DNA aqueous solution used for dialytical removal and enrichment of dioxin derivatives

In the present study, a dialytic method that uses a DNA aqueous solution to remove and enrich dioxins from polluted water was proposed. Circular dichroism (CD) and fluorescent spectra indicated that dibenzo-p-dioxin (DD), dibenzofuran (DF) and biphenyl (BP), which are dioxin derivatives, form complexes with DNA. Their experimental dialytic sorption coefficients were measured by quantifying the concentrations of DD, DF, and BP in aqueous solutions before and after dialysis of the DNA solution, and the values were 2.1×10⁵, 1.3×10⁵, and 1.5×10⁷, respectively. As a simulated water treatment model, DNA solution was dialyzed in an aqueous mixture of DD, DF, and BP for 96 h, the HPLC studies showed that the dioxin derivatives have been concentrated in the DNA solution about 200 times. The dialyzed DNA solution was reusable by an extraction with hexane.     

Fabrication of hyaluronic acid hydrogel beads for cell encapsulation

Hyaluronic acid (HA) hydrogel beads were prepared by photopolymerization of methacrylated HA and N-vinylpyrrolidone using alginate as a temporal spherical mold. Various fabrication conditions for preparing the hydrogel beads, such as the concentration of methacrylated HA and UV irradiation time, were optimized to control swelling properties and enzymatic degradability. A new concept for cell encapsulation is proposed in this paper. Viable cells were directly injected into the hydrogel beads using a microinjection technique. When bovine articular chondrocytes were injected into HA hydrogel beads and cultivated for 1 week, the cells could proliferate well within the HA beads. HA hydrogel beads could be potentially used as injectable cell delivery vehicles for regenerating tissue defects.     

Bacterial cellulose-chitosan composite hydrogel beads for enzyme immobilization

In this work, we report the preparation of bacterial cellulose (BC)-chitosan composite hydrogel beads by co-dissolution of BC and chitosan in 1-ethyl-3-methylimidazolium acetate and subsequent reconstitution with distilled water. The BC-chitosan hydrogel beads were used as enzyme supports for immobilizing Candida rugosa lipase by physical adsorption and covalent cross-linking. BC-chitosan hydrogel beads immobilized lipase more efficiently than microcrystalline cellulose (MCC)-chitosan hydrogel beads. The amount of protein adsorbed onto BCchitosan beads was 3.9 times higher than that adsorbed onto MCC-chitosan beads, and the catalytic activity of lipase was 1.9 times higher on the BC-chitosan beads. The lipase showed the highest thermal and operational stability when covalently cross-linked on BC-chitosan hydrogel beads. The half-life time of the lipase cross-linked on BC-chitosan bead at 60°C was 22.7 times higher than that of free lipase. Owing to their inherent biocompatibility and biodegradability, the BC-chitosan composite hydrogel beads described here could be used to immobilize proteins for various biomedical, environmental, and biocatalytic applications.     

Design and development of hydrogel beads for targeted drug delivery to the colon

The purpose of this research was to develop and evaluate a multiparticulate system of chitosan hydrogel beads exploiting pH-sensitive property and specific biodegradability for colon-targeted delivery of satranidazole. Chitosan hydrogel beads were prepared by the cross-linking method followed by enteric coating with Eudragit S100. All formulations were evaluated for particle size, encapsulation efficiency, swellability, and in vitro drug release. The size of the beads was found to range from 1.04±0.82 mm to 1.95±0.05 mm. The amount of the drug released after 24 hours from the formulation was found to be 97.67%±1.25% in the presence of extracellular enzymes as compared with 64.71%±1.91% and 96.52%±1.81% release of drug after 3 and 6 days of enzyme induction, respectively, in the presence of 4% cecal content. Degradation of the chitosan hydrogel beads in the presence of extracellular enzymes as compared with rat cecal and colonic enzymes indicates the potential of this multiparticulate system to serve as a carrier to deliver macromolecules specifically to the colon and can be offered as a substitute in vitro system for performing degradation studies. Studies demonstrated that orally administered chitosan hydrogel beads can be used effectively for the delivery of drug to the colon. Published: July 13, 2007     

DNA-cyclodextrin composite material for environmental applications

Recently, we prepared water-insoluble and nuclease-resistant DNA films by UV irradiation. The DNA films accumulated the harmful DNA-intercalating compounds, such as ethidium bromide and acridine orange. Additionally, the water-insoluble DNA films bound endocrine disruptors that had planar structures, such as dioxin derivatives, polychlorobiphenyl (PCB) derivatives, and benzo[a]pyrene. However, other harmful endocrine disruptors that lack a planar structure, such as bisphenol A and diethylstilbestrol, did not bind the water-insoluble DNA films. Therefore, we prepared the DNA-cyclodextrin composite material by mixing the DNA and the cyclodextrin-immobilized poly(allylamine) (PCD). As a result, these composite materials had properties of both the double-stranded DNA, such as intercalation, and the cyclodextrin, such as encapsulation of an organic molecule into the intramolecular cavity. Therefore, these materials could accumulate not only the harmful compounds with planar structures but also the nonplanar molecules, such as bisphenol A, diethylstilbestrol, and nonylphenol. These DNA-PCD composite materials may have the potential to be novel environmental materials that absorb harmful compounds.     

Simple and high-sensitivity detection of dioxin using dioxin-binding pentapeptide

The purposes of this study are to construct a simple dioxin detection system using peptides that bind to dioxin, and to test the system on real environmental samples. In this method, dioxin and N-NBD-3-(3',4'-dichlorophenoxy)-1-propylamine (NBD-DCPPA) are competitively bound to the peptides synthesized on beads. The fluorescence intensity of the bead decreases with increasing dioxin concentration. The concentration of dioxin is determined by measuring the fluorescence intensity using a fluorescence microscope equipped with a CCD camera. The fluorescence microscope system was equipped with a motor-driven stage and could be used with 96-well microplates and analytical software that automatically measured the fluorescence intensity of the bead images in the wells. Dioxin detection conditions, reaction temperature, number of beads and concentration of the organic solvent were optimized. About 0.5 nM (150 pg mL(-1)) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could be detected under the optimized conditions. Environmental soil samples were subjected to the detection system using the peptide beads. Although the results obtained correlated poorly with the toxicity equivalency quantity (TEQ) concentration obtained by a GC/MS method, our method is robust enough as a prescreening method to detect at least 250 pg-TEQ g(-1), the survey level for soil as stipulated in the law concerning special measures against dioxins in Japan.     

Dielectric properties of alginate beads and bound water relaxation studied by electrorotation

The electrical and dielectric properties of Ba²⁺ and Ca²⁺ cross‐linked alginate hydrogel beads were studied by means of single‐particle electrorotation. The use of microstructured electrodes allowed the measurements to be performed over a wide range of medium conductivity from about 5 mS/m to 1 S/m. Within a conductivity range, the beads exhibited measurable electrorotation response at frequencies above 0.2 MHz with two well‐resolved co‐ and antifield peaks. With increasing medium conductivity, both peaks shifted toward higher frequency and their magnitudes decreased greatly. The results were analyzed using various dielectric models that consider the beads as homogeneous spheres with conductive loss and allow the complex rotational behavior of beads to be explained in terms of conductivity and permittivity of the hydrogel. The rotation spectra could be fitted very accurately by assuming (a) a linear relationship between the internal hydrogel conductivity and the medium conductivity, and (b) a broad internal dispersion of the hydrogel centered between 20 and 40 MHz. We attribute this dispersion to the relaxation of water bound to the polysaccharide matrix of the beads. The dielectric characterization of alginate hydrogels is of enormous interest for biotechnology and medicine, where alginate beads are widely used for immobilization of cells and enzymes, for drug delivery, and as microcarriers for cell cultivation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 227–237, 1999     

Photo-cross-linked hydrogels with polysaccharide-poly(amino acid) structure: new biomaterials for pharmaceutical applications

The aim of this work has been the preparation and characterization of novel hydrogels with polysaccharide-poly(amino acid) structure having suitable physicochemical properties for pharmaceutical applications. In the first step, hyaluronic acid (HA) and alpha,beta-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA) have been derivatized with methacrylic anhydride (AMA), thus obtaining HA-AMA and PHM derivatives, respectively. In the second step, aqueous solutions of both these derivatives have been irradiated at 313 nm to obtain chemical hydrogels. The hydrogel obtained by irradiating for 15 min an aqueous solution containing 4% w/v of HA-AMA and 4% w/v of PHM resulted in the highest yield. Its swelling ability was dependent on the pH and nature of the external medium. Besides, this hydrogel undergoes a partial hydrolysis, especially in the presence of enzymes, such as esterase or hyaluronidase, but the entity of this degradation is lower than that observed for a hydrogel based on HA-AMA alone. The ability of this hydrogel to entrap drug molecules has been evaluated by using thrombin as a model drug. In vitro release studies and a platelet aggregation test demonstrated that the HA-AMA/PHM hydrogel is able to release thrombin in the active form, thus suggesting its suitability for the treatment of hemorrhages.     

Detection of Toxin-Producing Cyanobacteria by Use of Paramagnetic Beads for Cell Concentration and DNA Purification

Early detection of water blooms caused by potential toxin-producing cyanobacteria is important in environmental monitoring. We present a new nucleic acid-based method for detection of cyanobacteria in water that utilizes the same paramagnetic solid phase (beads) for both bacterial cell concentration and subsequent DNA purification. In the cell concentration step, the beads were attracted to a magnet after cell adsorption (in an alcohol- and salt-containing solution), and the supernatant was removed. For DNA purification, a buffer containing guanidine thiocyanate and Sarkosyl lysed the concentrated cells. The addition of alcohol precipitated the released DNA onto the same solid phase as was used for the cell concentration. Finally, to remove PCR inhibitors, the DNA was washed twice in alcohol while bound to the beads. All of the bead-DNA complex was used in the subsequent PCR amplification. The detection limit, as measured by 16S rDNA PCR amplification, was 50 cells in a 0.5-ml water sample, which is considerably lower than the limit (500 cells/ml) of toxic cyanobacteria tolerated in drinking water (New South Wales Blue-Green Algae Task Force, 1992). Testing of water from natural habitats showed a detection limit in the same range as that for the defined samples. The detection limits and the simplicity of the method (paramagnetic beads can be handled in automated systems) suggest that our method is suitable for routine environmental monitoring.     

Engineering lipobeads: properties of the hydrogel core and the lipid bilayer shell

We previously reported on a novel system termed Lipobead that consists of hydrogel beads encased within an anchored lipid bilayer. The hydrogel particles are formed by inverse suspension polymerization of dimethylacrylamide with N,N'-ethylenebis(acrylamide). During the polymerization stage, the water in oil emulsion is interfacially stabilized by small molecule surfactants as well as a small percentage of lipid functionalized with a vinyl group. The functionalized lipid becomes tethered to the bead surface and promotes the assembly of a lipid bilayer on the surface of the hydrogel beads. The presence of the functionalized lipid during polymerization dramatically alters the yield, average size, and size distribution of beads produced. This paper examines the effect of various chemical and physical processing parameters on the average size and size distribution of beads produced when lipid is a component of the surfactant mixture. Relationships between the processing parameters, average bead size, and size distribution were established. Macroscopic properties of the lipid bilayers of Lipobeads were also evaluated including phase transition temperature as well as permeability to the small polar molecule, adenosine triphosphate. It was established that the presence of functionalized lipid improves the organization of the bilayer on the Lipobead surface.     

Injectable and in situ gelling hydrogels for modified protein release

A novel injectable polysaccharide system based on calcium Alginate (Ca-Alg) hydrogel and two Dextran methacrylate derivatives (DexMA) was recently developed. The resulting Interpenetrating Polymer Network showed a synergistic mechanical behavior that can be exploited to target the hydrogel properties towards specific biomedical needs. In the present paper, hydrogels composed of 3% (w/v) Ca-Alg and Dextran (Mw 40 × 10⁴ and 500 × 10⁴), derivatized with methacrylic groups (derivatization degrees 5 and 30%) at concentrations 5% (w/v), were characterized. The data reported here evidenced that Mw and derivatization degree of Dex chains can deeply affect the mechanical as well a model protein (Horseradish peroxidase) delivery rate. The enzymatic activity of such model protein was never significantly altered by the adopted experimental conditions.     

Entrapment of microbial cells within polyurethane hydrogel beads with the advantage of low toxicity

Summary A new method is described for the entrapment of microbial cells in polyurethane (PUR) hydrogel beads. This hydrogel is produced from a hydrophilic pre-polymer blocked with bisulphite by adjusting the pH between 4 and 6.5. Bisulphite-blocked isocyanate has a substantially lower toxicity against living cells than unblocked (conventional) isocyanates. The poly(carbamoylsulfonate) (PCS) hydrogels have optimal elastic properties and therefore can be used for a matrix of biocatalysts in an agitated reactor as well as in a fluid-bed reactor. The results of ethanol fermentation ofSaccharomyces cerevisiae entrapped in PCS hydrogel beads, and of the denitrification activity of immobilizedParacoccus denitrificans are promising. In contrast, entrapped cells in conventional PUR hydrogels didn’t show any activity.     

Role of calcium alginate and mannitol in protecting bifidobacterium

Fourier transform infrared (FTIR) spectroscopy was carried out to ascertain the mechanism of Ca-alginate and mannitol protection of cell envelope components and secondary proteins of Bifidobacterium animalis subsp. lactis Bb12 after freeze-drying and after 10 weeks of storage at room temperature (25°C) at low water activities (a(w)) of 0.07, 0.1, and 0.2. Preparation of Ca-alginate and Ca-alginate-mannitol as microencapsulants was carried out by dropping an alginate or alginate-mannitol emulsion containing bacteria using a burette into CaCl(2) solution to obtain Ca-alginate beads and Ca-alginate-mannitol beads, respectively. The wet beads were then freeze-dried. The a(w) of freeze-dried beads was then adjusted to 0.07, 0.1, and 0.2 using saturated salt solutions; controls were prepared by keeping Ca-alginate and Ca-alginate-mannitol in aluminum foil without a(w) adjustment. Mannitol in the Ca-alginate system interacted with cell envelopes during freeze-drying and during storage at low a(w)s. In contrast, Ca-alginate protected cell envelopes after freeze-drying but not during 10-week storage. Unlike Ca-alginate, Ca-alginate-mannitol was effective in retarding the changes in secondary proteins during freeze-drying and during 10 weeks of storage at low a(w)s. It appears that Ca-alginate-mannitol is more effective than Ca-alginate in preserving cell envelopes and proteins after freeze-drying and after 10 weeks of storage at room temperature (25°C).     

Suppressive effects of flavonoids on dioxin toxicity

Dioxin type chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause a variety of toxicity. Most of the toxicity of TCDD has been attributed to a mechanism by which TCDD is bound to aryl hydrocarbon receptor (AhR) and transforms the receptor. Thus, suppression of the AhR transformation by food factors can suppress the dioxin toxicity. In this study, flavonoids at various concentrations were treated to a rat cytosolic fraction containing AhR before adding 1 nM TCDD. The transformed AhR was detected by an electrophoretic mobility shift assay with a DNA oligonucleotide consensus to dioxin response element. As the results, flavones and flavonols at dietary levels act as the antagonists for AhR and suppress the transformation. The antagonistic IC50 values were in a range between 0.14 and 10 microM, which are close to the physiological levels in human. These results suggest that a plant-based diet can prevent the dioxin toxicity.     

Enhanced efficacy of nitrifying biomass by modified PVA_SB entrapment technique

In this study, we developed a novel technique for preparing polyvinyl alcohol (PVA) hydrogel as an immobilizing matrix by the addition of sodium bicarbonate. This resulted in an increase in the specific surface area of PVA_sodium bicarbonate (PVA_SB) hydrogel beads to 65.23 m² g^?1 hydrogel beads, which was approximately 85 and 14 % higher than those of normal PVA and PVA_sodium alginate (PVA_SA) hydrogel beads, respectively. The D _e value of PVA_SB hydrogel beads was calculated as 7.49 × 10^?4 cm² s^?1, which was similar to the D _e of PVA_SA hydrogel beads but nearly 38 % higher than that of the normal PVA hydrogel beads. After immobilization with nitrifying biomass, the oxygen uptake rate and the ammonium oxidation rate of nitrifying biomass entrapped in PVA_SB hydrogel beads were determined to be 19.53 mg O₂ g MLVSS^?1 h^?1 and 10.59 mg N g MLVSS^?1 h^?1, which were 49 and 43 % higher than those of normal PVA hydrogel beads, respectively. Scanning electron microscopy observation of the PVA_SB hydrogel beads demonstrated relatively higher specific surface area and revealed loose microstructure that was considered to provide large spaces for microbial growth. This kind of structure was also considered beneficial for reducing mass transfer resistance and increasing pollutant uptake.     

Preparation and characterization of a novel pH-sensitive chitosan-g-poly (acrylic acid)/attapulgite/sodium alginate composite hydrogel bead for controlled release of diclofenac sodium

A series of pH-sensitive composite hydrogel beads composed of chitosan-g-poly (acrylic acid)/attapulgite/sodium alginate (CTS-g-PAA/APT/SA) was prepared as drug delivery matrices crosslinked by Ca²⁺ owing to the ionic gelation of SA. The structure and surface morphology of the composite hydrogel beads were characterized by FTIR and SEM, respectively. pH-sensitivity of these composite hydrogels beads and the release behaviors of drug from them were investigated. The results showed that the composite hydrogel beads had good pH-sensitivity. The cumulative release ratios of diclofenac sodium (DS) from the composite hydrogel beads were 3.76% in pH 2.1 solution and 100% in pH 6.8 solutions within 24 h, respectively. However, the cumulative release ratio of DS in pH 7.4 solution reached 100% within 2 h. The DS cumulative release ratio reduced with increasing APT content from 0 to 50 wt%. The drug release was swelling-controlled at pH 6.8.     

Ligand-dependent recruitment of the Arnt coregulator determines DNA recognition by the dioxin receptor.

The intracellular basic region/helix-loop-helix (bHLH) dioxin receptor mediates signal transduction by dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) and functions as a ligand-activated DNA binding protein directly interacting with target genes by binding to dioxin response elements. Here we show that the partially purified, ligand-bound receptor alone could not bind target DNA. In contrast, DNA binding by the receptor could be induced by addition of a cytosolic auxiliary activity which functionally and biochemically corresponded to the bHLH factor Arnt. While Arnt exhibited no detectable affinity for the dioxin response element in the absence of the dioxin receptor, it strongly promoted the DNA binding function of the ligand-activated but not the ligand-free receptor forms. Arnt also functionally reconstituted in vitro the DNA binding activity of a mutant, nuclear translocation-deficient dioxin receptor phenotype in cytosolic extracts from a dioxin-resistant hepatoma cell line. Importantly, coimmunoprecipitation experiments showed that Arnt physically interacted in solution with the ligand-activated dioxin receptor but failed to heterodimerize with the ligand-free, hsp90-associated receptor form. Mutational analysis suggested that the functional interaction between these two factors occurred via the bHLH motif of Arnt. These data suggest that dioxin receptor activity is governed by a complex pattern of combinatorial regulation involving repression by hsp90 and then by ligand-dependent recruitment of the positive coregulator Arnt. The dioxin receptor system also provides the first example of signal-controlled dimerization of bHLH factors.     

Encapsulation of the peptide Ac-Glu-Thr-Lys-Thr-Tyr-Phe-Trp-Lys-NH2 into polyvinyl alcohol biodegradable formulations—Effect of calcium alginate

It has been recently reported that the peptide Ac-Glu-Thr-Lys-Thr-Tyr-Phe-Trp-Lys-NH₂, analogue of the Glu1811-Lys1818 region of A3 light chain of blood coagulation factor VIII, presents in vitro significant anticoagulant activity. The encapsulation of this peptide into different polyvinyl alcohol formulations is examined here. The formulations were prepared using polyvinyl alcohol cross-linked with either boric acid or glutaraldehyde, giving a series of twelve different hydrogels. In case of PVA-boric acid method, a small percentage of sodium alginate was used in order to avoid bead's agglomeration. In that case, the most efficient encapsulation of the octapeptide (74%) was achieved with 0.2% (w/w) sodium alginate. It was also observed that the increase in sodium alginate percentage leads to beads with increased peptide release time, ranging from 60 to 90 min at 0.02% and 1% (w/w) sodium alginate respectively. The water holding of the PVA gels was estimated to be 27% regardless of the cross-linking reagent used, while it was increased with increasing sodium alginate concentration and reached about 60% for 1% sodium alginate. The longer octapeptide release, at 120 min, was observed with PVA-glutaraldehyde hydrogel, with encapsulation efficiency comparable to those obtained with boric acid, indicating that this hydrogel may be further used in drug delivery systems.     

Low-temperature electron microscopy for the study of polysaccharide ultrastructures in hydrogels. II. Effect of temperature on the structure of Ca2+-alginate beads

Calcium alginate beads were thermally treated at temperatures ranging from 25 degrees C to 130 degrees C for periods of up to 30 minutes. Important modifications to the structure of the alginate beads were shown to be a function of the temperature and period of incubation at each temperature. Modifications to the alginate beads included reduction in size, mechanical resistance, and molecular weight cut-off with increasing temperature and incubation period. Thus, heating 700 microm calcium alginate beads for 20 min at 130 degrees C resulted in a 23% reduction in diameter, 70% increase in mechanical resistance, and 67% reduction in molecular weight cut-off.Incubation of calcium alginate beads containing 2 x 10(6) kDa blue dextran for 20 min at 130 degrees C resulted in no detectable loss of either dye or alginate. This indicates the shrinkage of the beads was due to re-arrangement of the alginate chains within the beads, coupled with loss of water. This hypothesis was verified by direct visual observation of calcium alginate beads before and after thermal treatment using cryo-scanning electron microscopy (cryo-SEM). Unlike other microscopy methods cryo-SEM offers the advantage of extremely rapid freezing which preserves the original structure of the alginate network. As a result cryo-SEM is a powerful tool for studies of hydrogel and capsule structure and formation.Differential scanning calorimetry (DSC) showed that the water entrapped in 2% alginate beads was present in a single state, irrespective of the thermal treatment. This result is attributed to the low alginate concentration used to form the beads.