Cytocompatible drug delivery hydrogels based on carboxymethylagarose/chitosan pH-responsive polyelectrolyte complexes.

Several drugs are chemically unstable in the gastric environment and have low bioavailability restricted by intestinal absorption, which motivates the development of alternative routes for drug release, such as transdermal drug carriers for drug delivery to specific areas of the skin. Herein, novel polyelectrolyte complexes (PEC) consisting of carboxymethylagarose (CMA) and chitosan (CS) were prepared. pH-responsive CMA/CS hydrogels were obtained by mixing CMA and CS at various weight ratios. Swelling ratio was modulated by varying the CMA and CS weight ratio, and the highest swelling values were achieved for 2:1 wt% hydrogels at 25 °C and pH 6.0. PEC films were characterized by ATR-FTIR spectroscopy, TGA, DSC, and SEM. Results indicated that CMA and CS were successfully crosslinked by ionic complexation. As a model drug, diclofenac sodium (DS) was loaded in CMA/CS PECs. Association efficiency and loading capacity were ca. 69% and 79%, respectively, exhibiting 67% cumulative release after 72 h at 37 °C and pH 6.0 through Fickian diffusion mechanism. Viability assay of immortalized human keratinocyte (HaCat) cells showed ca. 100% survival in the presence of hydrogels and DS. Therefore, this work suggests that CMA/CS PECs can be applied as pH-responsive carriers for dermal drug delivery.

Evaluation of Trypanocidal and Antioxidant Activities of a Selected Series of 3-amidocoumarins.

BACKGROUND: Neglected diseases are becoming more prevalent due to globalization. This has inspired active research in the development of new drugs for the treatment of parasitic diseases such as Chagas disease. OBJECTIVES: With the aim of finding new trypanocidal agents, we report the in vitro evaluation of a new series of 3-amidocoumarins with or without hydroxyl substituents at position 4 of the coumarin ring. METHODS: Electrochemical and biological assays were performed in order to assess the antioxidant and trypanocidal potential of these compounds and to better understand the mechanisms involved in their activity. RESULTS: Most of the studied compounds showed high trypanocidal activity against both epimastigote and trypomastigote forms, with IC50 values in the low micromolar range. Some of them have greater activity and selectivity than the reference compound, nifurtimox. CONCLUSION: Compound 2 is the most active of this series, being also non-cytotoxic against murine RAW 264.7 macrophages. Electrochemical and radical scavenging experiments were carried out, providing new information about the profile of the best derivatives, and the potential therapeutic application of the new 3-amidocoumarins.

Characterization of poly-d-mannuronate and poly-l-guluronate block fractions from sodium alginate and preparation of hydrogels with poly(vinylalcohol).

Sodium salts of homopoly-d-mannuronic acid (MM) and of homopoly-l-guluronic acid (GG) from sodium alginates were characterized by NMR relaxometry. Determination of NMR spin-lattice and spin-spin relaxation times of water proton in homopolymeric block solutions and hydrogels indicated differences in homopolymeric blocks tertiary structure. Hydrogels of MM or GG blocks and poly(vinyl alcohol) (PVA) were prepared by freeze-thawing cycles method; their swelling properties and sensitivity to pH stimuli were assayed in control delivery of a model drug. MM/PVA hydrogels show better metformin release characteristics than GG/PVA hydrogels. It was found that release of the drug at pH 1.2 from hydrogels was minor to 5%. At the release equilibrium, 60 and 55% of the drug encapsulated were release from MM/PVA and GG/PVA hydrogels, respectively. Also, the release of metformin from hydrogels was studied by 1H NMR spectroscopy showing that 40 and 36% of drug were released after 4 h from MM/PVA and GG/PVA hydrogels, respectively.

Preparation and characterization of maleoylagarose/PNIPAAm graft copolymers and formation of polyelectrolyte complexes with chitosan.

A water soluble derivative in 98% yield with 23.1% incorporation of maleoyl groups was obtained by esterification of agarose with maleic anhydride. Graft copolymers were synthesized through vinyl groups of maleoylagarose with N-isopropylacrylamide using ceric ammonium nitrate or ammonium persulfate as initiator, by conventional method or microwave irradiation. High nitrogen content (4.6%) was obtained in the grafting process using ceric ammonium nitrate as initiator without microwave irradiation. Copolymers were characterized by FT-IR and NMR spectroscopies, TGA, DSC and morphological analysis by AFM and SEM microscopy, confirming the grafting of PNIPAAm onto polysaccharide backbone. Hydrogel films were obtained by ionic complexation between opposite charged groups of maleoylagarose-g-poly(N-isopropylacrylamide) and chitosan. The swelling of 1:1w/v maleoylagarose-g-PNIPAAm:chitosan film was higher than 2:1w/v film at 25 and 37°C. 53% release in vitro of diclofenac sodium from 1:1w/v maleoylagarose-g-PNIPAAm:chitosan was obtained at 37°C and pH 6.0 with <0.5 diffusional constant values.

In vitro release of metformin hydrochloride from sodium alginate/polyvinyl alcohol hydrogels.

Hydrogels, based on polysaccharides have found a number of applications as drug delivery carriers. In this work, hydrogels of full characterized sodium alginate (Mn 87,400g/mol) and commercial poly(vinyl alcohol) (PVA) sensitive to pH and temperature stimuli were obtained using a simple, controlled, green, low cost method based on freeze-thaw cycles. Stable hydrogels of sodium alginate/PVA with 0.5:1.5 and 1.0:1.0w/v concentrations showed very good swelling ratio values in distilled water (14 and 20g/g, respectively). Encapsulation and release of metformin hydrochloride in hydrogels of 1.0:1.0w/v sodium alginate/PVA was followed by UV spectroscopy. The hydrogel released a very low amount of metformin hydrochloride at pH 1.2; the highest release value (55%) was obtained after 6h at pH 8.0. Also, the release of metformin hydrochloride was studied by 1H NMR spectroscopy, the temporal evolution of methyl group signals of metformin showed 30% of drug release after 3h.

Chiroptical characterization of homopolymeric block fractions in alginates.

Homopolymannuronic and homopolyguluronic fractions were obtained by partial hydrolysis of the alkaline extracts from the brown seaweeds Ascoseira mirabilis, Desmarestia menziessi, Desmarestia ligulata and Durvillaea sp. collected in southern Chile. Full characterization of the fractions was achieved by FT-IR and NMR spectroscopy. Total hydrolysis with 90% formic acid of the homopolymeric fractions allowed the preparation of mannuronic and guluronic acids. Both monomers and homopolymeric fractions as neutral salts were studied by CD and ORD. Chiroptical spectra were similar in shape and sign to those previously published in the literature, and permitted to assign D configuration to mannuronic acid and L configuration to guluronic acid in alginic acids. Specific optical rotation values at the sodium D light for the homopolymannuronic (∼-100°) and homopolyguluronic (∼-110°) acid fractions were obtained. These high negative values are proposed for the assignment of the absolute configuration of monomers in homopolymeric fractions.

The effects of NO3(-) supply on Mazzaella laminarioides (Rhodophyta, Gigartinales) from southern Chile.

The effects of nitrate supply on growth, pigments, mycosporine-like amino acids (MAAs), C:N ratios and carrageenan yield were investigated in Mazzaella laminarioides cultivated under solar radiation. This species is economically important in southern Chile where an increase of nitrogen in coastal waters is expected as a consequence of salmon aquaculture activity. Apical segments were cultivated in enriched seawater with five different NO3(-) concentrations (0, 0.09, 0.18, 0.38 and 0.75 mm) during 18 days. Although phycoerythrin and phycocyanin content, as well as C:N ratios, were reduced in the control treatment (without NO3(-) supply), when compared to NO3(-) treatments, total MAA concentration, carrageenan yield and growth rates were similar in all tested conditions. Nevertheless, during the experiment, an important synthesis of mycosporine-glycine took place in a nitrate concentration-dependent manner, with accumulation being saturated around 0.18 mm of nitrate. These results indicate that exposure to high NO3(-) concentration of more than 100 times the values observed in the nature did not impair the photoprotection system, as determined by MAAs, nor did it have a deleterious effect on growth or carrageenan yield of M. laminarioides, a late successional species from Chile.

Preparation and characterization of hydrogels based on homopolymeric fractions of sodium alginate and PNIPAAm.

Graft copolymers were prepared by formation of an amide bond between poly-α-L-guluronic acid (MW 24,000), isolated from sodium alginate and the free amino group of PNIPAAm-NH(2). SEM micrographs revealed the formation of a macroscopic network on the surface of the grafted hydrogels with a porosity diameter of 10-20 µm. Semi-IPN hydrogels were prepared using different proportions of sodium poly-ß-D-mannuronate (MW 21,000), isolated from sodium alginate and cross-linked PNIPAAm-NH(2) polymers. SEM micrographs showed porosities of minor size (∼5 µm). Though both types of hydrogels are good water containers, the water retention capacity of graft copolymers is more than 50% higher than that of semi-IPN hydrogels. Both hydrogel types showed significant changes in swelling ratios between 20 and 45 °C: the swelling ratio decreases near the LCST of PNIPAAm. The water absorption and retention capacity of graft hydrogels increases with pH, reaching a maximum value at pH 7.0.

A sulfated galactan with antioxidant capacity from the green variant of tetrasporic Gigartina skottsbergii (Gigartinales, Rhodophyta).

The water soluble polysaccharide produced by the green variant of tetrasporic Gigartina skottsbergii was found to be composed of D-galactose and sulfate groups in a molar ratio of 1.0:0.65. (1)H and (13)C NMR spectroscopy studies of the desulfated polysaccharide showed a major backbone structure of alternating 3-linked ß-D-galactopyranosyl and 4-linked α-D-galactopyranosyl units, and minor signals ascribed to 3-O-methyl-substitution on the latter unit. Ethylation analysis of the polysaccharide indicated that the sulfate groups are mainly located at position O-2 of 4-linked α-D-galactopyranosyl residue and partially located at positions O-6 of the same unit and at position O-2 of 3-linked ß-D-galactopyranosyl residue, and confirmed the presence of 3-O-methyl-galactose in minor amounts (4.4%). The sulfated d-galactan presents a similar structure to λ carrageenan but with much lower sulfation at position O-6 of the α-residue and at position O-2 of ß-residue. The antioxidant capacity of the sulfated d-galactan was evaluated by the peroxyl radicals (ORAC method), hydroxyl radicals, chelating activity, and ABTS(+) assays. Kinetic results obtained in these assays were compared with those obtained for the commercial λ carrageenan. The antioxidant activity toward peroxyl radicals was higher for commercial λ carrageenan, this agrees with its higher content of sulfate group. The kinetics of the reaction of both polysaccharides with hydroxyl and ABTS(+) radicals showed a complex mechanism, but the antioxidant activity was higher for the polysaccharide from the green variant of tetrasporic Gigartina skottsbergii.

Antifungal activity of resveratrol against Botrytis cinerea is improved using 2-furyl derivatives.

The antifungal effect of three furyl compounds closely related to resveratrol, (E)-3,4,5-trimethoxy-ß-(2-furyl)-styrene (1), (E)-4-methoxy-ß-(2-furyl)-styrene (2) and (E)-3,5-dimethoxy-ß-(2-furyl)-styrene (3) against Botrytis cinerea was analyzed. The inhibitory effect, at 100 µg ml(-1) of compounds 1, 2, 3 and resveratrol on conidia germination, was determined to be about 70%, while at the same concentration pterostilbene (a dimethoxyl derivative of resveratrol) produced complete inhibition. The title compounds were more fungitoxic towards in vitro mycelial growth than resveratrol and pterostilbene. Compound 3 was the most active and a potential explanation of this feature is given using density functional theory (DFT) calculations on the demethoxylation/demethylation process. Compound 3 was further evaluated for its effects on laccase production, oxygen consumption and membrane integrity of B. cinerea. An increase of the laccase activity was observed in the presence of compound 3 and, using Sytox Green nucleic acid stain, it was demonstrated that this compound altered B. cinerea membrane. Finally, compound 3 partially affected conidia respiration.

Long-term protection against tobacco mosaic virus induced by the marine alga oligo-sulphated-galactan Poly-Ga in tobacco plants.

In order to study the antiviral effect of the oligo-sulphated galactan Poly-Ga, the leaves of tobacco plants Xhanti(NN) were sprayed with water (control), with increasing concentrations of Poly-Ga, for increasing numbers of treatments or cultivated for increasing times after treatment. Control and treated plants were infected with tobacco mosaic virus (TMV) and the numbers of necrotic lesions were measured in infected leaves. The number of necrotic lesions decreased with increasing concentrations of Poly-Ga, with increasing numbers of treatments and with increasing time after treatment, indicating a long-term protection against TMV that mimicks vaccination. In addition, control Xhanti(nn) plants and plants treated with Poly-Ga and cultivated for increasing times after treatment were infected with TMV in the middle part of the plant, and the levels of TMV-capsid protein (CP) transcripts were measured in apical leaves. TMV-CP transcripts decreased in distant leaves, indicating that Poly-Ga induces systemic protection against TMV. The activities of the defence enzymes phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX) and the amounts of several phenylpropanoid compounds (PPCs) were measured in control and treated plants without infection. A progressive increase in PAL activity was observed with increasing time after treatment, together with the accumulation of free and conjugated PPCs. In contrast, LOX activity remained unchanged. Interestingly, the increase in PAL activity showed a linear correlation with the decrease in necrotic lesions and the decrease in TMV-CP transcript level. Thus, Poly-Ga induced systemic and long-term protection against TMV in tobacco plants that is determined, at least in part, by a sustained activation of PAL and the accumulation of PPCs with potential antiviral activity.

Anthocyanin composition in aged Chilean Cabernet Sauvignon red wines.

Anthocyanins in aged Cabernet Sauvignon red wines were studied by HPLC-MS. The major anthocyanin in 6, 7, and 8year old red wine extracts was the dimer vitisin A produced by condensation of malvidin-3-O-glucoside mediated by pyruvic acid. In aged wine, the content of malvidin-3-O-glucoside decreased with age with a concomitant increase of vitisin A. The latter is accompanied by several condensation products with molecular weight up to 1500m/z. Differential pulse voltammetry indicated that aged wines have lower antioxidant capacity than young wine (400mV), but higher than malvidin-3-O-glucoside (483mV). Tafel's plots showed that the electrochemical process occurring in aged wines is different from young wines. Six, seven and eight year old wines show similar behaviour with plots of 234, 177 and 188mV/dec, respectively. These values are higher than the expected 120mV/dec corresponding to a first electronic transfer but smaller compared to the 523mV/dec corresponding to young wine.

FT-IR spectra of alginic acid block fractions in three species of brown seaweeds.

Sodium alginates obtained by alkaline extraction of Lessonia flavicans, Desmarestia ligulata and Desmarestia distans (Phaeophyta) from southern Chile were partially hydrolyzed with HCl. Each alginate gave three fractions that were characterized using FT-IR spectroscopy. The fractions soluble in 0.3M HCl presented in the fingerprint region four vibrations at around 960, 911, 890 and 815 cm(-1) that were assigned to heteropolymeric blocks. The fractions soluble at pH 2.85 showed bands at around 948, 888 and 820 cm(-1) attributed to homopolymannuronic acid blocks, the first band is resolved in the second-derivative spectra into two bands at 951 and 936 cm(-1). The fractions insoluble at pH 2.85 presented four bands at around 947, 903, 812 and 781 cm(-1), which were assigned to homopolyguluronic acid blocks. For some samples, the second derivative FT-IR spectra showed new bands indicating the presence of other structures, in low proportions. Structures deduced by FT-IR spectroscopy were corroborated by solution (1)H and (13)C NMR spectroscopy. Two-dimensional spectra were collected to confirm the fine structure of the hetero- and homopolymeric fractions. A geometrically optimized model for the disaccharide alpha-l-gulopyranuronosyl-(1-->4)-alpha-l-gulopyranuronic acid was calculated using density functional theory; good agreement was obtained between its corresponding calculated vibrations and the experimental bands assigned to homopolyguluronic acid blocks.

Salmonella enterica serovar Typhi O:1,9,12 polysaccharide-protein conjugate as a diagnostic tool for typhoid fever.

Serologic tests play an important role in diagnosis of typhoid fever. In an effort to develop a more defined reagent for these tests, purified Salmonella enterica serovar Typhi (ST) O:1,9,12 polysaccharide was conjugated to human serum albumin (HSA), and the conjugate was purified chromatographically to yield a reagent with 2 moles ST O polysaccharide per mole HSA. In 40 patients with bacteriologically confirmed typhoid fever, significant dot immunobinding titers (> or =20,000) were present in 28 (70%) tested with 100 ng of ST O antigen-HSA (ST O-HSA) conjugate, in 38 (95%) tested with 100 ng of ST lipopolysaccharide, and in 16 (40%) tested with purified unconjugated ST O chains. In sera from 22 patients with other nontyphoid fevers, 2 (9.1%) had such reactivities with 100 ng of ST O-HSA, 1 (4.5%) had such reactivity with 100 ng of ST lipopolysaccharide (4.5%), and none reacted with 100 ng of unconjugated ST O chains. None of the 17 healthy-control sera reacted significantly with any of the ST reagents. None of the patient or control sera reacted with unconjugated HSA. The sensitivity of dot immunobinding for typhoid fever was 70% with 100 ng of ST O-HSA, somewhat lower than that with 100 ng of ST lipopolysaccharide (95%) but similar to that of the Widal H agglutination test with a > or =1/160 cutoff (74%). Specificities of these tests were 91%, 95%, and 86%, respectively. These preliminary results suggest that ST O polysaccharide-protein conjugates could provide a nontoxic, easily quality-controlled synthetic reagent for analysis of human immune responses to ST as well as for the development of new diagnostics and vaccines for typhoid fever.

Structural analysis and antiviral activity of a sulfated galactan from the red seaweed Schizymenia binderi (Gigartinales, Rhodophyta).

Aqueous extraction of gametophytic Schizymenia binderi afforded a polysaccharide composed of galactose and sulfate groups in a molar ratio of 1.0:0.89 together with uronic acids (6.8 wt%) and minor amounts of other neutral sugars. Alkali-treatment of the polysaccharide afforded a polysaccharide devoid of 3,6-anhydrogalactose. 13C NMR spectroscopy of the desulfated alkali-treated polysaccharide showed a backbone structure of alternating 3-linked beta-D-galactopyranosyl and 4-linked alpha-galactopyranosyl units that are predominantly of the D-configuration and partly of the L-configuration. Methylation, ethylation and NMR spectroscopic studies of the alkali-treated polysaccharide indicated that the sulfate groups are located mainly at positions O-2 of 3-linked beta-D-galactopyranosyl residue and at position O-3 of 4-linked-alpha-galactopyranosyl residues, the latter is partially glycosylated at position O-2. The sulfated galactan from S. binderi exhibited highly selective antiviral activity against Herpes simplex virus types 1 and 2, with selectivity indices (ratio cytotoxicity/antiviral activity) >1000 for all assayed virus strains. This compound was shown to interfere with the initial adsorption of viruses to cells.

Ni(II) complexes with Schiff bases derived from amino sugars.

It was found by 1H and 13C NMR spectroscopy that the Schiff base, 2-deoxy-2-(2-hydroxybenzaldimino)-D-glucopyranose exhibits enol-imine-keto-amine and anomeric equilibria in methanolic, and in dimethyl sulfoxide solutions. The reaction of the Schiff base with nickel acetate gave the bidentate, mononuclear Ni(II) complex that was characterized by spectroscopic methods and by cyclic voltammetry. The coordination of the Schiff base to the metal is through the enol-imine tautomeric form, and the anomeric equilibrium remains in dimethyl sulfoxide solutions. This complex was also obtained by reaction of D-glucosamine with Ni(II) salicylaldehydate. The same reaction was employed for the synthesis of bis-N-[2-deoxy-D-galactopyranosyl-2-(2-hydroxybenzaldiminate)]Ni(II). The small paramagnetic shifts of the 1H NMR resonances of the complexes suggest that paramagnetic species are present in low proportions.