Hydrogels based on galactomannan and κ-carrageenan containing immobilized biomolecules for in vivo thermal-burn wound treatment.

Hydrogels based on natural polysaccharides have demonstrated efficacy in epithelial recovery from cutaneous burn wounds. Here, we prepared a double-network hydrogel consisting of galactomannan (from Cassia grandis seeds) and κ-carrageenan (commercially sourced), cross-linked with CaCl2, as a matrix for immobilizing lactoferrin and/or Cramoll, aiming at its applicability as dressings for second-degree burn wounds. The formulations obtained [H - hydrogel, HL - hydrogel + lactoferrin, HC - hydrogel + Cramoll and HLC - hydrogel + lactoferrin + Cramoll] were analyzed rheologically as well as in terms of their stability (pH, color, microbial contamination) for 90 days. The burn was created with an aluminum bar (97 ± 3 °C) in the dorsal region of Wistar rats and subsequently treated with hydrogels (H, HL, HC, HLC) and control saline solution (S). The burn was monitored for 3, 7 and 14 days to evaluate the efficacy of the hydrogels in promoting wound healing. The hydrogels did not reveal significant pH or microbiological changes; there was an increase in brightness and a reduction in opacity for H. The rheological analysis confirmed the gel-like viscoelastic signature of the systems without substantial modification of the basic rheological characteristics, however HLC proved to be more rigid, due to rheological synergy when combining protein biomolecules. Macroscopic analyses confirmed centripetal healing with wound contraction: S < H < HC < HL < HLC. Histopathological analyses showed that hydrogel-treated groups reduced inflammation, tissue necrosis and fibrosis, while promoting re-epithelialization with focal acanthosis, especially in HLC due to a positive synergistic effect, indicating its potential as a promising therapy in the repair of burns.

Polysaccharide composition of an anticoagulant fraction from the aqueous extract of Marsypianthes chamaedrys (Lamiaceae).

Marsypianthes chamaedrys (Lamiaceae) is a medicinal plant popularly used against envenomation by snakebite. Pharmacological studies have shown that extracts of M. chamaedrys have antiophidic, anti-inflammatory and anticoagulant properties, supporting the ethnopharmacological use. In this study, an aqueous extract of aerial parts of M. chamaedrys showed anticoagulant activity in the activated partial thromboplastin time assay (0.54 IU/mg). The bioassay-guided fractionation using ethanol precipitation and gel filtration chromatography on Sephadex G-50 and Sephadex G-25 resulted in a water-soluble fraction with increased anticoagulant activity (Fraction F2-A; 2.94 IU/mg). A positive correlation was found between the amount of uronic acids and the anticoagulant potential of the active samples. Chemical and spectroscopic analyses indicated that F2-A contained homogalacturonan, type I rhamnogalacturonan, type II arabinogalactan and α-glucan. UV and FT-IR spectra indicated the possible presence of ferulic acid. Pectic polysaccharides and type II arabinogalactans may be contributing to the anticoagulant activity of the aqueous extract of M. chamaedrys in the APTT assay.

Tunicate Heparan Sulfate Enriched in 2-Sulfated ß-Glucuronic Acid: Structure, Anticoagulant Activity, and Inhibitory Effect on the Binding of Human Colon Adenocarcinoma Cells to Immobilized P-Selectin.

Heparin or highly sulfated heparan sulfate (HS) has been described in different invertebrates. In ascidians (Chordata-Tunicata), these glycosaminoglycans occur in intracellular granules of oocyte accessory cells and circulating basophil-like cells, resembling mammalian mast cells and basophils, respectively. HS is also a component of the basement membrane of different ascidian organs. We have analyzed an HS isolated from the internal organs of the ascidian Phallusia nigra, using solution 1H/13C NMR spectroscopy, which allowed us to identify and quantify the monosaccharides found in this glycosaminoglycan. A variety of α-glucosamine units with distinct degrees of sulfation and N-acetylation were revealed. The hexuronic acid units occur both as α-iduronic acid and ß-glucuronic acid, with variable sulfation at the 2-position. A peculiar structural aspect of the tunicate HS is the high content of 2-sulfated ß-glucuronic acid, which accounts for one-third of the total hexuronic acid units. Another distinct aspect of this HS is the occurrence of high content of N-acetylated α-glucosamine units bearing a sulfate group at position 6. The unique ascidian HS is a potent inhibitor of the binding of human colon adenocarcinoma cells to immobilized P-selectin, being 11-fold more potent than mammalian heparin, but almost ineffective as an anticoagulant. Thus, the components of the HS structure required to inhibit coagulation and binding of tumor cells to P-selectin are distinct. Our results also suggest that the regulation of the pathway involved in the biosynthesis of glycosaminoglycans suffered variations during the evolution of chordates.

A unique fucosylated chondroitin sulfate type II with strikingly homogeneous and neatly distributed α-fucose branches.

Fucosylated chondroitin sulfates (FCSs) and sulfated fucans (SFs) are conspicuous components of the body wall of sea cucumbers (Holothuroidea). FCSs are composed of a central core of chondroitin sulfate (CS) decorated with branches of mono- or both mono- and disaccharides of α-fucose (FCS types I and II, respectively). FCSs type II have heterogeneous and irregularly distributed α-fucose branches; however, the novel FCS type II from Holothuria lentiginosa described herein via solution nuclear magnetic resonance has strikingly homogeneous α-fucose branches neatly distributed along its CS core. This FCS is built up of three distinct sequential units composed of the typical CS disaccharides of FCSs, rich in ß-galactosamine-4,6diS, decorated with branches of α-Fucp-2,4diS, α-Fucp-3,4diS or α-Fucp[1→3]α-Fucp-4S[1→ linked to the position 3- of the ß-glucuronic acid. Conformational analyses of these repetitive units revealed a fairly rigid structure despite of the high sulfate content of their α-fucose branches. We also determined the structure of the SF from H. lentiginosa as a repetitive tetrasaccharide sequence composed of →3]α-Fucp-2,4diS[1→3]α-Fucp[1→3]α-Fucp-2S[1→3]α-Fucp-2S[1→. Furthermore, we determined that the nonsulfated α-fucose units present in FCS type II did not interfere with their anticoagulant potencies and affinities to calcium. FCS is an autapomorphic molecular character of the class Holothuroidea and the composition of their α-fucose branches differs in a species-specific manner. Branches containing α-Fucp-2,4diS are the most common within the extant holothurians, being found in 90% of the FCSs characterized thus far.

Xyloglucan from Hymenaea courbaril var. courbaril seeds as encapsulating agent of l-ascorbic acid.

This study evaluated the l-ascorbic acid (AA) encapsulation in microparticles of xyloglucan (XAA) extracted from Hymenaea courbaril seeds by spray drying (SD) and its application in tilapia fish burgers. The encapsulation efficiency was 96.34±1.6% and the retention of the antioxidant activity was of 89.48±0.88% after 60days at 25°C. SEM images showed microspheres with diameters ranging from 4.4 to 34.0µm. FTIR spectrum confirmed the presence of AA in xyloglucan microparticles, which was corroborated by DSC and TGA. The release of ascorbic acid was found to be pH-dependent. The application of XAA in tilapia fish burger did not change the pH after heating and the ascorbic acid retention was greater compared to its free form. The results indicate that xyloglucan can be used to encapsulate AA by SD and suggest that XAA was able to reduce undesirable organoleptic changes in fish burgers.

Healing activity evaluation of the galactomannan film obtained from Cassia grandis seeds with immobilized Cratylia mollis seed lectin.

Galactomannan films from Cassia grandis seeds, associated or not with Cramoll 1,4, were used on topical wounds of rats for the evaluation of the healing process during 14days. All of the films were evaluated by cytotoxic assay, FTIR and lectin hemagglutinating activity (HA). Forty-five male rats were submitted to aseptic dermal wounds (Ø=0.8cm) and divided in groups (n=15): control, test 1, and test 2, treated respectively with saline, galactomannan film and film with immobilized Cramoll 1,4. Macroscopic evaluations were performed by clinical observations and area measurements, and microscopic analysis by histological criteria. Epithelial cell proliferation and differentiation was immunohistochemically assessed using CK14 and PCNA. The presence of CO peaks in the FTIR spectrum confirmed the immobilization of Cramoll 1,4 in the film, while the residual HA confirmed the stability of the lectin after immobilization with 90.94% of the initial HA. The films presented non-cytotoxicity and cell viability exceeding 80%. All of the animals presented re-epithelization around 10days, furthermore test 2 group showed a diffuse response at the stromal tissue and the basal layer associated with wounds completely closed with 11days of experiment. The results suggest a promising use of the films as topical wound curatives.

Sulfated Glycans in HIV Infection and Therapy.

The Human Immunodeficiency Virus (HIV) is the infectious agent causative of the Acquired Immune Deficiency Syndrome (AIDS). The number of HIV-infected people in the globe is incredibly large. Contrary to the big contamination rates, the incidence rate recently reported of AIDS-related deaths is fairly lower, less than 5% of the total infected population. The reduced rates of the AIDS-associated deaths rely primarily on the growing availability, variety and efficiency of the antiretroviral treatments. During the initial molecular events of the HIV infectivity, the glycoprotein gp120 on the HIV envelop must interact with CD4 and Heparan Sulfate (HS) proteoglycans on the surface of the host cells in order to enable HIV attachment, fusion and entry into these cells. In addition, the Trans-Activator Transcription (Tat), capable to enhance transcription and HIV virulence during infectivity, also binds to HS proteoglycans. The HS binding enables translocation of Tat proteins into the host cells. Certain chemokines and HS competitors such as exogenous glycosaminoglycans and other sulfated glycans, including those isolated from marine organisms, have been extensively studied as potential antiretroviral agents. This article is centered on revisiting the three above-mentioned functions of the sulfated glycans in the HIV infectivity and therapy: the essential roles played by HS in interactions with (1) gp120 during the HIV-host interaction, and (2) with Tat for its translocation into the host cells, and (3) the potential antiretroviral effects exerted by exogenous sulfated glycans of varying structures and origins.

Combined therapy using low level laser and chitosan-policaju hydrogel for wound healing.

We have evaluated the effect of POLI-CHI hydrogel based on policaju (POLI) from cashew tree (Anacardium occidentale L.) gum and chitosan (CHI), associated or not with Low level laser therapy (LLLT), in wound healing. Sixty male Wistar rats were assigned into four groups: POLI-CHI hydrogel (H); LLLT (L); POLI-CHI with LLLT (HL) and saline control (C). Macroscopic evaluations were carried out using clinical observations and area measurements, as well as microscopic analysis by histological criteria. H and HL presented more esthetical scar tissue and larger wound contraction compared to C. Histopathological analyzes showed: stronger presence of fibrin-leukocyte crust in L and HL at day 3; stronger collagen presence in H, L and HL; weak presence of focal necrosis at 7 and 14days in H; weak neutrophilic exudate in H, L and HL; regression of the vascular neoformation at 7days in H, and modulation of the same in L and HL. These results demonstrated that POLI-CHI contributed to more efficient healing process and modulation of the inflammation; furthermore, the combined use with LLLT subtle potentiated this process.

NMR structural biology of sulfated glycans.

Sulfated fucans, sulfated galactans, and glycosaminoglycans are extensively studied worldwide in terms of both structure and biomedical functions. Liquid-state nuclear magnetic resonance (NMR) spectroscopy is the most employed analytical technique in structural analysis of these sulfated glycans. This is due to the fact that NMR-based analyses enable a series of achievements such as (i) accurate structure characterization/determination; (ii) measurements of parameters regarding molecular motion (dynamics); (iii) assessment of the 3D structures (usually assisted by computational techniques of Molecular Modeling and/or Molecular Dynamics) of the composing monosaccharides (ring conformers) and the overall conformational states of the glycan chains either free in solution or bound to proteins; and (iv) analysis of the resultant intermolecular complexes with functional proteins through either the protein or the carbohydrate perspective. In this review, after a general introduction about the principal NMR parameters utilized for achieving this set of structural information, discussion is given on NMR-based studies of some representative sulfated fucans, sulfated galactans, and glycosaminoglycans. Due to the growing number of studies concerning both structure and function of sulfated glycans and the widely use of NMR spectroscopy in such studies, a review paper discussing (i) the most experiments employed for analysis, (ii) procedures used in data interpretation, and (iii) the general aspects of the sulfated glycans, is timely in the literature.

NMR-based conformation and dynamics of a tetrasaccharide-repeating sulfated fucan substituted by different counterions.

The sulfated fucan from the sea urchin Lytechinus variegatus is composed of the repetitive sequence [-3)-α-l-Fucp-4( OSO3-)-(1-3)-α-l-Fucp-2,4-di( OSO3-)-(1-3)-α-l-Fucp-2( OSO3-)-(1-3)-α-l-Fucp-2( OSO3-)-(1-]n . Conformation (of rings and chains) and dynamics of this tetrasaccharide-repeating sulfated fucan substituted by Na(+) , Ca(2+) , and Li(+) as counterions have been examined through experiments of liquid-state nuclear magnetic resonance spectroscopy. Scalar coupling and nuclear Overhauser effect (NOE)-based data have confirmed that all composing units occur as (1) C4 chair conformer regardless of the cation type, unit position within the repeating sequence, and sulfation type. Chain conformation determined by NOE signal pattern assisted by molecular modeling for a theoretical octasaccharide has shown a similar linear 3D structure for the three differently substituted forms. Data derived from spin-relaxation measurements have indicated a contribution of counterion type to dynamics. The calcium-based preparation has shown the highest mobility while the sodiated one showed the lowest mobility. The set of results from this work suggests that counterion type can affect the physicochemical properties of the structurally well-defined sulfated fucan. The counterion effect seems to impact more on the structural mobility than on average conformation of the studied sulfated glycan in solution.

Investigating a galactomannan gel obtained from Cassia grandis seeds as immobilizing matrix for Cramoll lectin.

Characterization, with emphasis on the rheological properties, of Cassia grandis seeds galactomannan gel containing immobilized Cramoll 1-4 is presented. The gels, with and without immobilized Cramoll 1-4, were evaluated along time by rheometry, pH, color, microbial contamination and lectin hemagglutinating activity (HA). Rheological determinations confirmed the gels to be very stable up to 30 days with variations occurring after this period. Rheological data also showed that the gel/Cramoll 1-4 immobilizing matrix loses its elastic modulus substantially after 60 days. Both gels presented no microbial contamination as well as a pH close to neutral. Colorimetric parameters demonstrated the gels transparency with occasional yellowness. The opacity of the galactomannan gel did not change significantly along the study; the same did not occur for the gel with immobilized Cramoll 1-4 as a statistically significant reduction of its opacity was observed. In what concerns immobilized Cramoll 1-4 HA, up to 90% of its initial HA was maintained after 20 days, with a decrease to 60% after 60 days. These results combined with the thickening and stabilizing characteristics of the galactomannan gel make this gel a promising immobilizing matrix for Cramoll 1-4 that can be further exploited for clinical and cosmetic applications.

Development and characterization of a new hydrogel based on galactomannan and κ-carrageenan.

A new hydrogel based on two natural polysaccharides was prepared in aqueous medium with 1.7% (w/v) galactomannan (from Cassia grandis seeds) and different concentrations of κ-carrageenan (0.3, 0.4 and 0.5%w/v), CaCl2 (0.0, 0.1 and 0.2M) and pH (5.0, 5.5 and 6.0), using a full factorial design based on rheological parameters. The best formulation was obtained with 1.7% (w/v) galactomannan and 0.5% (w/v) κ-carrageenan, containing 0.2M CaCl2 at pH 5.0. Nuclear magnetic resonance and scanning electron microscopy where used in order to characterize the hydrogel formulation. A shelf life study was carried out with this formulation along 90 days-period of storage at 4 °C, evaluating pH, color, microbial contamination and rheology. This hydrogel showed no significant changes in pH, no microbial contamination and became more translucent along the aging. Analyses by nuclear magnetic resonance and rheology showed a larger organization of the polysaccharides in the hydrogel matrix. The results demonstrated that this hydrogel was stable with possible applications in medical and cosmetic fields.

Development and characterization of hydrogels based on natural polysaccharides: policaju and chitosan.

The development of hydrogels based on natural polysaccharides was investigated by preparing mixtures of policaju/chitosan at weight ratios of 1:4 and 2:3. Utilizing dynamic light scattering (DLS) techniques for these mixtures, an increase on the hydrodynamic particle radius was observed varying their pH from 3.0 to 12.0. Furthermore, a reduction of ζ-potential was also observed for the same pH interval. Following rounds of drying/hydration cycles at a specific pH value, hydrogel matrices were formed. The pore size distribution of these formed hydrogels was examined using scanning electron microscopy. Further FT-IR analyses confirmed a physical interaction between the polysaccharides policaju and chitosan. Swelling experiments revealed water uptake values, after 24h of immersion in water, close to 270% for 1:4, and 320% for 2:3 hydrogels. Finally, rheological measurements were then conducted in order to confirm hydrogel viscoelastic features. These results indicate a promising road to biomaterials fabrication and biomedical applications.

Purification of a lectin from Canavalia ensiformis using PEG-citrate aqueous two-phase system.

A PEG/citrate aqueous two-phase system was tested in the partition of commercial Concanavalin A (Con A) and subsequently applied to the extraction and purification of Con A from the crude extract of Canavalia ensiformis seeds. Con A was successfully extracted to the bottom phase of a system composed of 22% (w/w) PEG8000 and 12% (w/w) citrate at pH 6.0. The obtained purification factor was 11.5 without any loss in the hemagglutinating activity. The purity of extracted lectin was confirmed by SDS-PAGE analysis.