Chitosan/copper nanocomposites: Correlation between electrical and antibacterial properties.

Correlation between electrical and antibacterial properties of chitosan/copper nanocomposites (CS/CuNPs) is investigated. We aim at achieving the minimum CuNPs concentration in a CS-matrix while keeping high antibacterial activity. UV-vis, TEM and XRD measurements confirms the formation of polygonal metallic CuNPs (ca. 30-50 nm). Interactions between NH2/OH groups of CS and CuNPs were determined by FTIR and XPS suggesting Cu chelation-induced mechanism during the CuNPs formation. DC electrical conductivity measurements reveals a percolation threshold at CuNPs volumetric concentration of ca. 0.143%. Antibacterial assays against Gram-positive bacteria and DC measurements helps correlate the antibacterial potency to the electron transfer between the negatively charged bacteria and CuNPs. Our study suggests that nanocomposite's maximum antibacterial activity is obtained below the electrical percolation threshold at extremely low CuNPs concentrations; this fact may prove useful in the design of nontoxic nanocomposites for biomedical applications.

Combined antibacterial/tissue regeneration response in thermal burns promoted by functional chitosan/silver nanocomposites.

We report the combined antibacterial/tissue regeneration responses to thermal burns promoted by functional chitosan/silver nanocomposites (CS/nAg) with ultralow silver content (0.018wt.%, 7-30nm). Our approach allows one to produce CS/nAg nanocomposites without silver nanoparticles (nAg) agglomeration, with bactericide potency higher than 1wt.% of nAg (ca. 10nm) content and, promoting the healing process in controlled thermal burns. CS/nAg films exhibit high antibacterial activity against S. aureus and P. aeruginosa after 1.5h of incubation, demonstrating the bacterial penetration into hydrated films and their interaction with nAg. Additionally, exceptional healing of induced thermal burns was obtained by increasing myofibroblasts, collagen remodeling, and blood vessel neoformation. These factors are associated with epiderma regeneration after 7days of treatment with no nAg release. Our results corroborate the controlled synthesis of nAg embedded in CS matrix with combined antibacterial/biocompatibility properties aiming to produce functional nanocomposites with potential use in wound dressing and health care applications.

Preparación y Caracterización de Nanocompositos Quitosano-Cobre con Actividad Antibacteriana para aplicaciones en Ingeniería de Tejidos / Preparation and characterization of Copper Chitosan Nanocomposites with Antibacterial Activity for Applications in Tissue Engineering

RESUMEN: El presente trabajo describe la preparación de nanocompositos formulados a partir de quitosano (QS)/nanopartículas de cobre (nCu) con características antibacterianas y aplicación potencial en ingeniería de tejidos. Para ello, se prepararon nanocompositos mediante mezclado en solución asistido con ultrasonido con el objetivo de incrementar la dispersión de la carga nanométrica en el biopolímero. El análisis de FTIR demostró que la presencia de nCu en la matriz de QS favorece la interacción del nCu con los grupos amino/hidroxilo de la molécula del QS. Se determinó mediante UV-Vis que los nanocompositos QS/nCu presentan absorción asociada con la presencia de nanopartículas y la posible liberación de iones Cu2+ en medio líquido. Mediante AFM se determinó que el QS hidratado forma una malla con microporos, que puede favorecer la penetración de bacterias en el nanocomposito y su interacción con las nCu. Finalmente, se determinó el efecto antibacteriano del material al contacto con la bacteria Staphylococcus aureus, en donde se presenta una actividad antibacteriana superior al 90% entre los 90 y 180 min de interacción. Dichos resultados sugieren que es posible obtener nanomateriales antibacterianos biocompatibles para su posible aplicación en ingeniería tisular.

ABSTRACT: The Present work describes the preparation of nanocomposites based on chitosan (QS)/copper nanoparticles (nCu) with antibacterial properties and potential application in tissue engineering. For this purpose, nanocomposites were prepared by solution blending with ultrasound assisted, aiming to increase the nanoparticles dispersion in the biopolymer. FTIR analyses demonstrates that nCu supported in QS increase their interaction of nanoparticles with amine/hydroxyl groups of QS molecule. UV-Vis analyses demonstrates that QS/nCu nanocomposites have an absorption signal associated with the presence of nanoparticles and the possible Cu2+ ions release in liquid media. AFM analyses shown that hydrated QS form a mesh with micro pores, improving the bacterial penetration and the direct contact with nCu. This behavior was corroborated by antibacterial assays, where QS/nCu nanocomposites shown an antibacterial activity higher than 90% between 90-180 minutes of interaction. Our results suggest that is possible to obtain combined antibacterial/biocompatible nanomaterials with potential application in tissue engineering.

Insights into Entamoeba histolytica virulence modulation.

Entamoeba histolytica is able to invade human tissues by means of several molecules and biological properties related to the virulence. Pathogenic amebas use three major virulence factors, Gal/GalNAc lectin, amebapore and proteases, for lyse, phagocytose, kill and destroy a variety of cells and tissues in the host. Responses of the parasite to host components such as mucins and bacterial flora influence the behavior of pathogenic amebas altering their expression of virulence factors. The relative virulence of different strains of E. histolytica has been shown to vary as a consequence of changes in conditions of in vitro cultivation which implies substantial changes in basic metabolic aspects and factors directly and indirectly related to amebic virulence. Comparison of E. histolytica strains with different virulence phenotypes and under different conditions of growth will help to identify new virulence factor candidates and define the interplay between virulence factors and invasive phenotype. Virulence attenuate mutants of E. histolytica are useful also to uncover novel virulence determinants. The comparison of biological properties and virulence factors between E. histolytica and E. dispar, a non-pathogenic species, has been a useful approach to investigate the key factors involved in the experimental presentation of amebiasis and its complex regulation. The molecular mechanisms that regulate these variations in virulence are not yet known. Their elucidation will help us to better understand the gene expression plasticity that enables the effective adaptation of the ameba to changes in growth culture conditions and host factors.

Antisense inhibition of expression of the light subunit (35 kDa) of the Gal/GalNac lectin complex inhibits Entamoeba histolytica virulence.

One of the under-represented genes identified by cDNA representational difference analysis (RDA) between avirulent Entamoeba histolytica strain Rahman and virulent strain HM-1:IMSS was the amoebic light (35 kDa) subunit of the Gal/GalNac lectin complex. This lectin complex, which mediates the adhesion of the parasite to the target cell, also contains a heavy (170 kDa) subunit, which has the carbohydrate-binding domain. Stable transfectants of the virulent strain in which the expression of the 35 kDa subunit was inhibited by antisense RNA were not significantly affected in their adhesion activity to mammalian or bacterial cells but were strongly inhibited in their cytopathic activity, cytotoxic activity and in their ability to induce the formation of liver lesions in hamsters. These findings suggest that the 35 kDa subunit may have a specific function in the pathogenic pathway and provides a new insight into the role of this component of the Gal/GalNac lectin complex in amoebic virulence.

Down regulation of Entamoeba histolytica virulence by monoxenic cultivation with Escherichia coli O55 is related to a decrease in expression of the light (35-kilodalton) subunit of the Gal/GalNAc lectin.

Entamoeba histolytica virulence is related to a number of amebic components (lectins, cysteine proteinases, and amebapore) and host factors, such as intestinal bacterial flora. Trophozoites are selective in their interactions with bacteria, and the parasite recognition of glycoconjugates plays an important role in amebic virulence. Long-term monoxenic cultivation of pathogenic E. histolytica trophozoites, strains HK-9 or HM-1:IMSS, with Escherichia coli serotype O55, which binds strongly to the Gal/GalNAc amebic lectin, markedly reduced the trophozoites' adherence and cytopathic activity on cell monolayers of baby hamster kidney (BHK) cells. Specific probes prepared from E. histolytica lectin genes as well as antibodies directed against the light (35-kDa) and heavy (170-kDa) subunits of the Gal/GalNAc lectin revealed a decrease in the transcription and expression of the light subunit in trophozoites grown monoxenically with E. coli O55. This effect was not observed when E. histolytica was grown with E. coli 346, a mannose-binding type I pilated bacteria. Our results suggest that the light subunit of the amebic lectin is involved in the modulation of parasite adherence and cytopathic activity.

Antisense inhibition of expression of cysteine proteinases affects Entamoeba histolytica-induced formation of liver abscess in hamsters.

Trophozoites of virulent Entamoeba histolytica transfected with the antisense gene encoding cysteine proteinase 5 (CP5) have only 10% of the CP activity but retain their cytopathic activity on mammalian monolayers. In the present study we found that the transfected trophozoites with low levels of CP activity were incapable of inducing the formation of liver lesions in hamsters.

Biochemical properties of a neuraminidase of Trichomonas vaginalis.

Trichomonas vaginalis possesses a membrane-associated neuraminidase activity that is released into culture medium during its growth in vitro. The neuraminidase shows an optimum pH of 4.5 and a Km of 0.15 mM for 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid as a substrate. This enzyme releases mainly alpha-2,3-linked sialic acid because it is able to liberate sialic acid from sialyllactose (mainly alpha-2,3) but not from mucin (alpha-2,6) or fixed erythrocytes (mainly alpha-2,6). The neuraminidase activity is strongly inhibited by 2,3-dehydro-2-deoxy-N-acetyl neuraminic acid, whereas EGTA and Ca2+ do not affect the activity. Gel filtration-fast protein liquid chromatography of culture supernatant displays a single peak of neuraminidase activity with molecular weight 52,000. The levels of neuraminidase activity are variable in fresh and long-term grown isolates of T. vaginalis, regardless of time in culture. However, there are 2 kinds of isolates, 1 group with high neuraminidase activity and able to secrete the enzyme during growth and the other with low neuraminidase activity. The results suggest that T. vaginalis possesses a membrane-associated neuraminidase that is present to a variable degree in different isolates.

Effect of intestinal bacteria on the virulence of Entamoeba histolytica.

The effect on amebal virulence of Escherichia coli, Clostridium symbiosum and a mixture of lactobacilli was investigated. Amebas from HM1 and HK axenic strains were incubated with a single bacterial strain or lactobacilli for short, intermediate, or long periods and analyzed for their erythrophagocytosis, hemolytic activity and cytotoxicity in vitro and hepatic abscesses formation in vivo. It was found that in vitro virulence was enhanced by all studied bacteria in HK9 but not in HM1 strains, which only showed an increase in cytotoxicity. The virulence in vivo was solely increased in trophozoites of HM1 strain by all tested bacteria.

In vitro activity of nalidixic acid and its iron (III) complex on Entamoeba histolytica.

The in vitro activity of the antibacterial agent nalidixic acid (HNal) and its iron (III) complex (FeNal) against Entamoeba histolytica HM1 strain trophozoites in axenic or monoxenic (associated with Clostridium symbiosum) cultures was investigated. Using a dilution test with TYI-S-33 medium, this protozoan was found to be susceptible to both drugs, but FeNal showed amoebicidal activity only at concentrations higher than those used with HNal.