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Biomedical subjects

Yuichi Yamasaki

Publications and source records attributed to Yuichi Yamasaki.

17 recordsLinked to original sources
PubMed2007

Preparation and characterization of polyion complex micelles with a novel thermosensitive poly(2-isopropyl-2-oxazoline) shell via the complexation of oppositely charged block ionomers.

Novel thermosensitive polyion complex (PIC) micelles were prepared in an aqueous medium based on the complexation of a pair of oppositely charged block ionomers, poly(2-isopropyl-2-oxazoline)-b-poly(amino acid)s (PiPrOx-b-PAA), containing thermosensitive PiPrOx segments. The controlled synthesis of PiPrOx-b-PAA was achieved via the ring-opening anionic polymerization of N-carboxyanhydrides (NCA) of either eta-benzyloxycarbonyl-l-lysine (Lys(Z)-NCA) or beta-benzyl-l-aspartate (BLA-NCA) with omega-amino-functionalized PiPrOx macroinitiators and the subsequent deprotection reaction under acidic or basic conditions. Gel permeation chromatography (GPC) and 1H NMR spectroscopy revealed that the syntheses of two block ionomers, poly(2-isopropyl-2-oxazoline)-b-poly(l-lysine) [PiPrOx-P(Lys)] and poly(2-isopropyl-2-oxazoline)-b-poly(aspartic acid) [PiPrOx-P(Asp)], proceeded almost quantitatively to give samples with a narrow molecular weight distribution (Mw/Mn </= 1.2). The mixing of these two oppositely charged block ionomers in an aqueous medium led to the spontaneous formation of PIC micelles, which was confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The PIC micelles were spherical particles with a narrow distribution in the range of the measured concentration (0.125-1 mg/mL) and were stable without any secondary aggregates. Furthermore, the PIC micelles had a constant cloud-point temperature (Tcp) of approximately 32 degrees C under physiological conditions regardless of the total concentration, suggesting that the concentration factor is almost negligible with respect to the Tcp of the micelles presumably because of the increased local concentration of the PiPrOx segments in the shell layer. These PIC micelles have a promising application as a size-regulated smart nanocontainer loading charged compounds as well as bearing a thermosensitive outer shell that is useful for physical affinity control.

Hot Temperature↗
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

Development of a fitting model suitable for the isothermal titration calorimetric curve of DNA with cationic ligands.

A novel curve fitting model was developed for the isothermal titration calorimetry (ITC) of a cationic ligand binding to DNA. The ligand binding often generates a DNA conformational change from an elongated random coil into a compact collapsed form that is referred to as "DNA condensation". The ligand binding can be classified into two regimes having different binding constants Ki, i.e., the binding to an elongated DNA chain with a binding constant K1 and with K2 that occurred during the conformational transition. The two-variable curve fitting models are usually bound by a strict regulation on the difference in the values of the binding constants K1 > K2. For the DNA condensation, however, the relationships for K1 and K2 are still unclear. The novel curve fitting model developed in this study takes into account this uncertainty on the relationship of the binding constants and is highly flexible for the two-variable binding constant system.

Calorimetry↗
PubMed2006

Semipermeable polymer vesicle (PICsome) self-assembled in aqueous medium from a pair of oppositely charged block copolymers: physiologically stable micro-/nanocontainers of water-soluble macromolecules.

A new entity of polymer vesicle with a polyion complex (PIC) membrane, a PICsome, was prepared by simple mixing of a pair of oppositely charged block copolymers, composed of biocompatible PEG and poly(amino acid)s, in an aqueous medium. Flow particle image analysis revealed the formation of spherical particles with a size range up to 10 mum. Observation by dark-field and confocal laser scanning microscopes clearly confirmed that the PICsome has a hollow structure with an inner-water phase, in which FITC-dextran emitting green fluorescence was successfully encapsulated simply by the simultaneous mixing with the block copolymers. Confocal laser scanning microscopic observation and spectral analysis revealed the smooth penetration of a low molecular weight fluorescent dye (TRITC; MW = 443.5) emitting red fluorescence into the FITC-dextran encapsulated PICsome to give the PICsome image with a merged color of yellows, indicating the semipermeable nature of the PICsome membrane. The PICsomes showed appreciable physiological stability even in the presence of serum proteins, suggesting their feasibility in biomedical fields such as carriers of therapeutic compounds and compartments for diagnostic enzymes.

Biocompatible Materials↗
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↗
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

Stabilization of lysozyme-incorporated polyion complex micelles by the omega-end derivatization of poly(ethylene glycol)-poly(alpha,beta-aspartic acid) block copolymers with hydrophobic groups.

To improve the stability of lysozyme-incorporated polyion complex (PIC) micelles in physiological condition, three types of hydrophobic groups, including phenyl (Phe), naphthyl (Nap), and pyrenyl (Py) terminal groups, were separately introduced to the omega-end of poly(ethylene glycol)-poly(alpha,beta-aspartic acid) block copolymers (PEG-P(Asp)). The goal was to enhance association forces between the enzyme, lysozyme, and PEG-P(Asp) carriers. Introduction of these hydrophobic groups significantly decreases micellar critical association concentration and increases the micellar tolerability against increasing NaCl concentrations. Particularly, PIC micelles formed from PEG-P(Asp) with Py groups was most stable against increasing NaCl concentrations up to 0.1 M. Significant deviation from a spherical shape for the micelles was also observed for the PEG-P(Asp)-Py system, consistent with an increased association number.

Aspartic Acid↗
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

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↗
PubMed2004

Supramolecular nanocarrier of siRNA from PEG-based block catiomer carrying diamine side chain with distinctive pKa directed to enhance intracellular gene silencing.

An siRNA nanocarrier formed through self-assembly of PEG-based block catiomer possessing two distinct amino groups with different pKa values in a side chain was developed. This design provided the carrier with a sufficient siRNA complexation and an assumed buffering capacity in the endosomes, allowing it to exhibit remarkable gene knockdown abilities as well as sufficient serum tolerability.

Cations↗
PubMed2004

Lactose-conjugated polyion complex micelles incorporating plasmid DNA as a targetable gene vector system: their preparation and gene transfecting efficiency against cultured HepG2 cells.

alpha-Lactosyl-poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) block copolymer (lactose-PEG-PAMA) was synthesized to construct a PIC micellar-type gene vector potentially useful for selective transfection of hepatic cells. Lactose-PEG-PAMA spontaneously formed a polyion complex (PIC) micelle with plasmid DNA (pDNA) encoding luciferase (pGL3-Luc) in aqueous solution without any precipitate formation. The lactosylated PIC micelle thus prepared achieved substantially higher transfection efficiency compared to the control PIC micelle without lactose moieties against HepG2 cells possessing asialoglycoprotein (ASGP) receptors recognizing the beta-d-galactose residue. This pronounced transfection efficacy of the lactosylated PIC micelle was inhibited by the addition of excess asialofetuin (ASF), a natural ligand against the ASGP receptor, indicating ASGP receptor-mediated endocytosis to be a major route of the cellular uptake of the lactosylated micelles. Notably, the lactosylated PIC micelle revealed enhanced transfection compared to the control PIC micelle at a lower dose of pDNA, demonstrating the feasibility of using the ligand-conjugated PIC micellar vector for gene delivery to targeted cells.

Cell Line, Tumor↗
PubMed2004

Block catiomer polyplexes with regulated densities of charge and disulfide cross-linking directed to enhance gene expression.

A block catiomer polyplex, showing a high stability in the extracellular medium and an efficient release of plasmid DNA (pDNA) in the intracellular compartment, was developed by controlling both the cationic charge and disulfide cross-linking densities of the backbone polycations. Poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) was thiolated using either of two thiolation reagents, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) or 2-iminothiolane (Traut's reagent), to investigate the effects of both the charge and disulfide cross-linking densities on the properties of the polyplexes. The introduction of thiol groups by SPDP proceeded through the formation of amide linkages to concomitantly decrease the cationic charge density of PLL segment, whereas Traut's reagent promoted the thiolation with the introduction of cationic imino groups to keep the charge density constant. These thiolated PEG-PLLs were complexed with pDNA to form the disulfide cross-linked block catiomer polyplexes, which had the size of approximately 100 nm. Both thiolation methods were similarly effective in introducing disulfide cross-links to prevent the polyplex from the dissociation through a counter polyanion exchange in the extracellular oxidative condition. On the other hand, the efficient release of pDNA responding to the reductive condition mimicking the intracellular environment was only achieved for the polyplex thiolated with SPDP, a system compensating for the decrease in the charge density with the disulfide cross-linking. This distinctive sensitivity toward oxidative and reductive environments was nicely correlated with the remarkable difference in the transfection efficiency between these two types of thiolated polyplexes (SPDP and Traut's reagent types): the former revealed approximately 50 times higher transfection efficiency toward 293T cells than the latter. Obviously, the balance between the densities of the cationic charge and disulfide cross-linking in the thiolated polyplex played a crucial role in the delivery and controlled release of entrapped pDNA into the microenvironment of intracellular compartment to achieve the high transfection efficiency.

Cell Line↗
PubMed2004

Nanospheres for DNA separation chips.

We report here a technology to carry out separations of a wide range of DNA fragments with high speed and high resolution. The approach uses a nanoparticle medium, core-shell type nanospheres, in conjunction with a pressurization technique during microchip electrophoresis. DNA fragments up to 15 kilobase pairs (kbp) were successfully analyzed within 100 s without observing any saturation in migration rates. DNA fragments migrate in the medium while maintaining their characteristic molecular structure. To guarantee effective DNA loading and electrofocusing in the nanosphere solution, we developed a double pressurization technique. Optimal pressure conditions and concentrations of packed nanospheres are critical to achieve improved DNA separations.

DNA↗
PubMed2004

In situ single cell observation by fluorescence resonance energy transfer reveals fast intra-cytoplasmic delivery and easy release of plasmid DNA complexed with linear polyethylenimine.

BACKGROUND: The investigation into the intracellular mechanisms for gene expression has acquired great impetus for the improvement of the transfection efficiency by a non-viral gene delivery system. METHODS: Intracellular trafficking as well as release of plasmid DNA (pDNA) complexed with polycations, including linear and branched polyethylenimine (LPEI, BPEI) and poly(L-lysine) (PLL), were explored under confocal microscopy using fluorescence resonance energy transfer (FRET) between a pair of donor-acceptor fluorescent dyes (fluorescein and Cy3) tagged on a single pDNA molecule. RESULTS: pDNA complexed with LPEI underwent a rapid escape from the endosomes, spreading uniformly into the cytoplasm with a substantial decrease in FRET efficiency due to the disintegration of LPEI/pDNA polyplex structure. pDNA complexed with BPEI also achieved a rapid escape from the endosomes. Nevertheless, the pDNA retained high FRET efficiency even after 24 h, indicating an appreciable stability of the BPEI/pDNA polyplex to keep pDNA in a condensed state. In the PLL/pDNA polyplexes, neither endosome escape nor pDNA decondensation was observed. These intracellular characteristics of polyplexes showed a clear correlation to their gene transfection efficiency: The LPEI/pDNA revealed a considerably higher and faster gene expression compared with BPEI/pDNA. Atomic force microscopy revealed that BPEI induced more effective condensation of pDNA than LPEI, being consistent with restricted cytoplasmic release of complexed pDNA. CONCLUSION: Fast endosomal escape and subsequent smooth disintegration of LPEI/pDNA in the cytoplasm are likely to be determining factors for the excellent transfection efficiency of this polyplex system. These properties may be particularly beneficial to achieve appreciably high gene expression in a prompt manner.

Cell Culture Techniques↗
PubMedDate not supplied

Polyion complex micelles of pDNA with acetal-poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) block copolymer as the gene carrier system: physicochemical properties of micelles relevant to gene transfection efficacy.

An acetal-poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) (acetal-PEG-PAMA) block copolymer spontaneously associated with plasmid DNA (pDNA) to form water-soluble complexes (polyion complex micelle: PIC micelle) in aqueous solution. Physicochemical characteristics and transfection efficiency of the PIC micelles thus prepared were studied here, focusing on the residual molar mixing ratio (N/P ratio) of AMA units in acetal-PEG-PAMA to the phosphate units in pDNA. With the N/P ratio increasing to unity, acetal-PEG-PAMA cooperatively formed complex micelles with pDNA through electrostatic interaction, allowing pDNA to condense effectively. Dynamic light scattering measurements revealed that the PIC micelle at N/P > or = 3 had a constant size of approximately 90-100 nm. Eventually, acetal-PEG-PAMA/pDNA micelles underwent no precipitation even after long-term storage for more than 1 month at all N/P ratios. The PIC micelles were stable even in the presence of excess polyanions, poly(vinyl sulfate), in contrast to polyplexes based on the PAMA homopolymer, yet this stabilization effect was highly dependent on the N/P ratio to reach a plateau at N/P = 3-4. This character may be attributed to the increased hydrophobicity in the vicinity of the complexed pDNA. Furthermore, the pDNA in the micelle was adequately protected from DNase I attack. The transfection ability of the PIC micelles toward 293 cells was remarkably enhanced with an increasing N/P ratio as high as 25. The zeta-potential of the micelles with a high N/P ratio was an appreciably large positive value, suggesting a noncooperative micelle formation. This deviated micellar composition with an excess cationic nature as well as the presence of free acetal-PEG-PAMA may play a substantial role in the enhanced transfection efficiency of the PIC micelle system in the high N/P ratio (approximately 25) region.

Cations↗
PubMedDate not supplied

Supramolecular assemblies for the cytoplasmic delivery of antisense oligodeoxynucleotide: polyion complex (PIC) micelles based on poly(ethylene glycol)-SS-oligodeoxynucleotide conjugate.

A novel cytoplasmic delivery system of antisense oligodeoxynucleotide (asODN) was developed by assembling a PEG-asODN conjugate with disulfide linkage (smart linkage) (PEG-SS-asODN) into polyion complex (PIC) micelles through the complexation with branched poly(ethylenimine) (B-PEI). The PIC micelle thus prepared showed a significant antisense effect against luciferase gene expression in HuH-7 cells, far more efficient than nonmicelle systems (asODN and PEG-SS-asODN in free form) and PIC micelle encapsulating the conjugate without the disulfide linkage. Use of poly(l-lysine) (PLL) instead of the B-PEI for PIC micellization led to a substantial decrease in the antisense effect. These results indicate that the PIC micelles formulated from PEG-SS-asODN conjugate and B-PEI is successfully transported from the endosomal compartment into the cytoplasm by the buffering effect of the B-PEI, releasing hundreds of active asODN molecules via cleavage of the disulfide linkage into the cellular interior, responding to a high glutathione concentration in the cytoplasmic compartment. Furthermore, the type of smart linkage (glutathione-sensitive SS linkage vs pH-sensitive linkage) in the conjugates substantially affected the antisense effect of the PIC micelles, depending on the nature of the counter polycation (B-PEI vs PLL).

Biocompatible Materials↗