Linear Polyethyleneimine-Based and Metal Organic Frameworks (DUT-67) Composite Hydrogels as Efficient Sorbents for the Removal of Methyl Orange, Copper Ions, and Penicillin V.

This research explores the integration of DUT-67 metal organic frameworks into polyethyleneimine-based hydrogels to assemble a composite system with enough mechanical strength, pore structure and chemical affinity to work as a sorbent for water remediation. By varying the solvent-to-modulator ratio in a water-based synthesis path, the particle size of DUT-67 was successfully modulated from 1 µm to 200 nm. Once DUT-67 particles were integrated into the polymeric hydrogel, the composite hydrogel exhibited enhanced mechanical properties after the incorporation of the MOF filler. XPS, NMR, TGA, FTIR, and FT Raman studies confirmed the presence and interaction of the DUT-67 particles with the polymeric chains within the hydrogel network. Adsorption studies of methyl orange, copper(II) ions, and penicillin V on the composite hydrogel revealed a rapid adsorption kinetics and monolayer adsorption according to the Langmuir's model. The composite hydrogel demonstrated higher adsorption capacities, as compared to the pristine hydrogel, showcasing a synergistic effect, with maximum adsorption capacities of 473 ± 21 mg L-1, 86 ± 6 mg L-1, and 127 ± 4 mg L-1, for methyl orange, copper(II) ions, and penicillin V, respectively. This study highlights the potential of MOF-based composite hydrogels as efficient adsorbents for environmental pollutants and pharmaceuticals.

Films of Poly(Hydroxybutyrate) (PHB) and Copper with Antibacterial Activity.

Poly(3-hydroxybutyrate), PHB, is a hydrophobic biopolymer with good mechanical and barrier properties. However, neat PHB is a semicrystalline polymer with a relative high degree of crystallinity and poor film properties. In this work, this biopolymer was plasticized with glycerol tributyrate and functionalized with copper (II) sulfate, allowing us to obtain biodegradable antimicrobial flexible films. Films with the minimum inhibitory concentration (MIC) of copper (II) sulfate presented a higher roughness than neat PHB films. The presence of plasticizer significantly improved the copper sulfate diffusion process, which was evidenced by a greater inhibition halo for plasticized materials compared to unplasticized ones, at the same salt concentration. Plasticized PHB with 2.5% copper (II) sulfate inhibited both Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomona aeruginosa) bacteria, as determined by the bacterial inhibition halo. In addition, neat PHB films and PHB containing copper (II) sulfate did not show in vitro cytotoxicity in the L-929 cell line. Thus, plasticized PHB functionalized with copper (II) sulfate can be used as biodegradable antimicrobial flexible films for different applications.

Recycling of hyoscyamine 6ß-hydroxylase for the in vitro production of anisodamine and scopolamine.

The tropane alkaloids hyoscyamine, anisodamine, and scopolamine are extensively used medicines. In particular, scopolamine has the greatest value in the market. Hence, strategies to enhance its production have been explored as an alternative to traditional field-plant cultivation. In this work, we developed biocatalytic strategies for the transformation of hyoscyamine into its products utilizing a recombinant Hyoscyamine 6ß-hydroxylase (H6H) fusion protein to the chitin-binding domain of the chitinase A1 from Bacillus subtilis (ChBD-H6H). Catalysis was carried out in batch, and recycling of H6H constructions was performed via affinity-immobilization, glutaraldehyde crosslinking, and adsorption-desorption of the enzyme to different chitin matrices. ChBD-H6H utilized as free enzyme achieved complete conversion of hyoscyamine in 3- and 22-h bioprocesses. Chitin particles demonstrated to be the most convenient support for ChBD-H6H immobilization and recycling. Affinity-immobilized ChBD-H6H operated in a three-cycle bioprocess (3 h/cycle, 30 °C) yielded in the first and third reaction cycle 49.8% and 22.2% of anisodamine and 0.7% and 0.3% of scopolamine, respectively. However, glutaraldehyde crosslinking decreased enzymatic activity in a broad range of concentrations. Instead, the adsorption-desorption approach equaled the maximal conversion of the free enzyme in the first cycle and retained higher enzymatic activity than the carrier-bound strategy along the consecutive cycles. The adsorption-desorption strategy permitted the reutilization of the enzyme in a simple and economical manner while exploiting the maximal conversion activity displayed by the free enzyme. This approach is valid since other enzymes present in the E. coli lysate do not interfere with the reaction. KEY POINTS: • A biocatalytic system for anisodamine and scopolamine production was developed. • Affinity-immobilized ChBD-H6H in ChP retained catalytic activity. • Enzyme-recycling by adsorption-desorption strategies improves product yields.

Comparison of three cyclodextrins to optimize bisphenol A extraction from source water: Computational, spectroscopic, and analytical studies.

Computationally and spectroscopically assisted analytical comparative investigation into the extraction of bisphenol A using three cyclodextrins, that is, α, ß, and γ respectively, were performed. A simple, self-tailored µ-solid-phase extraction podium was used to extract bisphenol A from water samples, and high-performance liquid chromatography-ultraviolet was used for the qualitative and quantitative analysis of bisphenol A. Density functional theory first principle calculations, attenuated total reflectance Fourier-transform infrared spectroscopy and Fourier-transform Raman spectroscopy data supports the analytical selection of ß-cyclodextrin as the adsorbent for bisphenol A extraction. Analytical optimization of various parameters including sample volume, sample pH, eluting solvent and its volume was performed to discover the most proper conditions for maximum extraction. Under the optimized conditions, a limit of detection value of 0.70 ng/ml and a limit of quantification value of 2.31 ng/ml was achieved with ß-cyclodextrin, with recovery (%) values over 98.40-102.50 in real source water samples. Overall, well assisted by comprehensive computational and spectroscopic studies, a novel, simple, sensitive and economic analytical method was developed for the extraction of bisphenol A from source water using cyclodextrin.

Solid phase extraction with rotating cigarette filter for determination of bisphenol A in source and drinking water: computational and analytical studies.

An ultrasound assisted solid phase extraction method using rotating cigarette filter is developed herein to preconcentrate and determine trace amount of bisphenol in source and drinking water. Qualitative and quantitative measurements were performed using high-performance liquid chromatography coupled with ultra violet detector. Sorbent-analyte interactions were thoroughly investigated computationally and experimentally using molecular dynamics simulations; and attenuated total reflectance Fourier transform infrared spectroscopy, and Raman spectroscopy, respectively. Various extraction parameters were investigated and optimized. Under the optimal conditions, the results were linear in a low scale range of 0.01-55 ng/mL with correlation coefficient of 0.9941 and a low limit of detection (0.04 ng/mL, signal/noise = 3:1). A good precision (intra-day relative standard deviation ≤ 6.05%, inter-day relative standard deviation ≤ 7.12%) and recovery (intra-day ≥ 98.41%, inter-day ≥ 98.04%)) are obtained. Finally, the proposed solid phase extraction method offered a low cost, simple, fast, and sensitive analytical method to determine trace amount of bisphenol A in source and drinking water samples with chromatographic detection.

Characterization of the Copper Transporters from Lotus spp. and Their Involvement under Flooding Conditions.

Forage legumes are an important livestock nutritional resource, which includes essential metals, such as copper. Particularly, the high prevalence of hypocuprosis causes important economic losses to Argentinian cattle agrosystems. Copper deficiency in cattle is partially due to its low content in forage produced by natural grassland, and is exacerbated by flooding conditions. Previous results indicated that incorporation of Lotus spp. into natural grassland increases forage nutritional quality, including higher copper levels. However, the biological processes and molecular mechanisms involved in copper uptake by Lotus spp. remain poorly understood. Here, we identify four genes that encode putative members of the Lotus copper transporter family, denoted COPT in higher plants. A heterologous functional complementation assay of the Saccharomyces cerevisiae ctr1∆ctr3∆ strain, which lacks the corresponding yeast copper transporters, with the putative Lotus COPT proteins shows a partial rescue of the yeast phenotypes in restrictive media. Under partial submergence conditions, the copper content of L. japonicus plants decreases and the expression of two Lotus COPT genes is induced. These results strongly suggest that the Lotus COPT proteins identified in this work function in copper uptake. In addition, the fact that environmental conditions affect the expression of certain COPT genes supports their involvement in adaptive mechanisms and envisages putative biotechnological strategies to improve cattle copper nutrition.

Pectin-decorated magnetite nanoparticles as both iron delivery systems and protective matrices for probiotic bacteria.

The goal of this work was to investigate biophysical stability of iron-pectin nanoparticles and analyze the feasibility of using them as delivery systems for the probiotic strain Lactobacillus plantarum CIDCA 83114. Iron oxide (Fe3O4) nanoparticles were synthesized from 0.25M FeCl2/0.5 M FeCl3.6H2O, and coated with citrus pectins. Their physico-chemical properties [FTIR, X-ray diffraction (XRD), ζ-potential, particle size, SEM, TEM] and their effect on bacterial stabilization (viability after freeze-drying/storage, stability when exposed to simulated gastro-intestinal conditions) were assessed. XRD indicated the almost exclusive presence of magnetite crystalline phases. FTIR spectra confirmed the adsorption of pectin on magnetite nanoparticles surface. SEM and TEM images evidenced agglomerated nanoparticles, and a morphological surface change after adsorption of pectin. DLS and ζ-potential results proved the solvation of the ionizable groups in the hydrophilic network which induced chain expansion and agglomeration. Iron from nanoparticles demonstrated to be non-toxic for microorganisms up to 1.00 mg/mL. Simulated saliva and gastric solutions prevented nanoparticles from dissolution. The higher pH of the intestinal conditions (solvated -COO- and Fe-O- groups) facilitated the dispersion and partial dissolution of nanoparticles. Pectins adsorption on magnetite nanoparticles significantly enhanced electrostatic repulsion, which aided the solvation of ionized iron forms. The soluble species diffused out from the aggregates, being detected in the simulated intestinal fluid. Regarding bacterial viability, no decays were observed neither when pectin-decorated nanoparticles were exposed to simulated fluids nor when stored at 4 °C for 60 days. The composites engineered in this work appear as adequate delivery systems for probiotic bacteria, whose target is the gut.

Smart release of antimicrobial ZnO nanoplates from a pH-responsive keratin hydrogel.

A smart antibacterial biomaterial based on a keratin hydrogel with pH-dependent behavior and Zinc Oxide nanoplates as biocide agent has been developed. The pH of a chronic wound is basic due to bacterial metabolism. Originally shrank at acid pH, keratin hydrogels swell upon contact with a bacterial contaminated media leading to the release of the nanoparticles. The material has been thoroughly characterized by infrared spectroscopy, Raman, scanning electron microscope, swelling behavior, Differential scanning calorimetry, Small-angle X-ray scattering, rheology, antimicrobial activity and cytotoxicity. The results show that 5% of Zinc Oxide nanoparticles concentration is the optimum for wound dressing applications.

Induction of ABCG2/BCRP restricts the distribution of zidovudine to the fetal brain in rats.

Safety concerns for fetus development of zidovudine (AZT) administration as prophylaxis of vertical transmission of HIV persist. We evaluated the participation of the ATP-binding cassette efflux transporter ABCG2 in the penetration of AZT into the fetal brain and the relevance for drug safety. Oral daily doses of AZT (60mg/kg body weight) or its vehicle were administered between post gestational days 11 (E11) and 20 (E20) to Sprague-Dawley pregnant rats. At E21, animals received an intravenous bolus of 60mg AZT/kg body weight in the presence or absence of the ABCG2 inhibitor gefitinib (20mg/kg body weight, ip) and AZT in maternal plasma and fetal brain were measured by HPLC-UV. ABCG2 protein expression in placenta and fetal brain, as well as mitochondrial function and ultrastructure in fetal brain were also analyzed. In utero chronic exposure to AZT markedly induced ABCG2 expression in placenta and fetal brain whereas did not significantly alter mitochondrial functionality in the fetal brain. The area-under-the-concentration-time-curve of AZT significantly decreased in fetal brains isolated from AZT-exposed fetuses compared to control group, but this effect was abolished by ABCG2 inhibition. Our results suggest that the absence of mitochondrial toxicity in the fetal brain after chronic in utero administration of AZT could be attributed to its low accumulation in the tissue caused, at least in part, by ABCG2 overexpression. We propose that any interference with ABCG2 activity due to genetic, pathological or iatrogenic factors would increase the amount of AZT reaching the fetal brain, which could increase the risk of toxicity of this drug on the tissue.

Innovative Immobilization Matrices.

We present a brief survey of some of the recent work of Professor Luis E. Díaz, performed together with his students and collaborators at the University of Buenos Aires. Dr Luis E. Díaz has been involved in research on biochemical and pharmaceutical sciences solving scientific and industry problems for over 40 years until he passed away. Prof. Díaz scientific interests included various topics from NMR spectroscopy to biomedicine but fundamentally he focused in various aspects of chemistry (analytical, organic, inorganic and environmental). This is not a complete survey but a sampling of prominent projects related to sol-gel chemistry with a focus on some of his recent publications.

Adsorption of Cd(II) and Pb(II) onto a one step-synthesized polyampholyte: kinetics and equilibrium studies.

A one step-synthesized polyampholyte, bearing carboxylate and 2-methylimidazole (2MI) groups, was tested as adsorbent for the removal of Pb(II) and Cd(II) from aqueous solutions. This material combines the benefits of synthetic polymers, such as high adsorption capacity and chemical stability, and the advantages of biosorbents in regard of costs and simplicity of the production. The short time needed to achieve the adsorption equilibrium indicated a chemical-reaction controlled process. A network expansion was predicted as a result of repulsive interaction between the fixed positive charges. Langmuir model presented the best fitting to isotherm equilibrium data, with a maximum adsorption capacity of 182 mg g(-1) for Cd(II) and 202 mg g(-1) for Pb(II). The metal removal was strongly dependent on pH, involving carboxylate and 2MI residues. An ion-exchange process for Pb(II) and Cd(II), combined with coordination for the later, were the most probable mechanism of interaction. The adsorption of 1.35 ppm Cd(II) was 72±6% in well-water, and the adsorption of 0.50 ppm Pb(II) was 62±5% in tap-water. The recovery figures for Cd(II) in 1% HNO(3) were optimal.

In vitro studies and preliminary in vivo evaluation of silicified concentrated collagen hydrogels.

Hybrid and nanocomposite silica-collagen materials derived from concentrated collagen hydrogels were evaluated in vitro and in vivo to establish their potentialities for biological dressings. Silicification significantly improved the mechanical and thermal stability of the collagen network within the hybrid systems. Nanocomposites were found to favor the metabolic activity of immobilized human dermal fibroblasts while decreasing the hydrogel contraction. Cell adhesion experiments suggested that in vitro cell behavior was dictated by mechanical properties and surface structure of the scaffold. First-to-date in vivo implantation of bulk hydrogels in subcutaneous sites of rats was performed over the vascular inflammatory period. These materials were colonized and vascularized without inducing strong inflammatory response. These data raise reasonable hope for the future application of silica-collagen biomaterials as biological dressings.

Removal of dyes from water using chitosan hydrogel/SiO2 and chitin hydrogel/SiO2 hybrid materials obtained by the sol-gel method.

This work describes the synthesis of chitosan hydrogel/SiO(2) and chitin hydrogel/SiO(2) hybrid mesoporous materials obtained by the sol-gel method for their use as biosorbents. Their adsorption capabilities against four dyes (Remazol Black B, Erythrosine B, Neutral Red and Gentian Violet) were compared in order to evaluate chitin as a plausible replacement for chitosan considering its efficiency and lower cost. Both chitin and chitosan were used in the form of hydrogels. This allowed full compatibility with the ethanol release from tetraethoxysilane. The hybrid materials were characterized by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray Spectroscopy (EDS), Nitrogen Adsorption Isotherms and (13)C solid-state Nuclear Magnetic Resonance. Adsorption experimental data were analyzed using Langmuir, Freundlich and Dubinin-Radushkevich isotherm models along with the evaluation of adsorption energy and standard free energy (ΔG(0)). The adsorption was observed to be pH dependent. The main mechanism of dye adsorption was found to be a spontaneous charge associated interaction, except for EB adsorption on chitin/SiO(2) matrix, which showed to involve a lower energy physical adsorption interaction. Aside from highly charged dyes the chitin containing matrix has similar or higher adsorption capacity than the chitosan one.

Effect of various parameters on viability and growth of bacteria immobilized in sol-gel-derived silica matrices.

Immobilized bacteria are being extensively used for metabolite production, biocatalysts, and biosensor construction. However, long-term viability and metabolic activity of entrapped bacteria is affected by several conditions such as their physiological state, the presence of high-osmolarity environments, porous structure and shrinkage of the matrix. The aim of this work was to evaluate the effect of various parameters on bacteria immobilized in sol-gel-derived silica matrices. With this purpose, we evaluated the stress of immobilization over bacteria cultures obtained from different growing states, the effect of cell density and bacteria capability to proliferate inside matrices. Best results to attain longer preservation times were obtained when we immobilized suspensions with an optimized bacterial number of 1 x 10(7) cfu/gel in the presence of LB medium using aqueous silica precursors. Furthermore, the impact of osmotic stress with the subsequent intracellular trehalose accumulation and the addition of osmolites were investigated. Shorter preservation times were found for bacteria immobilized in the presence of osmolites while trehalose accumulation in stressed cells did not produce changes on entrapped bacteria viability. Finally, nutrient addition in silica matrices was studied indicating that the presence of a carbon source without the simultaneous addition of nitrogen was detrimental for immobilized E. coli. However, when both carbon and nitrogen sources were present, bacteria were able to survive longer periods of time.

Antibody detection employing sol-gel immobilized parasites.

Immunofluorescence assay (IFA) and immunoperoxidase assay (IPA) are useful diagnostic techniques for specific antibody detection for different diseases. Both involve several alternatives for immobilization of cells, such as solvent or heat fixation. Non-covalent immobilization implies rigorous storage conditions at -20 degrees C to preserve the slides, and usually numerous cells are detached during the washing steps, which can lead to inconsistencies in the results. Sol-gel chemistry is usually used for coating different materials because of the mild conditions of the polymerization reaction and the ability to introduce functional groups to a wide variety of surfaces. We have developed a novel procedure for the attachment of Trypanosoma cruzi epimastigotes and Leishmania guyanensis promastigotes to a silicon oxide polymer covered glass surface. The film was prepared using standard microscope slides with tetraethoxysilane and 3-aminopropyl triethoxysilane as polymeric precursors. When acetone was used as the major coating solvent, the IFA showed the fluorescence of the attached parasites without matrix background interference. Similar results were observed when the IPA was evaluated. The sensitivity and specificity of the sol-gel immobilized parasite slides were comparable with the heat fixation technique. The performance of the coated slides was maintained for at least 2 months at 4 degrees C storage temperature. This immobilization method does not affect the molecular epitopes of the attached cells. Thus, homogeneous, ready to use, long lasting coated slides were obtained, which are appropriate for field conditions.

Plasmatic antioxidant capacity due to ascorbate using TEMPO scavenging and electron spin resonance.

BACKGROUND: Ascorbate is the most effective water-soluble antioxidant and its plasma concentration is usually measured by different methods including colorimetric assays, HPLC or capillary electrophoresis. Plasma antioxidant capacity is determined by indexes such as total reactive antioxidant potential, total antioxidant reactivity, oxygen radical absorbance capacity, etc. We developed an alternative method for the evaluation of the plasma antioxidant status due to ascorbate. METHODS: TEMPO kinetics scavenging analyzed by ESR spectroscopy was performed on plasma samples in different antioxidant situations. Plasma ascorbate concentrations were determined by capillary electrophoresis. Ascorbyl radical levels were measured by ESR. RESULTS: Plasma reactivity with TEMPO (PR-T) reflected plasma ascorbate levels. Average PR-T for normal plasmas resulted 85+/-27 micromol/l (n=43). PR-T during ascorbic acid intake (1 g/day) increased to an average value of 130+/-20 micromol/l (p<0.001, n=20). PR-T correlated with the plasmatic ascorbate levels determined by capillary electrophoresis (r=0.92), presenting as an advantage the avoiding of the deproteination step. Plasma ascorbyl radical levels increase from 16+/-2 to 24+/-3 nmol/l (p<0.005, n=14) after ascorbate intake. CONCLUSIONS: PR-T could be considered as a measure of the plasmatic antioxidant capacity due to the plasma ascorbate levels and could be useful to investigate different antioxidant situations.

Efficient preservation in a silicon oxide matrix of Escherichia coli, producer of recombinant proteins.

The aim of this work was to study the use of silicon oxide matrices for the immobilization and preservation of recombinant-protein-producing bacteria. We immobilized Escherichia coli BL21 transformants containing different expression plasmids. One contained DNA coding for a T-cell receptor beta chain, which was expressed as inclusion bodies in the cytoplasm. The other two encoded bacterial superantigens Staphylococcal Enterotoxin G and Streptococcal Superantigen, which were expressed as soluble proteins in the periplasm. The properties of immobilization and storage stability in inorganic matrices prepared from two precursors, silicon dioxide and tetraethoxysilane, were studied. Immobilized E. coli was stored in sealed tubes at 4 and 20 degrees C and the number of viable cells and level of recombinant protein production were analyzed weekly. Different tests showed that the biochemical characteristics of immobilized E. coli remained intact. At both temperatures selected, we found that the number of bacteria in silicon dioxide-derived matrix was of the same order of magnitude (10(9) cfu ml(-1)) as before immobilization, for 2 months. After 2 weeks, cells immobilized in an alkoxide-derived matrix decreased to 10(4) cfu ml(-1) at 4 degrees C, and no viable cells were detected at 20 degrees C. We found that immobilized bacteria could be used as a starter to produce recombinant proteins with yields comparable to those obtained from glycerol stocks: 15 mg l(-1) for superantigens and 2 mg l(-1) for T-cell receptor beta chain. These results contribute to the development of methods for microbial cell preservation under field conditions.