-based block catiomers, which form a nanoscaled core-shell polyplex with biocompatible PEG palisades. In this study, a series of PEG-based block catiomers with different amine functionalities were newly prepared by a simple and affordable synthetic procedure based on an aminolysis reaction, and their utility as gene carriers was investigated. This study revealed that the block catiomers carrying the ethylenediamine unit at the side chain are capable of efficient and less toxic transfection even toward primary cells, highlighting critical structural factors of the cationic units in the construction of polyplex-type gene vectors. Moreover, the availability of the polyplex micelle for transfection with primary osteoblasts will facilitate its use for bone regeneration in vivo mediated by nonviral gene transfection.

Animals↗
PubMed2006

Photochemical enhancement of transgene expression by polymeric micelles incorporating plasmid DNA and dendrimer-based photosensitizer.

The development of synthetic gene carriers has recently received much attention; however, they might lack the ability to control the transgene expression. The use of external stimuli for enhancement of the transgene expression may be a promising approach for the site-directed transfection in vivo. In this regard, a new technology of "photochemical internalization (PCI)" has recently been reported, in which the endosomal escape of gene carriers is assisted by photodamage of the endosomal membrane with co-incubating photosensitizers. To apply this technology for systemic gene delivery, the development of appropriate carrier systems for both the plasmid DNA (pDNA) and photosensitizer is of crucial importance. Also, the photocytotoxicity accompanied by the photochemical enhancement of the gene expression may need to be reduced. In this study, the combinational formulation of polymeric micelles incorporating pDNA and a dendrimer-based photosensitizer (DP) (dendrimer phthalocyanine (DPc)) was applied in the PCI-mediated transfection in vitro and then, estimating its potential utility for in vivo applications. The PCI using the polymeric micelle system achieved a remarkable photochemical enhancement of the transgene expression while maintaining an approximate 80% cell viability over a wide range of the DPc concentrations and light doses. Thus, this system may be promising for in vivo PCI-mediated gene delivery.

Cell Survival↗
PubMed2006

[Nano-engineering for biomedical applications].

Recently, medical applications of nanotechnology have received much attention. Among them, advances in polymer chemistry have significantly contributed to this field. Here, we review nano-engineering of functional polymers for diagnostic and therapeutic purposes, which have been carried out mainly by our group. Functional polymer-coated nanoparticles can be used for quick detection of biomarkers and DNA separation. Multiarray of hepatocyte spheroids on a microfabricated polymer-brush surface can maintain the hepatocyte viability and liver-specific functions, offering a new high-throughput screening method of pharmacologically and toxicologically active compounds for drug discovery. Polymeric micelles are promising drug carriers, of which the critical parameters such as the size, drug loading and release can be controlled by engineering the constituent block copolymers. Thus, polymeric nano-device will be the leading technology in this field.

Biomedical Engineering↗
PubMed2005

Light-induced gene transfer from packaged DNA enveloped in a dendrimeric photosensitizer.

The control of gene transfection in the body is a core issue in gene therapy. Photochemical internalization is a technology that allows light-induced delivery of DNA, drugs or other biological factors directly inside cells. Usually it requires that a photosensitizer be added to the drug-delivery system to photochemically destabilize the endosomal membrane. Here we present a system for in vivo DNA delivery in which these two components are assembled into one structure. This is a ternary complex composed of a core containing DNA packaged with cationic peptides and enveloped in the anionic dendrimer phthalocyanine, which provides the photosensitizing action. The ternary complex showed more than 100-fold photochemical enhancement of transgene expression in vitro with reduced photocytotoxicity. In an animal experiment, subconjuctival injection of the ternary complex followed by laser irradiation resulted in transgene expression only in the laser-irradiated site. This work demonstrates a new biomedical application for dendrimers, and the first success in the photochemical-internalization-mediated gene delivery in vivo.

Animals↗
PubMed2005

Freeze-dried formulations for in vivo gene delivery of PEGylated polyplex micelles with disulfide crosslinked cores to the liver.

A stable, freeze-dried formulation consisting of a core-shell-type polyplex with a poly(ethylene glycol) (PEG) shell (polyplex micelles) was prepared from a polyion complex of plasmid DNA (pDNA) and thiolated PEG-poly(L-lysine) block copolymers. The use of lyoprotectants was avoided by crosslinking the core with disulfide bonds. The crosslinked polyplex micelles (CPMs) showed excellent stability during freeze-drying and reconstitution processes, which is in sharp contrast with the formation of visible agglomerates from the non-crosslinked polyplex micelles (NCPMs) after a similar process. A thiolation degree higher than 13% of the lysine residues was required to achieve sufficient tolerability of the CPMs during the freeze-drying/reconstitution cycle. Dynamic light scattering measurements and atomic force microscopy observations demonstrated that the original size and shape of the CPMs with a thiolation degree of higher than 13% were maintained even after the freeze-drying. Furthermore, the CPMs reconstituted from the freeze-dried state achieved a transfection efficiency as high as that of the original samples. The intravenous injection of the CPM with a thiolation degree of 37% into mice via the orbital vein led to an appreciably uniform gene expression of a yellow fluorescence protein variant (Venus) in the liver, while no gene expression was observed in the case of the free pDNA injection. The procedure of disulfide crosslinking of the polyplex micell core allows the preparation of non-viral gene vectors as a powder formulation without the use of any lyoprotectants. This achievement is certainly useful for pharmaceutical applications and exhibits many advantages, including easy concentration adjustments of dosing samples, long-term storage stability, and large-scale production reproducibility.

Animals↗
PubMed2005

Multifunctional polymeric micelles with folate-mediated cancer cell targeting and pH-triggered drug releasing properties for active intracellular drug delivery.

A new type of multifunctional polymeric micelle drug carrier for active intracellular drug delivery was prepared and characterized in this study. The micelle is a nano-supramolecular assembly with a spherical core-shell structure, and its surface and core were modified with piloting molecules for cancer cells and pH-sensitive drug binding linkers for controlled drug release, respectively. In order to prepare such micelles, self-assembling amphiphilic block copolymers, folate-poly(ethylene glycol)-poly(aspartate hydrazone adriamycin) [Fol-PEG-P(Asp-Hyd-ADR)], were specially designed and synthesized by installing a molecular promoter to enhance intracellular transport, folate (Fol), at the end of the shell-forming PEG chain and conjugating the anticancer drug, adriamycin (ADR), to the side chain of the core-forming PAsp segment through an acid-sensitive hydrazone bond. Because folate-binding proteins (FBP) are selectively overexpressed on the cancer cell membranes, the folate-bound micelles (FMA) can be guided to the cancer cells in the body, and after the micelles enter the cells, hydrazone bonds are cleaved by the intracellular acidic environment (pH 5-6) so that the drug release profile of the micelles is controlled pH-dependently. In this regard, FBP-binding selectivity of the prepared FMA was evaluated by surface plasmon resonance (SPR) measurements. The tetrazolium dye method (MTT assay) using human pharyngeal cancer cells (KB cell) revealed that FMA significantly improved cell growth inhibitory activity in spite of a short exposure time due to the selective and strong interaction between folate molecules and their receptors. Subsequent flow cytometric analysis showed that cellular uptake of FMA significantly increased. Consequently, these findings would provide one of the most effective approaches for cancer treatment using intracellular environment-targeting supramolecular drug carriers.

Cell Division↗
PubMed2005

PEGylated polyplex micelles from triblock catiomers with spatially ordered layering of condensed pDNA and buffering units for enhanced intracellular gene delivery.

An A-B-C type triblock copolymer, tandemly aligning two types of polycations with different pKa values in a single polymer strand, was developed for the construction of novel polyplex micelles, satisfying a high DNA condensing ability as well as a proton buffering activity directed to elevating gene transfection. The micelle might feature the distinctive three-layered structure, where an inner polyplex layer of condensed pDNA with poly(l-lysine) (pKa approximately 9.4) as the C segment is successively wrapped with an intermediate layer of poly[(3-morpholinopropyl)aspartamide] (B segment) with a comparatively low pKa of approximately 6.2, to provide a buffering effect, and an outer PEG layer (A segment) as a biocompatible palisade.

Aspartic Acid↗
PubMed2005

Lactosylated poly(ethylene glycol)-siRNA conjugate through acid-labile beta-thiopropionate linkage to construct pH-sensitive polyion complex micelles achieving enhanced gene silencing in hepatoma cells.

The remarkably enhanced gene silencing in hepatoma cells was achieved by assembling lactosylated-PEG-siRNA conjugates bearing acid-labile beta-thiopropionate linkages into polyion complex (PIC) micelles through the mixing with poly(l-lysine). The PIC micelles with clustered lactose moieties on the periphery were successfully transported into hepatoma cells in a receptor-mediated manner, releasing hundreds of active siRNA molecules into the cellular interior responding to the pH decrease in the endosomal compartment. Eventually, almost 100 times enhancement in gene silencing activity compared to that of the free conjugate was achieved for the micelle system, facilitating the practical utility of siRNA therapeutics.

Carcinoma, Hepatocellular↗
PubMed2005

Preparation and biological properties of dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt)-loaded polymeric micelles.

Block copolymer micelles, containing dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt), the oxaliplatin parent complex, were prepared through polymer-metal complex formation of DACHPt with poly(ethylene glycol)-poly(glutamic acid) block copolymer [PEG-P(Glu)] in distilled water. By dynamic light scattering (DLS) measurement, the micelle size was determined to be 40 nm with narrow distribution. The release of platinum complexes from the micelle core was measured in phosphate buffer saline (pH 7.4) at 37 degrees C. DACHPt-loaded micelle showed a sustained release rate of platinum after an induction period of 12 h. In the same conditions, the kinetic stability of DACHPt-loaded micelle was measured. The micelle was found to be very stable, keeping the initial size, for 240 h. Against murine colon adenocarcinoma 26 (C-26) cells, DACHPt-loaded micelle exhibited considerable in vitro cytotoxicity, lower than oxaliplatin but increasing with exposure time as a result of the release of platinum complexes from the micelle. In vivo biodistribution assay performed on tumor-bearing mice demonstrated that the micelle showed prolonged blood circulation due to its high stability and high tumor accumulation for a prolonged time.

Animals↗
PubMed2005

Nanotechnology-based photodynamic therapy for neovascular disease using a supramolecular nanocarrier loaded with a dendritic photosensitizer.

Photodynamic therapy (PDT) for exudative age-related macular degeneration (AMD) was evaluated using a supramolecular nanomedical device, that is, a novel dendritic photosensitizer (DP) encapsulated by a polymeric micelle formulation. The characteristic dendritic structure of the DP prevents aggregation of its core sensitizer, thereby inducing a highly effective photochemical reaction. With its highly selective accumulation on choroidal neovascularization (CNV) lesions, this treatment resulted in a remarkably efficacious CNV occlusion with minimal unfavorable phototoxicity.

Animals↗
PubMed2005

Smart polymeric micelles as nanocarriers for oligonucleotides and siRNA delivery.

The development of in vivo delivery systems for oligonucleotides and siRNA is strongly desired to achieve their clinical applications. Recently, polyplex micelles, which are formed through an electrostatic interaction between nucleic acid compounds (DNA and RNA) and poly(ethylene glycol) (PEG)-polycation block copolymers, have received much attention due to their nanometric-scaled size and excellent biocompatibility. Here, three types of newly engineered block copolymers were developed to construct polyplex micelles useful for oligonucleotides and siRNA delivery: (1) PEG-polycation diblock copolymers possessing diamine side-chain with distinctive pKa for siRNA encapsulation into polyplex micelles with high endosomal escaping ability, (2) Lactosylated PEG-(oligonucleotide or siRNA) conjugate through acid-labile beta-thiopropionate linkage to construct pH-sensitive PIC micelles, and (3) PEG-poly(methacrylic acid) block copolymer for the construction of organic/inorganic hybrid nanoparticles encapsulating siRNA.

Hydrogen-Ion Concentration↗
PubMed2005

New drug delivery for corneal neovascularization using polyion complex micelles.

PURPOSE: To introduce photodynamic therapy (PDT) for corneal neovascularization (NV) by polyion complex (PIC) micelles, a novel drug delivery system. METHODS AND RESULTS: Development of specific drug delivery systems to corneal NV sites is an important part of next-generation photodynamic therapy. Nanocarriers consisting of PIC micelles bound by polyethylene glycol (PEG) shell exhibit good stability, high drug-loading capacity, and excellent potential for controlled drug release. Encapsulation of the photosensitizer dendrimer porphyrin (DP) into PIC micelles (DP-micelles) conveys adequate stability and increased photocytotoxicity without compromising photophysical properties of DP stability. We assessed the accumulation of DP-micelles and observed that the DP-micelles were incorporated into the corneal NV area. CONCLUSIONS: PIC micelles possess attractive features of selective accumulation at the corneal NV site. PDT using DP-micelles appears to be effective and safe for the treatment of corneal NV.

Animals↗
PubMed2005

[Medical applications of nanotechnology: polymeric micelles for drug delivery].

Recently, medical applications of nanotechnology have received much attention. Among them, drug delivery systems (DDSs) are expected to be the leading technology in diagnostic and therapeutic medicine. An increasing number of DDSs have been approved for clinical use or are currently being studied in clinical trials. Polymeric micelles, self-assemblies of block copolymers, are one of the most refined and promising modalities of DDSs, since the critical parameters such as size, drug loading, and release can be controlled by engineering the constituent block copolymers. It has been demonstrated that polymeric micelles show unique disposition characteristics in the body suitable for drug targeting (e.g., prolonged blood circulation and significant tumor accumulation). Also, recent advances in polymer synthesis have allowed the development of polymeric micelles with integrated smart functions such as environmental sensitivity and targetability. Thus polymeric micelles might have great potential for nanotechnology-based DDSs for clinical use.

Antineoplastic Agents↗