F. M. Gama

BC nanofibres: In vitro study of genotoxicity and cell proliferation

Nanomaterials have unusual properties not found in the bulk materials, which can be exploited in numerous applications such as biosensing, electronics, scaffolds for tissue engineering, diagnostics and drug delivery. However, research in the past few years has turned up a range of potential health hazards, which has given birth to the new discipline of nanotoxicology. Bacterial cellulose (BC) is a promising material for biomedical applications, namely due its biocompatibility. Although BC has been shown not to be cytotoxic or genotoxic, the properties of isolated BC nanofibres (NFs) on cells and tissues has never been analysed. Considering the toxicity associated to other fibre-shaped nanoparticles, it seems crucial to evaluate the toxicity associated to the BC-NFs. In this work, nanofibres were produced from bacterial cellulose by a combination of acid and ultrasonic treatment. The genotoxicity of nanofibres from bacterial cellulose was analysed in vitro, using techniques previously demonstrated to detect the genotoxicity of fibrous nanoparticles. The results from single cell gel electrophoresis (also known as comet assay) and the Salmonella reversion assays showed that NFs are not genotoxicity under the conditions tested. A proliferation assay using fibroblasts and CHO cells reveals a slight reduction in the proliferation rate, although no modification in the cell morphology is observed.

Biocompatibility of poly(lactic acid) with incorporated graphene-based materials

The incorporation of graphene-based materials has been shown to improve mechanical properties of poly(lactic acid) (PLA). In this work, PLA films and composite PLA films incorporating two graphene-based materials - graphene oxide (GO) and graphene nanoplatelets (GNP) - were prepared and characterized regarding not only biocompatibility, but also surface topography, chemistry and wettability. The presence of both fillers changed the films surface topography, increasing the roughness, and modified the wettability - the polar component of surface free energy increased 59% with GO and decreased 56% with GNP. Mouse embryo fibroblasts incubated with both fillers exceeded the IC(50) in both cases with a concentration of 10 μg mL(-1). No variations in cell proliferation at the surface of the composite films were observed, except for those containing GO after 24 h incubation, which presented higher cell proliferation than pristine PLA films. Platelet adhesion to PLA and PLA/GNP films was lower in the presence of plasma proteins than when no proteins were present. Furthermore, incorporation of GNP into PLA reduced platelet activation in the presence of plasma proteins. The results indicated that low concentrations of GO and GNP may be incorporated safely in PLA to improve aspects relevant for biomedical applications, such as mechanical properties.

Characterization of cellulose surface free energy

Junta Nacional de Investigacao Cientifica e Tecnologica (JNICT) - INTERREG II/BM/03/REG II/3/97.

Polymeric nanogels as vaccine delivery systems

UNLABELLED Polymeric nanogels find a relevant field of application in the formulation of a new generation of therapeutic and preventive vaccines, aiming at the fine-tuned modulation of the immune response. Intrinsic properties of polymeric nanogels, such as material chemistry, size and shape, surface charge, and hydrophobicity or hydrophilicity, may be determining factors in shaping the induced immune response. These materials can thus work as synthetic adjuvants, which can also be conjugated with immunostimulants. Polymeric nanogels protect vaccine antigens from degradation in vivo and, surface-conjugated with antibodies or specific ligands, could increase active targeting specificity. This review covers the recent published data concerning the modulation of innate and adaptive immune responses by engineered polymeric nanogels and their potential application as delivery systems in vaccination. FROM THE CLINICAL EDITOR In this review, the utility of polymeric nanogels is discussed as adjuvants and protective agents for enhanced vaccination with more robust immune response and a more uniform outcome.

Development of a hybrid dextrin hydrogel encapsulating dextrin nanogel as protein delivery system.

Dextrin, a glucose polymer with low molecular weight, was used to develop a fully resorbable hydrogel, without using chemical initiators. Dextrin was first oxidized (oDex) with sodium periodate and then cross-linked with adipic acid dihidrazide, a nontoxic cross-linking molecule. Furthermore, a new bidimensional composite hydrogel, made of oxidized dextrin incorporating dextrin nanogels (oDex-nanogel), was also developed. The oDex hydrogels showed good mechanical properties and biocompatibility, allowing the proliferation of mouse embryo fibroblasts 3T3 cultured on top of the gel. The gelation time may be controlled selecting the concentrations of the polymer and reticulating agent. Both the oDex and oDex-nanogel hydrogels are biodegradable and present a 3-D network with a continuous porous structure. The obtained hybrid hydrogel enables the release of the dextrin nanogel over an extended period of time, paralleling the mass loss curve due to the degradation of the material. The dextrin nanogel allowed the efficient incorporation of interleukin-10 and insulin in the oDex hydrogel, providing a sophisticated system of controlled release. The new hydrogels present promising properties as an injectable carrier of bioactive molecules. Both proteins and poorly water-soluble low-molecular-weight drugs are efficiently encapsulated in the nanogel, which performs as a controlled release system entrapped in the hydrogel matrix.

Studies on the properties of Celluclast/Eudragit L-100 conjugate

A cellulase from Trichoderma reesei was immobilized on Eudragit L-100, a reversibly soluble polymer depending on the pH of the medium. The solubility of the modified cellulase was studied at different pH values. By changing the pH, the adsorption equilibrium of the derivatized proteins is switched towards the liquid phase, thus making recycling possible. This method allows for improved stability, without major loss of specific activity. The adsorption of cellulase on Eudragit lowers the enthalpy of denaturation, but affects only slightly the denaturation temperature. The use of carbodiimide was ineffective on linking the enzymes covalently to the polymer, since the immobilization process was found to be only mediated by non-covalent forces.

Improving the affinity of fibroblasts for bacterial cellulose using carbohydrate‐binding modules fused to RGD

The attachment of cells to biomedical materials can be improved by using adhesion sequences, such as Arg-Gly-Asp (RGD), found in several extracellular matrix proteins. In this work, bifunctional recombinant proteins, with a Cellulose-Binding Module (CBM), from the cellulosome of Clostridium thermocellum and cell binding sequences-RGD, GRGDY-were cloned and expressed in E.coli. These RGD-containing cellulose binding proteins were purified and used to coat bacterial cellulose fibres. Its effect on the cell adhesion/biocompatibility properties was tested using a mouse embryo fibroblasts culture. Bacterial cellulose (BC) secreted by Gluconacetobacter xylinus (=Acetobacter xylinum) is a material with unique properties and promising biomedical applications. CBMs adsorbs specifically and tightly on cellulose. Thus, they are a useful tool to address the fused RGD sequence (or other bioactive peptides) to the cellulose surface, in a specific and simple way. Indeed, fibroblasts exhibit improved ability to interact with bacterial cellulose sheets coated with RGD-CBM proteins, as compared with cellulose treated with the CBM, that is, without the adhesion peptide. The effect of the several fusion proteins produced was analyzed.

Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds

The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.

Enzymatic upgrade of old paperboard containers

The use of enzymes in secondary fiber (old paperboard containers) upgrading was investigated. The following aspects were analyzed: (i) the effect of several enzymes and (non-hydrolytic) cellulose-binding domains on the pulp and paper properties; (ii) factors influencing enzymatic treatment of secondary fiber: enzyme dosage and reaction time; and (iii) enzyme action on fractions with different fiber-length. In general, all the tested enzymatic preparations were able to improve the pulp drainability. In most cases this improvement was obtained at the expense of paper strength. The use of cellulose-binding domains allowed for the simultaneous increase in drainability and strength properties.

Proving the suitability of magnetoelectric stimuli for tissue engineering applications

A novel approach for tissue engineering applications based on the use of magnetoelectric materials is presented. This work proves that magnetoelectric Terfenol-D/poly(vinylidene fluoride-co-trifluoroethylene) composites are able to provide mechanical and electrical stimuli to MC3T3-E1 pre-osteoblast cells and that those stimuli can be remotely triggered by an applied magnetic field. Cell proliferation is enhanced up to ≈ 25% when cells are cultured under mechanical (up to 110 ppm) and electrical stimulation (up to 0.115 mV), showing that magnetoelectric cell stimulation is a novel and suitable approach for tissue engineering allowing magnetic, mechanical and electrical stimuli.