Intracellular inducible alkylation system that exhibits antisense effects with greater potency and selectivity than the natural oligonucleotide.
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Alkylation↗
Biomedical subjects
Yukio Nagasaki
Publications and source records attributed to Yukio Nagasaki.
Preparation of a novel PEG-clay hybrid as a DDS material: dispersion stability and sustained release profiles.
An advanced hybrid drug carrier has been developed using porous nanocrystals of a swelling clay mineral conjugated with a block copolymer containing poly(ethylene glycol) and polyamine segments. Synthetic hectorite (Laponite) modified with (alpha-acetal-poly(ethylene glycol)-block-[poly(2-(N,N-dimethylamino) ethyl methacrylate)] (Acetal-PEG-b-PAMA) produced a homogeneous dispersion of organic-inorganic hybrid in an aqueous solution, which showed flocculation-resistive properties with an elevated ionic strength. The zeta-potential measurement revealed that nonionic PEG brush layers are formed on the surface of the clay nanocrystals since negative charge of the clay surface was completely neutralized by the positive charge of the cationic PAMA segment and the entire surface charge is successfully shielded by the effect of nonionic PEG segment in the block copolymer. This charge neutralization is in good agreement with the dispersion stability in solutions of high ionic strength. The average particle size of the PEG-modified hybrid particle was estimated to be 120 nm by a dynamic light scattering (DLS) method. When pyrene was used as the model compound of hydrophobic drug, it was incorporated into the nanopore in the clay mineral without showing any remarkable expansion of the basal spacings. Fluorescence spectra and powder X-ray diffraction patterns demonstrated that pyrene molecules are captured in an amorphous state in the range of low pyrene content (<5%), while excimer formation was seen at the higher pyrene concentration (>5%). The PEG-clay hybrid act as a carrier for sustained release of hydrophobic substances due to the high affinity (K = 1.52 x 10(4)) between the drug and clay surface.
Algorithms↗
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↗
A reactive poly(ethylene glycol) layer to achieve specific surface plasmon resonance sensing with a high S/N ratio: the substantial role of a short underbrushed PEG layer in minimizing nonspecific adsorption.
A reactive poly(ethylene glycol) (PEG)-brushed layer was constructed on a surface plasmon resonance (SPR) sensor chip using a heterobifunctional PEG possessing an acetal group at one end and a mercapto group at the other end (alpha-acetal-omega-mercapto-PEG). The density of the PEG brushed layer substantially increased with repetitive adsorption/rinse cycles of the PEG on the sensor chip, allowing dramatic reduction of nonspecific protein adsorption. Notably, formation of a short, filler layer of PEG (2 kDa) in the preconstructed longer PEG brushed layer (5 kDa) achieved almost complete prevention of nonspecific protein adsorption. The acetal group located at the distal end of the tethered PEG was converted to an aldehyde group by the acid treatment, followed by the installation of biocytin hydrazide through Schiff base formation. SPR sensing of streptavidin was done with a very high S/N ratio even in a proteinous medium using the biotinylated PEG (5 kDa) tethered chip with an inert filler layer of short PEG (2 kDa). Furthermore, the specific affinity of streptavidin for the biotinylated PEG was highly influenced by the length of the filler PEG and was significantly reduced when the length of the filler PEG was longer than that of the biotinylated PEG. This result clearly revealed the substantial importance of the steric factor on biospecific interaction at the distal end of tethered PEG on the sensor surface.
Adsorption↗
Smart polyion complex micelles for targeted intracellular delivery of PEGylated antisense oligonucleotides containing acid-labile linkages.
A novel pH-sensitive and targetable antisense ODN delivery system based on multimolecular assembly into polyion complex (PIC) micelles of poly(L-lysine) (PLL) and a lactosylated poly(ethylene glycol)-antisense ODN conjugate (Lac-PEG-ODN) containing an acid-labile linkage (beta-propionate) between the PEG and ODN segments has been developed. The PIC micelles thus prepared had clustered lactose moieties on their peripheries and achieved a significant antisense effect against luciferase gene expression in HuH-7 cells (hepatoma cells), far more efficiently than that produced by the nonmicelle systems (ODN and Lac-PEG-ODN) alone, as well as by the lactose-free PIC micelle. In line with this pronounced antisense effect, the lactosylated PIC micelles showed better uptake than the lactose-free PIC micelles into HuH-7 cells; this suggested the involvement of an asialoglycoprotein (ASGP) receptor-mediated endocytosis process. Furthermore, a significant decrease in the antisense effect (27 % inhibition) was observed for a lactosylated PIC micelle without an acid-labile linkage (thiomaleimide linkage); this suggested the release of the active (free) antisense ODN molecules into the cellular interior in response to the pH decrease in the endosomal compartment is a key process in the antisense effect. Use of branched poly(ethylenimine) (B-PEI) instead of the PLL for PIC micellization led to a substantial decrease in the antisense effect, probably due to the buffer effect of the B-PEI in the endosome compartment, preventing the cleavage of the acid-labile linkage in the conjugate. The approach reported here is expected to be useful for the construction of smart intracellular delivery systems for antisense ODNs with therapeutic value.
Cell Line, Tumor↗
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↗
Characterization of aldehyde-PEG tethered surfaces: influence of PEG chain length on the specific biorecognition.
A functionalized poly(ethylene glycol) (PEG) layer possessing a reactive aldehyde group at the free end of the tethered PEG chain was constructed by simple coating on the substrate, using the acetal-PEG/poly(DL-lactide) block copolymer, followed by the hydrolysis of the acetal end group by an acid treatment. The reactivity of the aldehyde group at the distal end of the PEG tethered chain was evaluated via a reductive amination using 4-amino-2,2,6,6-tetramethylpiperidinyloxy as the model compound. Further conjugation of the aldehyde group with sugar moieties has demonstrated an increased recognition ability with lectins with an increasing PEG chain length, which was attributable to the mobility of the chain end. These results provide a novel idea for highly sensitive biorecognition, suggesting a method to create highly selective biosensing surfaces that are able to prevent the undesired nonspecific adsorption of biocomponents.
Aldehydes↗
A design of nanosized PEGylated-latex mixed polymer solution for microchip electrophoresis.
We report here advanced microchip electrophoresis using a nanoparticle doped polymer solution that enables greater separation of DNA. The proposed system is simple and effective without any new apparatus or complicated procedures. Various amounts and sizes (80 nm, 110 nm, and 193 nm) of polymer nanoparticle solutions (PEGylated-latex) were mixed with a conventional polymer solution for microchip electrophoresis. When a 0.49 wt% hydroxyl propyl methyl cellulose (HPMC) buffer solution was mixed with a 2.25 wt% 80 nm-PEGylated-latex a higher separation efficiency and a higher mobility of a wider molecular range of dsDNA (10 bp to 2 kbp) was achieved under low viscosity conditions (<5.5 cP) than in conventional 0.7% HPMC. The separation performance was dependent on the particle size and concentration. Furthermore, the effectiveness of the larger PEGylated-latex (193 nm) was not as high as the smaller one (80 to 110 nm). The observed separation improvement by polymer solution with latex-nanoparticles seems to derive from the balance between wider polymer mesh size and the structural obstacles of particles in the buffer.
DNA↗
Novel molecular recognition via fluorescent resonance energy transfer using a biotin-PEG/polyamine stabilized CdS quantum dot.
A novel functionally PEGylated quantum dot (QD) was prepared by a coprecipitation method in the presence of the biotin-PEG/polyamine block copolymer. When CdCl2 and Na2S were mixed in aqueous media in the presence of the biotin-PEG-b-poly(2-(N,N-dimethylamino)ethyl methacrylate) [biotin-PEG/PAMA], a CdS QD with a size of ca. 5 nm was prepared. The polyamine segment was anchored on the surface of the formed CdS nanoparticle, whereas the PEG segment was tethered on the surface to form a hydrophilic palisade, thus improving the dispersion stability in aqueous media even under a high salt concentration condition. An effective fluorescent resonance energy transfer (FRET) was observed by the specific interaction of the biotin-PEG/PAMA stabilized CdS QD with TexasRed-labeled streptavidin of the physiological ionic strength of 0.15 M. The extent of the energy transfer was in proportion to the concentration of the TexasRed-streptavidin. This FRET system using the PEGylated CdS QD coupled with fluorescent-labeled protein can be utilized as a highly sensitive bioanalytical system.
Biotin↗
Thermal characterization of poly(ethylene glycol)-poly(D,L-lactide) block copolymer micelles based on pyrene excimer formation.
Poly(ethylene glycol)--poly(D,L-lactide) (PEG-PDLLA) block copolymers were prepared by anionic ring-opening polymerization, resulting in block sizes effectively controlled by initial monomer/initiator ratios and low molecular weight distributions (<1.12). A pyrene derivative (1-pyrenyl carbonyl cyanide--Py) was conjugated to the end of the hydrophobic block (PDLLA) in a quantitative manner, with coupling efficiencies >95%. The so-obtained PEG-PDLLA-Py copolymers displayed fluorescent properties that were associated with the pyrene monomers, when placed in good solvents for both the hydrophilic and hydrophobic blocks. When placed in selective solvents, these copolymers self-assembled into micelles in the 30-nm range, also with low particle size distributions (<0.09), within which Py could be readily entrapped in the hydrophobic PDLLA core. Py entrapment resulted in the formation of excimers, as evident from fluorescence measurements. Observation of excimer formation/dissociation further conveyed information on the physicochemical properties of the core. Thermal characterization of these systems showed that an increase in the temperature resulted in changes in the properties of excimer fluorescence, an occurrence attributed to a higher mobility of the otherwise glassy PDLLA. This, in turn, greatly affected the inter-molecular distance between pyrene molecules, a crucial factor for excimer formation. The glass transition of the PDLLA block, approximately 38 degrees C, defined the onset for increasing chain mobility and whence excimer dissociation. Excimer fluorescence appeared to be time-dependent. Based on these observations, chain exchange processes were clearly evidenced through the time-dependent dissociation of excimers into unimers, a process that was influenced by changes in temperature.
Lactates↗
Two-dimensional multiarray formation of hepatocyte spheroids on a microfabricated PEG-brush surface.
A two-dimensional microarray of ten thousand (100 x 100) hepatocyte heterospheroids, underlaid with endothelial cells, was successfully constructed with 100 microm spacing in an active area of 20 x 20 mm on microfabricated glass substrates that were coated with poly(ethylene glycol) brushes. Cocultivation of hepatocytes with endothelial cells was essential to stabilize hepatocyte viability and liver-specific functions, allowing us to obtain hepatocyte spheroids with a diameter of 100 microm, functioning as a miniaturized liver to secret albumin for at least one month. The most important feature of this study is that these substrates are defined to provide an unprecedented control of substrate properties for modulating cell behavior, employing both surface engineering and synthetic polymer chemistry. The spheroid array constructed here is highly useful as a platform of tissue and cell-based biosensors and detects a wide variety of clinically, pharmacologically, and toxicologically active compounds through a cellular physiological response.
Animals↗
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↗
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↗
Preparation of functionally Pegylated gold nanoparticles with narrow distribution through autoreduction of auric cation by alpha-biotinyl-PEG-block-[poly(2- (N,N-dimethylamino)ethyl methacrylate)].
PEGylated gold nanoparticles with biotin moieties installed at the distal end of the PEG tethered chains were prepared by the autoreduction of HAuCl4 catalyzed by alpha-biotinyl-PEG-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (biotinyl-PEG/PAMA) in aqueous medium at room temperature. The size of the gold nanoparticles was controllable in a range of 6-13 nm by changing the initial Au3+/polymer ratio, while retaining their narrow size distribution. The dispersion stability of the nanoparticles in aqueous medium was extremely high even under the condition of salt concentration as high as I = 2.0. Biotinyl-PEG/PAMA-anchored gold nanoparticles underwent specific aggregation in the presence of streptavidin, revealing their promising utility as colloidal sensing systems applicable under biological condition.
Letter↗
Synthesis and evaluation of the functional oligonucleotides-PEG conjugates.
We have previously demonstrated that the phenylsulfide and phenylsulfoxide derivatives of 2-amino-6-vinylpurine exhibit efficient cross-linking with high selectivity towards cytidine through synchronous activation within duplex. In this study, we synthesized their conjugates with PEG for possible application to in vitro as well as in vivo antisense agents. Thus, the ODN (2) with the antisense sequence toward luciferase was synthesized, having the 2-amino-6-vinylpurine nucleoside analog at the reactive site for targeting C and the amino linker at the 3' terminal. The vinyl group was transformed to the corresponding substituted phenylsulfide derivatives (3), and then the amino group of the 3' end was converted to the thiopropionyl group (4). The thiol nucleophile of 4 was reacted to the acid-responsive PEG2 in good isolated yields (55-62%).
Chromatography, High Pressure Liquid↗
A core-shell-type fluorescent nanosphere possessing reactive poly(ethylene glycol) tethered chains on the surface for zeptomole detection of protein in time-resolved fluorometric immunoassay.
To increase the sensitivity and to depress the nonspecific binding in biochemical assays, a new core-shell-type fluorescent nanosphere (106.7 nm) covalently conjugated with antibody was prepared. The core-shell-type nanosphere was constructed by dispersion radical polymerization of styrene in the presence of heterotelechelic poly(ethylene glycol) (PEG) macromonomer, which has a polymerizable vinylbenzyl group at one end and a primary amino group at the other chain end and used as well as a surfactant. The resulting nanosphere had PEG tethered chains on the surface, which possesses a primary amino group at the distal end of the PEG chain (NH(2) nanosphere). The fluorescent NH(2) nanosphere was constructed by incorporating fluorescent europium chelates with beta-diketonate ligands in the core of the NH(2) nanosphere by means of a physical entrapment method. The primary amino groups on the fluorescent NH(2) nanosphere were then converted to maleimide groups using a hetero cross-linker. The resulting nanosphere had maleimide groups on the surface (maleimide nanosphere), onto which proteins having SH group in the molecule could be covalently conjugated quantitatively without any denaturation of the proteins under the milder reaction condition. The applicability of the fluorescent nanosphere was tested in a model sandwich immunoassay for alpha-fetoprotein (AFP) determination. Anti-human AFP Fab' fragment was covalently conjugated onto the maleimide nanosphere (Fab' nanosphere), and it was used for the solid-phase time-resolved fluorometric immunoassay of AFP. The detection limit (mean + 2 SD) was 0.040 pg/mL or 57.1 zmol (57.1 x 10(-)(21) mol, M(w,AFP) = 70000) for AFP. The imprecision (concentration CV) over the whole assay range was 1.1% (100 pg/mL) - 17.1% (0.1 pg/mL), even though with this conjugation of antibody to the nanosphere, the nonspecific binding was practically negligible (0.0008%) and even when approximately 1.9 x 10(9) particles of the Fab' nanosphere were applied to the microtitration well.
Ethylene Glycol↗
PEGylated nanoparticles for biological and pharmaceutical applications.
The utility of polymeric micelles formed through the multimolecular assembly of block copolymer was comprehensively described as novel core-shell typed colloidal carriers for drug and gene targeting. Particularly, novel approaches for the formation of functionalized poly(ethylene glycol) (PEG) layers as hydrophilic outer shell were focused to attain receptor-mediated drug and gene delivery through PEG-conjugated ligands with a minimal non-specific interaction with other proteins. Surface organization of block copolymer micelles with cross-linking core was also described from a standpoint of the preparation of a new functional surface-coating with a unique macromolecular architecture. The micelle-attached surface and the thin hydrogel layer made by layered micelles exhibited nonfouling properties and worked as the reservoir for hydrophobic reagents. Furthermore, the potential utility of multimolecular assembly derived from heterobifunctional PEGs and block copolymers were explored to systematically modify the properties of metal and semiconductor nanostructures by controlling their structure and their surface properties, making them extremely attractive for use in biological and biomedical applications.
Animals↗
Temperature-related change in the properties relevant to drug delivery of poly(ethylene glycol)-poly(D,L-lactide) block copolymer micelles in aqueous milieu.
The block copolymers of poly(ethylene glycol) and poly(D,L-lactide) (PEG-PDLLA), the latter having a glass transition temperature (T(g)) around the physiological condition, was self-assembled into polymer micelles with a narrow and unimodal distribution in aqueous milieu either by dialysis or by the ultrasonication-aided dispersion method. The 1H NMR measurement of the PEG-PDLLA micelles in D(2)O revealed a gradual increase in the chain mobility of PDLLA segment in the core of the micelles at a temperature range above the T(g) of PDLLA. The critical association concentration (c.a.c.) of the PEG-PDLLA micelles was determined at various temperatures (25-55 degrees C) using pyrene as a probe to monitor the change in the polarity of the microenvironment in the micelle. An Arrhenius plot of the c.a.c. (ln(c.a.c.) versus 1/T) exhibited a break near T(g) of PDLLA. In sharp contrast with the linear decrease in ln(c.a.c.) versus 1/T in the region above the T(g), there was observed an almost constant c.a.c. (7-8 mg/l) regardless of the temperature change below the T(g). Furthermore, the chain exchange reaction between micelles was investigated based on the migration of the end-tagged block copolymers (alpha-lactosyl-PEG-PDLLA and omega-pyrenyl-PEG-PDLLA). The change in the binding affinity of the fluorescent micelles toward the RCA-1 lectin immobilized column was monitored with time to estimate the chain exchange. Consequently, appreciable acceleration in the chain exchange rate was revealed by increasing the surrounding temperature indicating the core mobility to be a substantial factor for inter-micellar chain migration. These results indicate that the engineering of the thermal characteristics of the core-forming segment of the block copolymer should be one of the crucial factors for optimizing the properties of the polymer micelles used for drug delivery.
Deuterium Oxide↗