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.

Effects including photobiomodulation of galactomannan gel from Cassia grandis seeds in the healing process of second-degree burns.

The epithelium recovery of skin-burned wounds has been currently achieved by several therapies, for example, hydrogel-based dressings and photobiomodulation therapy (PBMT). Thus, this work aimed to evaluate the healing activity of Cassia grandis seeds' galactomannan gel, associated or not with PBMT, in second-degree burns. Sixty male Wistar rats were assigned to four groups: Control (CG), Gel (GG), Laser/PBMT (LG), and Laser+Gel (GLG). Burns were made with an aluminum bar (90 °C), and submitted to clinical observations diary and area measurements at specific days. Microscopic analysis was based on histological criteria. The results showed that GG, LG, and GLG had a higher contraction rate (p < 0.05) than CG on the 14th experimental day, not differing from each other (∼95 %). At 21 days, all groups showed complete contraction (p > 0.05). Considering the histological results, LG and GLG showed excellent pro-wound healing properties after 14 days; at 21 days, all groups showed wound recovery compared to previous days. In view of the macroscopic and microscopic observations, the isolated treatments (Gel or Laser) effectively accelerated healing; however, the association (Laser+Gel) promoted re-epithelialization and stromal remodeling with better evolution of epithelium recovery due to the positive synergistic effect, thus emerging as a promising therapeutic alternative in the repair of burns.

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.

Lectin-Carbohydrate Interactions: Implications for the Development of New Anticancer Agents.

Lectins are a large group of proteins found in animals, plants, fungi, and bacteria that recognize specific carbohydrate targets and play an important role in cell recognition and communication, host-pathogen interactions, embryogenesis, and tissue development. Recently, lectins have emerged as important biomedical tools that have been used in the development of immunomodulatory, antipathogenic, and anticancer agents. Several lectins have been shown to have the ability to discriminate between normal cells and tumor cells as a result of their different glycosylation patterns. Furthermore, the specific binding of lectins to cancer cells has been shown to trigger mechanisms that can promote the death of these abnormal cells. Here, we review the importance of lectins-carbohydrates interactions in cancer therapy and diagnosis. We examine the use of lectins in the modification of nanoparticles (liposomes, solid lipid nanoparticles and other polymers) for anticancer drug delivery. The development of drug delivery systems (liposomes, alginate/chitosan microcapsules, alginate beads) carrying some antitumor lectins is also discussed. In these cases, the processes of cell death induced by these antitumor lectins were also showed (if available). In both cases (lectin-conjugated polymers or encapsulated lectins), these new pharmaceutical preparations showed improved intracellular delivery, bioavailability and targetability leading to enhanced therapeutic index and significantly less side effects.

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.

Immobilization of bioactive compounds in Cassia grandis galactomannan-based films: Influence on physicochemical properties.

Galactomannan extracted from Cassia grandis seeds was used for the production of films containing different concentrations of the bioactive compounds lactoferrin (LF), bioactive peptides (BAPs), and phytosterols. SEM, FTIR, mechanical and thermal properties, colour, moisture content (MC), solubility, water vapour permeability (WVP), and contact angle (CA) were performed evaluating the effect of increasing concentrations of bioactive compounds on the films' physicochemical properties. The immobilization of bioactive compounds leads to films with roughness on their surface, as observed by SEM. The thermal events demonstrated that bioactive compounds avoided the establishment of more hydrogen bonds when compared to galactomannan control film; this behaviour was also confirmed by FTIR. All the studied films had a strong whiteness tendency as well as a yellowish appearance. The addition of Lf reduced MC and solubility values and leads to an increase of WVP and CA values, while the addition of BAPs and phytosterols did not changed the films solubility. The mechanical properties were affected by the addition of bioactive compounds, which improved the stiffness of the films. Galactomannan-based films from C. grandis showed to be a promising structure for the immobilization of biomolecules, pointing at a significant number of possible applications in food and pharmaceutical industries.

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.

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.

Structure and rheological properties of a xyloglucan extracted from Hymenaea courbaril var. courbaril seeds.

Hymenaea courbaril var courbaril seed xyloglucan was efficiently extracted with 0.1M NaCl, followed by ethanol precipitation (yield=72±5% w/w). Its amorphous structure was identified by the pattern of X-ray diffraction. The monosaccharide composition was determined by GC/MS analysis of the alditol acetates and showed the occurrence of glucose:xylose:galactose:arabinose (40:34:20:6). One-(1D) and two-dimensional-(2D) NMR spectra confirmed a central backbone composed by 4-linked ß-glucose units partially branched at position 6 with non-reducing terminal units of α-xylose or ß-galactose-(1→2)-α-xylose disaccharides. The xyloglucan solution was evaluated by dynamic light scattering and presents a polydisperse and practically neutral profile, and at 0.5 and 1.0% (w/v) the solutions behave as a viscoelastic fluid. The polysaccharide did not show significant antibacterial or hemolytic activities. Overall our results indicate that xyloglucan from H. courbaril is a promising polysaccharide for food and pharmaceutical industries.

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.

Characterization and rheological study of the galactomannan extracted from seeds of Cassia grandis.

Galactomannan extracted from seeds of Cassia grandis with 0.1M NaCl, followed by ethanol precipitation, presented a yield of 36 ± 8%. The polysaccharide has a constant mannose/galactose ratio (2.44:1). Methylation analysis, one and two dimensional NMR spectroscopy confirmed that the polysaccharide has a central core composed of 4-linked ß-mannose units, with branches of galactose, linked to the carbohydrate core through α(1-6) linkage. The amorphous nature of the galactomannan was confirmed by X-ray diffraction. Rheological characterization exhibited Newtonian plateaus followed by shear-thinning zones characteristic of polymer solutions up to 1.5% (w/v) and above this value the system exhibited yield stress associated with a weak gel. Adjusting stress-strain curves confirmed a 1.6% (w/v) as the galactomannan concentration value for the sol-gel transition. These results indicate that the galactomannan extracted from C. grandis seeds presents rheological characteristics suitable for applications in pharmaceutical, biomedical, cosmetic and food industries.

Fermentation medium for collagenase production by Penicillium aurantiogriseum URM4622.

Medium composition and culture conditions for maximal collagenase production by Penicillium aurantiogriseum URM4622 were optimized using a response surface approach. A full two-level design on three factors (initial medium pH, soybean flour concentration, and temperature) was employed to identify the most significant fermentation parameters for collagenase production, and a subsequent central composite design (CCD) was used to find the optimal levels of the two most significant factors (initial medium pH and soybean flour concentration). The design results indicated that the initial medium pH and the temperature had significant negative main effects, whereas the substrate concentration had a positive effect on the collagenase production. The maximum collagenolytic activity predicted by the fitted response surface was expected to occur at pH 7.21, 1.645% soybean flour concentration and 24°C. Three replicate experiments were run at these conditions and yielded an activity response of 283.36 ± 1.33 U, which not only is the highest obtained in this study but also represents a 5-fold increase over the lowest response observed in the initial design. Since all experiments were carried out with an inexpensive substrate, the final results point out to a cost-effective medium for collagenase production with potential industrial-scale 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.