Magnesium-doped zinc oxide electrochemically grown on fluorine-doped tin oxide substrate.

Nanostructures of magnesium (Mg) doped Zinc oxide (ZnO) were successfully deposited on conducting fluorine-doped tin oxide (FTO) coated glass plates by cathodic electrochemical deposition method at different potentials and temperature conditions. The deposited samples were characterized by XRD and SEM techniques to confirm their structures, morphologies and optical properties. These measurements show that Mg doped ZnO has a wurtzite structure and that the strongest intensity of the (002) peak is found at 60 degrees C and -1.0 V. Tunable transmittance of Mg doped ZnO has a band gap energy from 3.45 eV to 3.82 eV, which is the direct evidence of doping.

Establishment and characterization of embryonic stem-like cells from porcine somatic cell nuclear transfer blastocysts.

This study was aimed to establish embryonic stem (ES)-like cells from blastocysts derived from somatic cell nuclear transfer (SCNT) in pig. Somatic cells isolated from both day-30 fetus and neonatal cloned piglet were used for donor cells. A total of 60 blastocysts (46 and 14 derived from fetal and neonatal fibroblast donor cells, respectively) were seeded onto a mitotically inactive mouse embryonic fibroblast (MEF) monolayer and two ES-like cell lines, one from each donor cell type, were established. They remained undifferentiated over more than 52 (fetal fibroblast-derived) and 48 (neonatal fibroblast-derived) passages, while retaining alkaline phosphatase activity and reactivity with ES specific markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-4, TRA-1-60 and TRA-1-81. These ES-like cells maintained normal diploid karyotype throughout subculture and successfully differentiated into embryoid bodies that expressed three germ layer-specific genes (ectoderm: beta-III tubulin; endoderm: amylase; and mesoderm: enolase) after culture in leukemia inhibitory factor-free medium. Microsatellite analysis confirmed that they were genetically identical to its donor cells. Combined with gene targeting, our results may contribute to developing an efficient method for producing transgenic pigs for various purposes.

Demetylation of the sex-determining region Y gene promoter and incidence of disorder of sex development in cloned dog males.

Canine cloning is occasionally accompanied by abnormal sexual development. Some male donor cells produce cloned pups with female external genitalia and complete male gonadal dysgenesis, which is classified as an XY disorder of sex development (XY DSD). In this study, we examine the potential of 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to reduce the phenotypic abnormality XY DSD in somatic cell nuclear transfer (SCNT)- derived pups. We used a 9-year-old normal male German Shepherd dog as a cell donor. Donor cells were treated with 10 nM 5-aza-dC for 4 days before being used for SCNT. At the same stage of cell development, significantly lower levels of DNA methylation of the sex-determining region Y (SRY) promoter was observed in the treated donor cells compared to that in the untreated cells (95.2% versus 53.3% on day 4 for the control and treated groups, respectively). No significant differences were observed in the control or treatment groups concerning fusion rate, pregnancy rate (30 days or entire period), the number of pups, or the incidence of XY DSD. However, more XY DSD dogs were observed in the control group (31.25%) than in the treatment group (14.29%). Hypermethylation of the SRY promoter was observed in the XY DSD cloned pups in both the treatment (84.8%) and control groups (91.1 ± 1.4%) compared to the methylation level in the phenotypically normal male pups of the treatment (23.2 ± 20.9%) and control groups (39.1 ± 20.1%). These results suggest that 5-aza-dC treatment of donor cells can reduce the methylation level of the SRY promoter in donor cells, and thus, 5-aza-dC is advantageous for reducing the incidence of XY DSD in canine cloning.

Apicidin, the histone deacetylase inhibitor, suppresses Th1 polarization of murine bone marrow-derived dendritic cells.

Apicidin is a fungal metabolite shown to exhibit anti-proliferative, anti-invasive, and anti-inflammatory properties by the inhibition of histone deacetylase (HDAC). However, the effects of apicidin on the maturation and immunostimulatory function of dendritic cells (DCs) remain unknown. In this study, we investigated whether apicidin modulates surface molecule expression, cytokine production, endocytosis capacity, and underlying signaling pathways in murine bone marrow-derived DCs. We observed that apicidin significantly attenuated surface molecule expression in LPS-stimulated DCs, suppressed production of interleukin (IL)-12 and proinflammatory cytokines (IL-6 and TNF-alpha) by DCs, and reduced IFN-gama production by T cells. The apicidin-treated DCs were found to be highly efficient in antigen capture via mannose receptor-mediated endocytosis. Apicidin also inhibited LPS-induced MAPK activation and NF-kB nuclear translocation in DCs. Moreover, the apicidin-treated DCs were incapable of inducing Th1 responses and normal cell-mediated immune responses. These novel findings not only provide new insights into the immunopharmacological role of apicidin in terms of its effects on DCs, but also broaden current perspectives of the immunopharmacological functions of apicidin, and have implications for the development of therapeutic adjuvants for the treatment of DC-related acute and chronic diseases.

Simple preparation of nanoparticles coated with carbohydrate-carrying polymers.

Nanoparticles bearing carbohydrate chains on the surface can be prepared by the simple diafiltration method. The nanoparticles prepared by the present method displayed high yield, no-aggregation formation, small size, narrow size distribution, and one-step procedure. Also, the high density carbohydrate chains on the particles can be recognized by liver cells.

Evaluation of an intestinal pressure-controlled colon delivery capsules prepared by a dipping method.

A new method for preparation of large amounts of empty pressure-controlled colon delivery capsules (PCDCs) by a dipping method has been developed. Empty PCDCs are composed of two polymer membranes. The inner one was a water-insoluble polymer membrane, ethylcellulose (EC). The outer one was an enteric polymer membrane, hydroxypropylmethylcellulose phthalate (HPMCP) or hydroxypropylmethylcellulose acetate succinate (HPMCAS). By consequently dipping into an ethanolic EC solution and an alkalized enteric polymer solution, empty PCDCs were obtained after both the capsule body and cap were adjusted to the size of #2 capsules. With each enteric polymer, two types of empty PCDCs of different thickness were prepared. Fluorescein (FL) was formulated with suppository base, PEG1000, and used as a model drug. FL/PEG1000 suspension was introduced into empty PCDCs which were then sealed with enteric polymer solution. The PCDCs were evaluated by an in vivo experiment using beagle dogs. After oral administration of the test PCDC preparations containing 30 mg of FL, blood samples were obtained from the jugular vein and serum FL levels were measured. The thickness of the EC membrane layer varied in both the capsule body and cap. HPMCAS PCDCs had 62.1+/-5.0 (S.E.) microm (body) and 49.7+/-3.3 microm (cap) with thicker ones and 55.7+/-6.6 microm (body) and 46.8+/-6.2 microm (cap) with thinner ones. HPMCP PCDCs had 28.1+/-3.3 microm (body), 30.9+/-1.0 microm (cap) with thinner ones and 43.1+/-9.8 microm (body), 42.4+/-8.2 microm (cap) with thicker ones. The mean T(i) values, the first appearance time, of FL in the serum of HPMCAS PCDCs were 2.0+/-0.7 h for thicker ones and 3.8+/-0.5 h for thinner ones, while the mean T(i) values of HPMCP PCDCs were 2.0+/-0.0 h for thinner ones and 3.5+/-0.7 h for thicker ones. Since the colon arrival time in beagle dogs was 3.5+/-0.3 h as determined by a sulfasalazine test, thinner HPMCAS PCDCs and thicker HPMCP PCDCs were thought to deliver FL to the colon.

Clonazepam release from core-shell type nanoparticles in vitro.

Block copolymers consisting of poly(gamma-benzyl L-glutamate) (PBLG) as the hydrophobic block and poly(ethylene oxide) (PEO) as the hydrophilic block were synthesized and characterized. Core-shell type nanoparticles of the block copolymers (abbreviated as GE) were prepared by the diafiltration method. The particle size diameter obtained by dynamic light scattering of GE-1 (PBLG content: 60.5 mol%), GE-2 (PBLG content: 40.0 mol %), GE-3 (PBLG content: 124.4 mol %) copolymer was 309.9 +/- 160.9, 251.9 +/- 220.6 and 200.5 +/- 177.1nm, respectively. The shape of the nanoparticles by SEM or TEM was almost spherical. The critical micelle concentration of the block copolymers obtained by fluorescence spectroscopy was dependent on the chain length of hydrophobic PBLG. The micelle structure of the copolymers nanoparticle was very stable against sodium dodecyl sulfate. Clonazepam (CZ) was loaded onto the core part of the nanoparticle as the crystalline state. Release of CZ from the nanoparticles in vitro was dependent on the drug loading contents and PBLG chain length.

Protection against dextran sulfate sodium-induced colitis by microspheres of ellagic acid in rats.

Ellagic acid (EA), a naturally occurring plant phenol, has the antioxidant and anti-inflammatory activities. In the present study, we examined the effect of EA contained in microspheres on the ulcerative colitis induced experimentally in rats by dextran sulfate sodium (DSS). Experimental colitis was induced in male Fisher 344 rats by daily treatment with 3% DSS solution in drinking water for 7 days. EA of microspheres (mcEA: 1 approximately 10 mg/kg as EA contents) was administered p.o. twice daily for 6 days. In a preliminary study, we found that these microsphere capsules, when administered p.o., are effectively dissolved in the proximal to the ileo-cecal junction and distributed to the terminal ileum and the colon. The ulceration area, colon length, and mucosal myeloperoxidase (MPO) activity as well as thiobarbituric acid-reactive substances (TBARS) were measured on 7th day after the onset of DSS treatment. The DSS treatment for 7 days caused severe mucosal lesions in the colon, accompanied with the increases of MPO activity and TBARS as well as the decreases of body weight gain and colon length. Administration of mcEA reduced the severity of DSS-induced colitis in a dose-dependent manner, and a significant effect was observed at 10 mg/kg, the ED50 being 2.3 mg/kg. This mcEA treatment also significantly mitigated changes in various biochemical parameters in the colonic mucosa induced by DSS. Although plain EA (without using microspheres) was also effective in reducing the severity of DSS-induced colitis, this effect was much less potent as compared with that of mcEA; the ED50 was about 15 times higher than that of mcEA. In addition, a significant effect on DSS-induced colitis was also obtained by intra-rectal administration of superoxide dismutase, an anti-oxidative agent. These results suggest that EA prevents the ulcerative colitis induced by DSS, probably by radical scavenging and/or anti-oxidative actions. The microspheres used in this study may be useful for delivering an orally administered drug specifically to the colon.

Self-assembled hydrogel nanoparticles composed of dextran and poly(ethylene glycol) macromer.

Biodegradable hydrogel nanoparticles were prepared from glycidyl methacrylate dextran (GMD) and dimethacrylate poly(ethylene glycol) (DMP). GMD was synthesized by coupling of glycidyl methacrylate to dextran in the presence of 4-(N,N-dimethylamino)pyridine (DMAP) using dimethylsulfoxide (DMSO) as an aprotic solvent. DMP was synthesized from poly(ethylene glycol) (PEG) and methacryloyl chloride. GMD/DMP (abbreviated as DP) hydrogel was prepared by radical polymerization of GMD and DMP using ammonium peroxydisulfate (APS) as an initiator and UV curing. DP hydrogel nanoparticles were obtained by diafiltration method using DMSO solution. The GMD and DMP were characterized by fourier transform infrared spectroscopy. Fluorescence probe technique was used to investigate the self-assembly of DP in water using pyrene as a hydrophobic probe. The critical association concentration (CAC) was determined to be 5.6 x 10(-2) g/l. The shape of DP hydrogel nanoparticles was spherical when observed by transmission electron microscope (TEM). The size range of DP hydrogel nanoparticles was about 20 approximately 50 nm. The hydrodynamic size of DP hydrogel nanoparticles was measured by photon correlation spectroscopy (PCS) and gradually increased with time in PBS (0.1 M, pH 7.4). Drug release study was performed using clonazepam (CNZ) as a hydrophobic model drug. In vitro release rate of CNZ from the DP hydrogel nanoparticles was dependent on the existence of dextranase and the pH of the release medium.

Thermo-responsive self-assembled polymeric micelles for drug delivery in vitro.

Cholic acid (CA) was coupled with amine-terminated poly (N-isopropylacrylamide) (ATPNIPAAm) using N,N'-dicyclohexyl carbodiimide as a coupling agent. Self-assembled CA/PNIPAAm conjugate (abbreviated as CN) micelles were prepared by diafiltration method in water. The CN micelles exhibited the lower critical solution temperature (LCST) at 31.5 degrees C. The CN micelles were observed as spherical shapes and their dried sizes were ranged between 30 and 50 nm by the transmission electron microscope (TEM) images. Hydrated micelle sizes measured by photon correlation spectroscopy (PCS) were ranged 337.5+/-67.8 nm. And reversible size changes of CN micelles were observed with two point temperature 10 and 40 degrees C, respectively. From the fluorescence spectra, fluorescence intensity of pyrene in the CN micelles was increased and red-shifted as the concentration of CN increased, indicating the formation of self-assembled polymeric micelles in water. The critical micelle concentration (CMC) was evaluated as 8.9 x 10(-2) g/l. Much more indomethacin (IN) was released from th CN micelles at 10 than at 40 degrees C due to the thermo-sensitivity of the PNIPAAm in the CN polymer.

Core-shell type polymeric nanoparticles composed of poly(L-lactic acid) and poly(N-isopropylacrylamide).

Poly(L-lactic acid)/poly(N-isopropylacrylamide) (abbreviated as LN) block copolymers were synthesized and the LN nanoparticles were prepared by simple diafiltration method. The thermal transition of the LN nanoparticles was at 32.3 degrees C, the lower critical solution temperature (LCST) of the polymer. The fluorescence spectroscopy data showed that LN was self-assembled in water to form core-shell structure nanoparticles, and the critical association concentration (CAC) value was estimated as 1.3x10(-2) g/l. From the transmission electron microscope observations, the LN nanoparticles were spherically shaped and ranged in size between 30 and 50 nm below the LCST. The hydrated size was measured by photon correlation spectroscopy, and reversible size changes were investigated by the factor of temperature. The release of indomethacin from the LN nanoparticles was thermo-sensitive due to the unique characteristic of poly(N-isopropylacrylamide).

Effect of solvent on the preparation of surfactant-free poly(DL-lactide-co-glycolide) nanoparticles and norfloxacin release characteristics.

The surfactant-free nanoparticles of poly(DL-lactide-co-glycolide) (PLGA) were prepared by dialysis method without surfactant and physicochemical properties such as particle size and drug contents were investigated against used initial solvent. The size of PLGA nanoparticles and drug contents were significantly changed by used initial solvent. The size of PLGA nanoparticles prepared from dimethylacetamide (DMAc), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) as a initial used solvent was smaller than that of acetone. Selected initial solvent used to dissolve the copolymer significantly affects the size of nanoparticles and drug contents. It was shown that PLGA nanoparticles have spherical shapes from the results of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations. It was thought that surfactant-free nanoparticles of PLGA entrapping norfloxacin (NFX) has nice drug loading capacity without free-drug on the surface of nanoparticles through the analysis of X-ray powder diffraction. From these results, it was showed the potential that the PLGA nanoparticles could be formed successively by dialysis method without surfactant. Release kinetics of NFX used as a model drug was governed by not only drug contents but also particle size parameter. The higher the drug contents and the larger the particle size resulted in slower the drug release.

Adriamycin release from flower-type polymeric micelle based on star-block copolymer composed of poly(gamma-benzyl L-glutamate) as the hydrophobic part and poly(ethylene oxide) as the hydrophilic part.

Star-block copolymer based on PBLG as the hydrophobic part and PEO as the hydrophilic one (as abbreviated GEG) was synthesized and characterized. Polymeric micelle was prepared by the diafiltration method. From the measurement of photon correlation spectroscopy, the nanoparticle sizes of GEG-1, GEG-2 and GEG-3 were 106.5+/-59.2, 43.8+/-0.7 and 13.5+/-1.0 nm in number average, respectively, indicating of the formation of polymeric micelle. Also, the nanoparticle sizes were dependent on the PBLG chain length, i.e. the more PBLG content in the copolymer, the larger the particle size. From the observation of transmission electron microscope(TEM), GEG-2 block copolymer had almost spherical shapes with size range about 20-70 nm, that was similar to particle size measurement. Fluorescence spectroscopy measurement indicated that GEG block copolymers associated in water to form polymeric micelles and critical micelle concentration (CMC) values of the block copolymers decreased with increasing PBLG chain length in the block copolymer. Characteristic peaks of the protons of the benzyl group in the PBLG and the methylene protons adjacent to the benzyl group of the PBLG segment in the GEG-2 nanoparticles appeared in 7.2 approximately 7.4 and 5.0 approximately 5.2 ppm, respectively, and disappeared in D(2)O, indicating the restricted motions of these protons within the micellar core and the very rigid structure of the PBLG core in the GEG polymeric micelles. Release of ADR from the polymeric micelles in vitro was slower in longer PBLG chain length and higher loading contents of ADR.

Clonazepam release from core-shell type nanoparticles of poly(epsilon-caprolactone)/poly(ethylene glycol)/poly(epsilon-caprolactone) triblock copolymers.

The triblock copolymer based on poly(epsilon-caprolactone) (PCL) as hydrophobic part and poly(ethylene glycol) (PEG) as hydrophilic one was synthesized and characterized. Core-shell type nanoparticles of poly(epsilon-caprolactone)/poly(ethylene glycol)/poly(epsilon-caprolactone) (CEC) block copolymer were prepared by a dialysis technique. According to the amphiphilic characters, CEC block copolymer can self-associate at certain concentration and their critical association concentration (CAC) was determined by fluorescence probe technique. CAC value of the CEC-2 block copolymer was evaluated as 0.0030 g/l. CAC values of CEC block copolymer decreased with the increase of PCL chain length, i.e. the shorter the PCL chain length, the higher the CAC values. From the observation of transmission electron microscopy (TEM), the morphologies of CEC-2 core-shell type nanoparticles were spherical shapes. Particle size of CEC-2 nanoparticles was 32.3+/-17.3 nm as a monomodal and narrow distribution. Particle size, drug loading, and drug release rate of CEC-2 nanoparticles were changed by the initial solvents and the molecular weight of CEC. The degradation behavior of CEC-2 nanoparticles was observed by 1H NMR spectroscopy. It was suggested that clonazepam (CNZ) release kinetics were dominantly governed by diffusion mechanism.

Application of Eudragit P-4135F for the delivery of ellagic acid to the rat lower small intestine.

Based on the assumption that the delivery of ellagic acid to its site of action would show an antiinflammatory activity in inflammatory bowel disease (IBD), we have prepared microspheres using a new pH-sensitive polymer, Eudragit P-4135F (P-4135F), to deliver ellagic acid to the lower small intestine in rats. The microspheres were spherical in shape and the mean diameters were approximately 100-150 microm. The amount of ellagic acid released from the microspheres decreased by increasing the formulated amount of P-4135F. The release characteristics of ellagic acid were pH-dependent. By considering the factors loading efficiency and microsphere particle size distribution, ellagic acid-2 microspheres (P-4135F/ellagic acid = 1.65) were selected for further investigation. In a dissolution study, more than 95% ellagic acid was released within 0.5 h in pH 7.4 and 8.0 buffers. The release percent of ellagic acid was less than 40% in pH 6.8 and 7.0 and was less than 10% in pH 5.6 and 5.9. To observe the dissolution sites of the microspheres in the rat small intestine fluorescein was formulated in the microspheres as a tracer drug along with ellagic acid (50 mg kg(-1)). After intraduodenal administration of fluorescein-labelled microspheres to rats, the plasma fluorescein level started to increase at 0.5 h, by which time the microspheres had reached the middle part of the ileum. Microspheres started to dissolve within 1.0 h and the peak plasma fluorescein concentration was observed at 3.0 h, when the majority of the administered microspheres were dissolved in the terminal ileum. These results suggested that P-4135F microspheres could deliver ellagic acid to the lower part of the small intestine, and that the released ellagic acid would be distributed into the caecum and the ascending colon.

Drug release from thermo-responsive self-assembled polymeric micelles composed of cholic acid and poly(N-isopropylacrylamide).

Cholic acid, conjugated with amine-terminated poly(N-isopropylacrylamide) (abbreviated as CA/ATPNIPAAm), was synthesized by a N, N'-dicyclohexyl carbodiimide (DCC)-mediated coupling reaction. Self-assembled CA/ATPNIPAAm micelles were prepared by a diafiltration method in aqueous media. The CA/ATPNIPAAm micelles exhibited a lower critical solution temperature (LCST) at 31.5 degrees C. Micelle sizes measured by photon correlation spectroscopy (PCS) were approximately 31.6+/-5.8 nm. The CA/ATPNIPAAm micelles were spherical and their thermal size transition was observed by transmission electron microscope (TEM). A fluorescence probe technique was used for determining the micelle formation behavior of CA/ATPNIPAAm in aqueous solutions using pyrene as a hydrophobic probe. The critical micelle concentration (CMC) was evaluated as 8.9 x 10(-2) g/L. A drug release study was performed using indomethacin (IN) as a hydrophobic model drug. The release kinetics of IN from the CA/ATPNIPAAm micelles revealed a thermo-sensitivity by the unique character of poly(N-isopropylacrylamide) i.e. the release rate was higher at 25 degrees C than at 37 degrees C.

Clonazepam release from core-shell type nanoparticlesin vitro.

AB-type amphiphilic copolymers (abbreviated as LE) composed of poly (L-leucine) (PLL) as the A component and poly (ethylene oxide) (PEO) as the B component were synthesized by the ring-opening polymerization of L-leucine N-carboxy-anhydride initiated by methoxy polyoxyethylene amine (Me-PEO-NH(2)) and characterized. Core-shell type nanoparticles were prepared by the diafiltration method. Particle size distribution obtained by dynamic light scattering was dependent on PLL composition and the size for LE-1, LE-2 and LE-3 was 369.6+/-267, 523.4+/-410 and 561.2+/-364 nm, respectively. Shapes of the nanoparticles observed by transmission electron microscope (TEM) were almostly spherical. The critical micelle concentration (CMC) of the nanoparticles determined by a fluorescence probe technique was dependent on the composition of hydrophobic PLL, and the CMC for LE-1, LE-2 and LE-3 was 2. 0x10(-6), 1.7x10(-6) and 1.5x10(-6) (mol/l), respectively. Clonazepam release from core-shell type nanoparticles in vitro was dependent on PLL composition and drug loading content.

Albumin release from biodegradable hydrogels composed of dextran and poly(ethylene glycol) macromer.

Biodegradable hydrogels based on glycidyl methacrylate dextran (GMD) and dimethacrylate poly(ethylene glycol) (DMP) were proposed for colon-specific drug delivery. GMD was synthesized by coupling of glycidyl methacrylate with dextran in the presence of 4-(N,N-dimethyl-amino)pyridine (DMAP) using dimethylsulfoxide as a solvent. Methacrylate-terminated poly (ethylene glycol) (PEG) macromer was prepared by the reaction of PEG with methacryloyl chloride. GMD/DMP hydrogels were prepared by radical polymerization of phosphate buffer solution (0.1M, pH 7.4) of GMD and DMP, using ammonium peroxydisulfate (APS) and UV as initiating system. The synthetic GMD, DMP, and GMD/DMP hydrogels were characterized by fourier transform infrared (FT-IR) spectroscopy. The FITC-albumin loaded hydrogels were prepared by adding FITC-albumin solution before UV irradiation. Swelling capacity of GMD/DMP hydrogels was controlled not only by molecular weight of dextran, but also by incorporation ratio of DMP Degradation of the hydrogels has been studied in vitro with dextranase. FITC-albumin release from the GMD/DMP hydrogels was affected by molecular weight of dextran and the presence of dextranase in the release medium.

Clonazepam release from poly(DL-lactide-co-glycolide) nanoparticles prepared by dialysis method.

Aim of this work is to prepare poly(DL-lactide-co-glycolide) (PLGA) nanoparticles by dialysis method without surfactant and to investigate drug loading capacity and drug release. The size of PLGA nanoparticles was 269.9 +/- 118.7 nm in intensity average and the morphology of PLGA nanoparticles was spherical shape from the observation of SEM and TEM. In the effect of drug loading contents on the particle size distribution, PLGA nanoparticles were monomodal pattern with narrow size distribution in the empty and lower drug loading nanoparticles whereas bi- or trimodal pattern was showed in the higher drug loading ones. Release of clonazepam from PLGA nanoparticles with higher drug loading contents was slower than that with lower loading contents.