Hyaluronic acid-amphotericin B nanocomplexes: a promising anti-leishmanial drug delivery system.

The development of an effective amphotericin B (AmB) formulation to replace actual treatments available for leishmaniasis, which present serious drawbacks, is a challenge. Here we report the development of hyaluronic acid-amphotericin B self-assembled nanocomplexes (HA-AmB), processed by freeze-drying (FD) or nano spray-drying (SD), using a simple process that favors the non-covalent drug-polysaccharide association in an amorphous state. These water-soluble formulations, which presented a nanometric size (300-600 nm), high colloidal stability (zeta potential around -39 mV) and an AmB loading (15-18%) in aggregated and super aggregated states, demonstrated less in vitro cytotoxic and hemolytic effects compared to the free-drug. A significant decrease in the number of intramacrophagic L. infantum amastigotes upon treatment (IC50 of 0.026 and 0.030 µM for HA-AmB FD and HA-AmB SD, respectively) was also observed, and the best selectivity index (SI) was observed for the HA-AmB SD nanocomplex (SI of 651). Intravenous administration of the HA-AmB SD nanocomplex for 3 alternate days showed an effective parasite reduction in the spleen and liver of C57BL/6 mice without signs of toxicity commonly observed upon free-AmB treatment. Although lower than that achieved with AmBisome® in the liver, the observed parasite reduction for the nanocomplex was of a similar order of magnitude. The efficacy, stability, safety and low cost of the HA-AmB SD nanocomplex highlight its potential as an alternative treatment for leishmaniasis.

Targetability of hyaluronic acid nanogel to cancer cells: In vitro and in vivo studies.

We have, in previous work developed, characterized and evaluated the biocompatibility of an engineered hyaluronic acid nanogel. Here we assess the targetability of a hyaluronic acid nanogel towards CD44 overexpressing cells, in vitro and in vivo. Results obtained by flow cytometry and confocal fluorescence microscopy shows that nanogel is greatly internalized by non-small cancer lung cells (A549 cells), that overexpress CD44 receptors. The biodistribution and tumor targetability of the nanogel labelled with a near-infrared (NIR) probe were performed, in mice, through a non-invasive imaging system. Results revealed nanogel high targetability towards an induced subcutaneous A549 tumor. Nanogels pharmacokinetics was evaluated also in healthy animals, and Alexa Fluor 680 labelled nanogel exhibited higher accumulation in liver, kidneys and skin. Also, a comparative biodistribution study was performed, using two NIR imaging probes, Cy5.5 and Alexa Fluor 680.

Processing and size range separation of pristine and magnetic poly(l-lactic acid) based microspheres for biomedical applications.

Biodegradable poly(l-lactic acid) (PLLA) and PLLA/CoFe2O4 magnetic microspheres with average sizes ranging between 0.16-3.9µm and 0.8-2.2µm, respectively, were obtained by an oil-in-water emulsion method using poly(vinyl alcohol) (PVA) solution as the emulsifier agent. The separation of the microspheres in different size ranges was then performed by centrifugation and the colloidal stability assessed at different pH values. Neat PLLA spheres are more stable in alkaline environments when compared to magnetic microspheres, both types being stable for pHs higher than 4, resulting in a colloidal suspension. On the other hand, in acidic environments the microspheres tend to form aggregates. The neat PLLA microspheres show a degree of crystallinity of 40% whereas the composite ones are nearly amorphous (17%). Finally, the biocompatibility was assessed by cell viability studies with MC3T3-E1 pre-osteoblast cells.

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.

Dextrin-based nanomagnetogel: in vivo biodistribution and stability.

The biodistribution profile of a new dextrin nanomagnetogel, which consists of γ-Fe2O3 superparamagnetic nanoparticles loaded within a polymeric matrix of modified dextrin, was studied in mice. The nanomagnetogel bear a monomodal size distribution profile (average diameter 110 nm) close to neutral surface charge and higher relaxivity (r2 = 215-248 mM(-1) s(-1) and r2/r1 = 13-11) than those of commercial formulations (r2 = 160-177 mM(-1) s(-1) and r2/r1 = 4-7). Also, the observed blood half-life-approximately 4 h-is superior to that of similar commercially available formulations, which remain for a few minutes in circulation. PEGylation resulted in 1.7- and 1.2-fold lower accumulation in the liver and spleen, respectively, within the first 24 h. Noteworthy, a good correlation was obtained between the amount of polymer (quantified by scintigraphy) in the spleen, 48 h after administration, and the amount of iron physically loaded through hydrophobic interactions (quantified by ICP) indicating the absence of iron leakage from the polymeric matrix. This study provides evidence of the in vivo stability of a self-assembled nanomagnetogel, a relevant feature which is seldom reported in the literature.

Neuronal cells' behavior on polypyrrole coated bacterial nanocellulose three-dimensional (3D) scaffolds.

In this work, polypyrrole (PPy) was in situ polymerized onto the surface of bacterial nanocellulose (BNC) produced by Gluconacetobacter xylinus, by chemical oxidation in aqueous medium using ammonium persulfate. Composites (BNC/PPy) were produced with varying concentrations of pyrrole (Py). The produced BNC/PPy membranes were used as a template for the seeding of PC12 rat neuronal cells. Cell suspensions were directly seeded onto the surfaces of the BNC/PPy membranes. The Py concentration affected the behavior of neuronal cells that adhered and grew significantly more on BNC/PPy comparatively to BNC. Scanning electron microscopy (SEM) micrographs revealed that PC12 cells adhered on the surface of the BNC and BNC/PPy membranes. Conductive PPy coatings on nanofibers acting as an active interface for tissue engineering may be used to regulate cell activity through electrical stimulations.

New dextrin nanomagnetogels as contrast agents for magnetic resonance imaging.

This study aims at the production and characterization of a "nanomagnetogel" consisting of superparamagnetic iron oxide nanoparticles (γ-Fe2O3) stabilized within a hydrophobized-dextrin nanogel. The nanomagnetogel obtained was extensively characterized with respect to physico-chemical (transmission electron microscopy, cryo-scanning electron microscopy, dynamic light scattering, small angle X-ray scattering), magnetic (relaxometry, MIAplex) and biocompatibility (interaction with cells) properties. The obtained nanomagnetogel formulation, with about 4 mM of iron and a diameter of 100 nm, presents relevant features as a promising magnetic resonance imaging (MRI) contrast agent, noteworthy superparamagnetic behavior, high stability, narrow size distribution and potential for magnetic guidance to target areas by means of an external magnetic field. High values of transverse relaxivity make the nanomagnetogel a promising T2 contrast agent, allowing enhanced lesion detectability through magnetic resonance imaging. The nanomagnetogel demonstrated non-toxicity for 3T3 fibroblast cultures and was efficiently internalized by bone marrow-derived macrophages, therefore having potential as a contrast agent for MRI of the organs associated with the reticuloendothelial system (spleen, liver). The production of the nanomagnetogel is simple and easy to scale up, thus offering great technological potential.

Studies on the biodistribution of dextrin nanoparticles.

The characterization of biodistribution is a central requirement in the development of biomedical applications based on the use of nanoparticles, in particular for controlled drug delivery. The blood circulation time, organ biodistribution and rate of excretion must be well characterized in the process of product development. In this work, the biodistribution of recently developed self-assembled dextrin nanoparticles is addressed. Functionalization of the dextrin nanoparticles with a DOTA-monoamide-type metal chelator, via click chemistry, is described. The metal chelator functionalized nanoparticles were labelled with a gamma-emitting (153)Sm(3+) radioisotope and the blood clearance rate and organ biodistribution of the nanoparticles were obtained. The effect of PEG surface coating on the blood clearance rate and organ biodistribution of the nanoparticles was also studied.

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.

Enzymatic versus chemical deinking of non-impact ink printed paper.

Enzymatic versus chemical deinking is examined for MOW and photocopy prints. Several enzymatic preparations and two fibre/ink particle separation methods are tested. Deinking was monitored by image analysis and standard pulp and paper characterisation procedures. The effectiveness of the fibre/ink particle separation method depends on the ink particle's size: for smaller particles a washing step is recommended whereas for larger particles, the use of flotation is necessary. The enzymatic treatment is a competitive alternative for MOW and photocopy paper deinking. However, the process requires the selection of an adequate enzymatic preparation for each paper grade.

Characterisation and application of glycanases secreted by Aspergillus terreus CCMI 498 and Trichoderma viride CCMI 84 for enzymatic deinking of mixed office wastepaper.

Two enzymatic extracts obtained from xylan-grown Aspergillus terreus CCMI 498 and cellulose-grown Trichoderma viride CCMI 84 were characterised for different glycanase activities. Both strains produce extracellular endoxylanase and endoglucanase enzymes. The enzymes optimal activity was found in the temperature range of 45-60 degrees C. Endoglucanase systems show identical activity profiles towards temperature, regardless of the strain and inducing substrate. Conversely, the endoxylanases produced by both strains showed maximal activity at different pH values (from 4.5 to 5.5), being the more acidic xylanase produced by T. viride grown on cellulose. The endoglucanase activities have an optimum pH at 4.5-5.0. The endoxylanase and endoglucanase activities exhibited high stability at 50 degrees C and pH 5.0. Mannanase, beta-xylosidase, and amylase activities were also found, being the first two activities only present for T. viride extract. These two enzymatic extracts were used for mixed office wastepaper (MOW) deinking. When the enzymatic extract from T. viride was used, a further increase of 24% in ink removal was obtained by comparison with the control. Both enzymes contributed to the improvement of the paper strength properties and the obtained results clearly indicate that the effective use of enzymes for deinking can also contribute to the pulp and paper properties improvement.

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.

Selective enzyme-mediated extraction of capsaicinoids and caratenoids from chili guajillo puya (Capsicum annum L.) using ethanol as solvent.

The selective extraction of capsaicinoids and carotenoids from chili guajillo "puya" flour was studied. When ethanol was used as solvent, 80% of capsaicinoids and 73% of carotenoids were extracted, representing an interesting alternative for the substitution of hexane in industrial processes. Additionally, when the flour was pretreated with enzymes that break the cell wall and then dried, extraction in ethanol increased to 11 and 7% for carotenoid and capsaicinoid, respectively. A selective two-stage extraction process after the treatment with enzymes is proposed. The first step uses 30% (v/v) ethanol and releases up to 60% of the initial capsaicinoids, and the second extraction step with industrial ethanol permits the recovery of 83% of carotenoids present in the flour.

Bleach boosting and direct brightening by multiple xylanase treatments during peroxide bleaching of kraft pulps.

The effects of multiple xylanase treatments were assessed during the peroxide bleaching of three pulps: Douglas-fir (kraft); Western hemlock (oxygen delignified kraft); and trembling Aspen (kraft). The addition of a xylanase treatment stage, either before or after the peroxide bleaching stage(s), resulted in the enhanced brightening of all pulps. A higher brightness was achieved using two enzyme treatments, one before and one after the peroxide stage(s). Both bleach boosting and direct brightening seemed to contribute to the enhancement of the peroxide bleaching. Compared to xylanase prebleaching, xylanase posttreatment of peroxide bleached pulps solubilized less lignin and chromophores and made smaller amounts of these materials alkaline soluble. Nevertheless, the final brightness achieved by xylanase posttreatment was similar or superior to that achieved with xylanase prebleaching of the corresponding unbleached pulps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 312-318, 1997.

Cellulose morphology and enzymatic reactivity: A modified solute exclusion technique.

An expeditious and accurate simplification of Stone and Scallan's solute exclusion technique was developed, thereby avoiding several sources of experimental error coupled with the determination of cellulose pore volume. Using this method, it is shown that cellulolytic enzymes do not enter into the micropores of five studied celluloses. These results suggestes that hydrolysis occurs initially at the external surface of the fibers. This surface area was calculated with the help of adsorption isotherms of bovine serum albumin. The obtained values for the different samples agree with the microscopically observed cellulose morphology. The correlation obtained by several authors relating cellulose porosity and its digestibility is explained as a consequence of the lower crystallinity and easier fragmentation of the more porous celluloses during hydrolysis. (c) 1994 John Wiley & Sons, Inc.