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.

A fluorescent quantum dot conjugate to probe the interaction of Enterolobium contortisiliquum trypsin inhibitor with cancer cells.

Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology.

Chemical composition and biological activities of the essential oil from Eugenia stipitata McVaugh leaves.

In the present study, the volatile components and cytotoxic, antibacterial, antioxidant, and antiprotozoal activities of the essential oil obtained from the leaves of Eugenia stipitata McVaugh (Myrtaceae) grown in the Brazilian Northeast region (Araripe) were investigated. The essential oil was obtained by hydrodistillation. The leaves of E. stipitata provided an oil yield of 0.13 ± 0.01% (w/w). The volatile compounds in the essential oil of E. stipitata were analysed using gas chromatography, and the volatile chemical composition was mainly composed of ß-eudesmol (15.28%), γ-eudesmol (10.85%), elemol (10.21%) and caryophyllene oxide (6.65%). The essential oil of E. stipitata was highly selective against Leishmania braziliensis and L. infantum promastigotes. The essential oil exhibited good antibacterial activity. E. stipitata essential oil showed low free-radical scavenging activity. Our results suggest that the E. stipitata essential oil is a relevant source of the primary compounds required for the development of antibacterial and antiprotozoal drugs.

Investigation of factors related to biofilm formation in Providencia stuartii.

Providencia stuartii is one of the Enterobacteriaceae species of medical importance commonly associated with urinary infections, which can also cause other ones, including uncommon ones, such as liver abscess and septic vasculitis. This bacterium stands out in the expression of intrinsic and acquired resistance to antimicrobials. Besides, it uses mechanisms such as biofilm for its persistence in biotic and abiotic environments. This study investigated the cellular hydrophobicity profile of clinical isolates of P. stuartii. It also analyzed genes related to the fimbrial adhesin in this species comparing with other reports described for other bacteria from Enterobacteriaceae family. The investigated isolates to form biofilm and had a practically hydrophilic cell surface profile. However, fimH and mrkD genes were not found in P. stuartii, unlike observed in other species of Enterobacteriaceae. These results show that P. stuartii has specificities regarding its potential for biofilm formation, which makes it difficult to destabilize the infectious process and increases the permanence of this pathogen in hospital units.

Bauhinia monandra leaf lectin (BmoLL) conjugated with quantum dots as fluorescent nanoprobes for biological studies: application to red blood cells.

Carbohydrates perform important physiological functions in eukaryotic and prokaryotic cells. Indeed, alterations in glycan patterns may be associated with disorders. The analysis of these sugars can be reached using nanoprobes composed by lectins associated with fluorescent nanoparticles. This study reports the conjugation of a galactose-binding lectin (BmoLL) isolated from Bauhinia monandra leaves with quantum dots (QDs) by adsorption. QDs-BmoLL conjugates showed bright fluorescence and the hemagglutination assay revealed that the lectin preserved its carbohydrate-binding ability after the conjugation. To evaluate the efficiency/specificity of the bioconjugate, ABO human red blood cells (RBCs) were used as biological models and the labeling was analyzed by flow cytometry. Among ABO blood groups, higher labeling (71.7 ± 5.9%) was detected for B-type RBCs, whose antigens have galactose in their structure. The specificity of labeling was confirmed since A- and O-types RBCs incubated with QDs-BmoLL, as well as B-type cells incubated with previously galactose-inhibited conjugates, were labeled below 6%. In AB-type RBCs, which simultaneously have B and A (N-acetylgalactosamine) antigens on their membrane, the labeling was ca. 14.1 ± 4.8%. Therefore, a successful conjugation was reached and QDs-BmoLL conjugates can be considered promising fluorescent nanoprobes for biological investigations.

Evaluating glucose and mannose profiles in Candida species using quantum dots conjugated with Cramoll lectin as fluorescent nanoprobes.

Glycoconjugates found on cell walls of Candida species are fundamental for their pathogenicity. Laborious techniques have been employed to investigate the sugar composition of these microorganisms. Herein, we prepared a nanotool, based on the fluorescence of quantum dots (QDs) combined with the specificity of Cramoll lectin, to evaluate glucose/mannose profiles on three Candida species. The QDs-Cramoll conjugates presented specificity and bright fluorescence emission. The lectin preserved its biological activity after the conjugation process mediated by adsorption interactions. The labeling of Candida species was analyzed by fluorescence microscopy and quantified by flow cytometry. Morphological analyses of yeasts labeled with QDs-Cramoll conjugates indicated that C. glabrata (2.7 µm) was smaller when compared to C. albicans (4.0 µm) and C. parapsilosis sensu stricto (3.8 µm). Also, C. parapsilosis population was heterogeneous, presenting rod-shaped blastoconidia. More than 90% of cells of the three species were labeled by conjugates. Inhibition and saturation assays indicated that C. parapsilosis had a higher content of exposed glucose/mannose than the other two species. Therefore, QDs-Cramoll conjugates demonstrated to be effective fluorescent nanoprobes for evaluation of glucose/mannose constitution on the cell walls of fungal species frequently involved in candidiasis.

Titanium dioxide nanotubes functionalized with Cratylia mollis seed lectin, Cramoll, enhanced osteoblast-like cells adhesion and proliferation.

An alternative to accelerate the osseointegration on titanium dioxide nanotubes (TNTs) used in osseointegrated implants is through the functionalization of these nanostructured surfaces with biomolecules. In this work, we immobilized a lectin with recognized mitogenic activity, the Cramoll lectin, extracted from Cratylia mollis seeds, on surfaces modified by TNTs. For the immobilization of Cramoll on TNTs surfaces, we used the Layer-by-Layer technique (LbL) by growing five alternate layers of poly(allylamine) hydrochloride (PAH) and poly(acrylic) acid (PAA); lastly we incubated the lectin, at different concentrations, with the TNTs-LbL. Before and after the immobilization procedures, the substrate surfaces were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and, electrochemical impedance spectroscopy (EIS). We also evaluated the Cramoll activity after immobilization on TNTs by using the lectin interaction with ovalbumin. The lectin did not lose its biological activity, even after immobilization onto nanotubular arrays. In addition, we observed an increase osteoblast-like cell adhesion on the TNTs-LbL-Cramoll system when compared to the bare TNTs surfaces. Moreover, a significative cell proliferation was identified on the substrates when Cramoll was immobilized at concentrations of 80, 160 and 320 µg/mL after 48 h of incubation by using the resazurin assay. Our results suggest that Cramoll was efficiently immobilized on a nanotubular array and this new platform presents a great potential to be tested in implantology.

Lectins as mitosis stimulating factors: Briefly reviewed.

Lectins are carbohydrate binding proteins that can stimulate cell proliferation. This property makes these biomolecules capable of being used as mitogen reagents to study the interaction with lymphocytes allowing evaluation of immunomodulatory action, since B and T lymphocytes are related to humoral and innate immunity, respectively. Isolated cells from spleen, which include lymphocytes, are widely applied as a model in screening lectin mitogenic capacity. This mitotic stimulus is initiated by interaction of the lectin with T-cell receptor on cell surface. This brief review article aims to explain how cell proliferation, especially lymphocytes, can be achieved through lectin induction. Additionally, this work intends to highlight the main colorimetric and radiographic techniques to encourage the scientific community in searching for new mitogenic lectins.

Quantum dot-Cramoll lectin as novel conjugates to glycobiology.

The optical properties of quantum dots (QDs) make them useful tools for biology, especially when combined with biomolecules such as lectins. QDs conjugated to lectins can be used as nanoprobes for carbohydrate expression analysis, which can provide valuable information about glycosylation changes related to cancer and pathogenicity of microorganisms, for example. In this study, we evaluated the best strategy to conjugate Cramoll lectin to QDs and used the fluorescent labeling of Candida albicans cells as a proof-of-concept. Cramoll is a mannose/glucose-binding lectin with unique biological properties such as immunomodulatory, antiparasitic, and antitumor activities. We probed covalent coupling and adsorption as conjugation strategies at different pH values. QDs conjugated to Cramoll at pH7.0 showed the best labeling efficiency in the fluorescence microscopy analysis. Moreover, QD-Cramoll conjugates remained brightly fluorescent and preserved identical biological activity according to hemagglutination assays. Flow cytometry revealed that approximately 17% of C. albicans cells were labeled after incubation with covalent conjugates, while approximately 92% of cells were labeled by adsorption conjugates (both at pH7.0). Inhibition assays confirmed QD-Cramoll specificity, which reduced the labeling to at most 3%. Therefore, the conjugates obtained by adsorption (pH7.0) proved to be promising and versatile fluorescent tools for glycobiology.

Functionalization of titanium dioxide nanotubes with biomolecules for biomedical applications.

Titanium (Ti) and its alloys are extensively used in the manufacture of implants because they have biocompatibility. The production of a nanostructured surface can be achieved by means of titanium dioxide nanotubes (TNTs) which can have dimensions equivalent to the nanometric components of human bone, in addition to increasing the efficiency of such implants. The search is ongoing for ways to improve the performance of these TNTs in terms of their functionalization through coating these nanotubular matrices with biomolecules. The biocompatibility of the functionalized TNTs can be improved by promoting rapid osseointegration, by preventing the adhesion of bacteria on such surfaces and/or by promoting a more sustained local release of drugs that are loaded into such TNTs. In addition to the implants, these nanotubular matrices have been used in the manufacture of high-performance biosensors capable of immobilizing principally enzymes on their surfaces, which has possible use in disease diagnosis. The objective of this review is to show the main techniques of immobilization of biomolecules in TNTs, evidencing the most recent applications of bioactive molecules that have been functionalized in the nanotubular matrices for use in implants and biosensors. This surveillance also proposes a new class of biomolecules that can be used to functionalize these nanostructured surfaces, lectins.

Treatment with pCramoll Alone and in Combination with Fluconazole Provides Therapeutic Benefits in C. gattii Infected Mice.

Cryptococcus gattii is one of the main causative agents of cryptococcosis in immunocompetent individuals. Treatment of the infection is based on the use of antimycotics, however, the toxicity of these drugs and the increase of drug-resistant strains have driven the search for more effective and less toxic therapies for cryptococcosis. pCramoll are isolectins purified from seeds of Cratylia mollis, a native forage plant from Brazil, which has become a versatile tool for biomedical application. We evaluated the effect of pCramoll alone and in combination with fluconazole for the treatment of mice infected with C. gatti. pCramoll alone or in combination with fluconazole increased the survival, reduced the morbidity and improved mice behavior i.e., neuropsychiatric state, motor behavior, autonomic function, muscle tone and strength and reflex/sensory function. These results were associated with (i) decreased pulmonary and cerebral fungal burden and (ii) increased inflammatory infiltrate and modulatory of IFNγ, IL-6, IL-10, and IL-17A cytokines in mice treated with pCramoll. Indeed, bone marrow-derived macrophages pulsed with pCramoll had increased ability to engulf C. gattii, with an enhanced production of reactive oxygen species and decrease of intracellular fungal proliferation. These findings point toward the use of pCramoll in combination with fluconazole as a viable, alternative therapy for cryptococcosis management.

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.

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.

Cratylia mollis lectin nanoelectrode for differential diagnostic of prostate cancer and benign prostatic hyperplasia based on label-free detection.

The research for new biomarkers of cancer has studied the role of fetuin glycoprotein on the metastatic disease diagnosis. Cratylia mollis is a lectin with high finity to fetuin, and used here to differentiate prostate cancer and benign prostatic hyperplasia. A label-free electrochemical nanosensor based on assembled carboxylated carbon nanotubes (COOH-CNTs) and poly-L-lysine (PLL) film was developed and applied to serum samples of prostate cancer positive for Gleason score. The electrode analytical response to fetuin in PBS samples, obtained by square wave voltammetry, exhibited a linear range from 0.5 to 25µgmL(-1), with a high correlation coefficient (r=0.994, p<0.001) and low limit of detection (0.017µgmL(-1)). The lectin nanoelectrode showed a good repeatability (1.24% RSD) and reproducibility (4.24% RSD). A pool of serum samples from prostate cancer patients with known the Gleason score were tested showing a significant statistically correlation. Thus, the lectin nanoelectrode was able to distinguish the degree of staging prostate cancer, providing the diagnostic differentiation of benign and malign hyperplasia. To the best of our knowledge, it is the first biosensor for this application using a lectin.

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.

A lectin from Bothrops leucurus snake venom raises cytosolic calcium levels and promotes B16-F10 melanoma necrotic cell death via mitochondrial permeability transition.

BlL, a galactose-binding C-type lectin purified from Bothrops leucurus snake venom, exhibits anticancer activity. The current study was designed to elucidate the cellular mechanisms by which BlL induces melanoma cell death. The viabilities of B16-F10 melanoma cells and HaCaT keratinocytes treated with BlL were evaluated. Necrotic and apoptotic cell death, cytosolic Ca(2+) levels, mitochondrial Ca(2+) transport and superoxide levels were assessed in B16-F10 melanoma cells exposed to BlL. We found that treatment with BlL caused dose-dependent necrotic cell death in B16-F10 melanoma cells. Conversely, the viability of non-tumorigenic HaCaT cells was not affected by similar doses of BlL. BlL-induced B16-F10 necrosis was preceded by a significant (2-fold) increase in cytosolic calcium concentrations and a significant (3-fold) increase in mitochondrial superoxide generation. It is likely that BlL treatment triggers B16-F10 cell death via mitochondrial permeability transition (MPT) pore opening because the pharmacological MPT inhibitors bongkrekic acid and Debio 025 greatly attenuated BlL-induced cell death. Experiments evaluating mitochondrial Ca(2+) transport in permeabilized B16-F10 cells strongly supported the hypothesis that BlL rapidly stimulates cyclosporine A-sensitive Ca(2+)-induced MPT pore opening. We therefore conclude that BlL causes selective B16-F10 melanoma cell death via dysregulation of cellular Ca(2+) homeostasis and Ca(2+)-induced opening of MPT pore.

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.