Synthesis of magnetic nanoparticles functionalized with histidine and nickel to immobilize His-tagged enzymes using ß-galactosidase as a model.

The aim of this study was to synthesize iron magnetic nanoparticles functionalized with histidine and nickel (Fe3O4-His-Ni) to be used as support materials for oriented immobilization of His-tagged recombinant enzymes of high molecular weight, using ß-galactosidase as a model. The texture, morphology, magnetism, thermal stability, pH and temperature reaction conditions, and the kinetic parameters of the biocatalyst obtained were assessed. In addition, the operational stability of the biocatalyst in the lactose hydrolysis of cheese whey and skim milk by batch processes was also assessed. The load of 600 Uenzyme/gsupport showed the highest recovered activity value (~50%). After the immobilization process, the recombinant ß-galactosidase (HisGal) showed increased substrate affinity and greater thermal stability (~50×) compared to the free enzyme. The immobilized ß-galactosidase was employed in batch processes for lactose hydrolysis of skim milk and cheese whey, resulting in hydrolysis rates higher than 50% after 15 cycles of reuse. The support used was obtained in the present study without modifying chemical agents. The support easily recovered from the reaction medium due to its magnetic characteristics. The iron nanoparticles functionalized with histidine and nickel were efficient in the oriented immobilization of the recombinant ß-galactosidase, showing its potential application in other high-molecular-weight enzymes.

Immobilization of ß-Galactosidases on Magnetic Nanocellulose: Textural, Morphological, Magnetic, and Catalytic Properties.

We describe a process for obtaining nanocrystalline cellulose (NC) by either acidic (H-NC) or alkaline treatment (OH-NC) of microcrystalline cellulose, which was subsequently bonded to magnetic nanoparticles (H-NC-MNP and OH-NC-MNP) and used as support for the immobilization of Aspergillus oryzae (H-NC-MNP-Ao and OH-NC-MNP-Ao) and Kluyveromyces lactis (H-NC-MNP-Kl and OH-NC-MNP-Kl) ß-galactosidases. The mean size of magnetic nanocellulose particles was approximately 75 nm. All derivatives reached saturation magnetizations of 7-18 emu/g, with a coercivity of approximately 4 kOe. Derivatives could be applied in batch hydrolysis of lactose either in permeate or in cheese whey for 30× and it reached hydrolysis higher than 50%. Furthermore, using a continuous process in a column packed-bed reactor, the derivative OH-NC-MNP-Ao had capacity to hydrolyze over 50% of the lactose present in milk or whey after 24 h of reaction. Fungal ß-galactosidases immobilized on magnetic nanocellulose can be applied in lactose hydrolysis using batch or continuous processes.

Self-assembled carbohydrate-based vesicles for lectin targeting.

This study examined the physicochemical interactions between vesicles formed by phosphatidylcholine (PC) and glycosylated polymeric amphiphile N-acetyl-ß-d-glucosaminyl-PEG900-docosanate (C22PEG900GlcNAc) conjugated with Bauhinia variegata lectin (BVL). Lectins are proteins or glycoproteins capable of binding glycosylated membrane components. Accordingly, the surface functionalization by such entities is considered a potential strategy for targeted drug delivery. We observed increased hydrodynamic radii (RH) of PC+C22PEG900GlcNAc vesicles in the presence of lectins, suggesting that this aggregation was due to the interaction between lectins and the vesicular glycosylated surfaces. Furthermore, changes in the zeta potential of the vesicles with increasing lectin concentrations implied that the vesicular glycosylated surfaces were recognized by the investigated lectin. The presence of carbohydrate residues on vesicle surfaces and the ability of the vesicles to establish specific interactions with BVL were further explored using atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) analysis. The results indicated that the thickness of the hydrophilic layer was to some extent influenced by the presence of lectins. The presence of lectins required a higher degree of polydispersity as indicated by the width parameter of the log-normal distribution of size, which also suggested more irregular structures. Reflectance Fourier transform infrared (HATR-FTIR), differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR) and ultraviolet-visible (UV-vis.) analyses revealed that the studied lectin preferentially interacted with the choline and carbonyl groups of the lipid, thereby changing the choline orientation and intermolecular interactions. The protein also discretely reduced the intermolecular communication of the hydrophobic acyl chains, resulting in a disordered state.

Interaction of jack bean (Canavalia ensiformis) urease and a derived peptide with lipid vesicles.

Ureases are metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. Jack bean (Canavalia ensiformis) produces three isoforms of urease (Canatoxin, JBU and JBURE-II). Canatoxin and JBU display several biological properties independent of their ureolytic activity, such as neurotoxicity, exocytosis-inducing and pro-inflammatory effects, blood platelets activation, insecticidal and antifungal activities. The Canatoxin entomotoxic activity is mostly due to an internal peptide, named pepcanatox, released upon the hydrolysis of the protein by insect cathepsin-like digestive enzymes. Based on pepcanatox sequence, Jaburetox-2Ec was produced in Escherichia coli. JBU and its peptides were shown to permeabilize membranes through an ion channel-based mechanism. Here we studied the JBU and Jaburetox-2Ec interaction with platelet-like multilamellar liposomes (PML) using Dynamic Light Scattering and Small Angle X-ray Scattering techniques. We also analyzed the interaction of JBU with giant unilamellar vesicles (GUVs) using Fluorescence Microscopy. The interaction of vesicles with JBU led to a slight reduction of hydrodynamic radius, and caused an increase in the lamellar repeat distance of PML, suggesting a membrane disordering effect. In contrast, Jaburetox-2Ec decreased the lamellar repeat distance of PML membranes, while also diminishing their hydrodynamic radius. Fluorescence microscopy showed that the interaction of GUVs with JBU caused membrane perturbation with formation of tethers. In conclusion, JBU can interact with PML, probably by inserting its Jaburetox "domain" into the PML external membrane. Additionally, the interaction of Jaburetox-2Ec affects the vesicle's internal bilayers and hence causes more drastic changes in the PML membrane organization in comparison with JBU.

A novel globular protein electrospun fiber mat with the addition of polysilsesquioxane.

The aim of this work has been to elaborate well defined gliadin nanofibers with incorporation of inorganic molecules, such as polyhedral oligomeric silsesquioxane (POSS). Nanofibers were obtained by electrospinning processing, controlling the relevant parameters such as tip-to-collector distance, voltage and feed rate. The fiber mats were characterized by SEM, confocal images, DSC, viscosity, FTIR and conductivimetry analysis. FTIR spectra showed characteristic absorption bands related to the presence of POSS-NH(2) within the matrices. SEM micrographs showed that gliadin fibers decreased their dimensions as the amount of POSS-NH(2) increased in the spinning solution. The electrical conductivity of gliadin solutions diminished as the concentration of POSS-NH(2) was increased. Besides, confocal micrographs revealed that POSS-NH(2) might be dispersed as nanocrystals into gliadin and gluten fibers. The dimension of gluten nanofibers was also affected by the POSS-NH(2) concentration, but conversely, this dependence was not proportional to the POSS-NH(2) amount. Somehow, the interaction between gliadin and POSS-NH(2) in aqueous TFE affected the solution viscosity and, as a consequence, higher jet instabilities and thinner fiber dimensions were obtained.

Wormlike micellar aggregates of saponins from Ilex paraguariensis A. St. Hil. (mate): a characterisation by cryo-TEM, rheology, light scattering and small-angle neutron scattering.

This work reports the physico-chemical characterisation of the micellar structures formed by a saponin fraction obtained from an important South American species, Ilex paraguariensis (mate). The mate saponin-enriched fraction (MSF) mainly comprises triterpenic glycosides and was obtained from mate green fruits through solid-phase extraction. The physico-chemical studies focused on the determination of the critical micellar concentration (CMC), the size and shape of the micelles, using conventional transmission electronic microscopy (TEM), as well as Cryo-TEM, light scattering and small-angle neutron scattering. The rheological behaviour of the solutions up to 4 wt% was also determined using a controlled-strain rheometer. Finally, the MSF ability to solubilise poorly water-soluble drugs was assayed using carbamazepine and flurbiprofen as basic and weak acidic drug models. Small spherical micelles of around 20 Å radius were observed in the presence of elongated structures with lengths of more than 500 nm, possessing a well-defined CMC of 0.41 g/L. MSF solutions ranging from 0.25 to 4% (w/v) demonstrated a viscoelastic behaviour independent of the concentration. MSF could improve the solubility of carbamazepine in the range of 0.13 to 1.5% (w/v).

Structure of self-organized diblock copolymer solutions in partially miscible solvents.

A diblock copolymer dissolved in a mixture of partially miscible solvents creates a self-organized microemulsion with a morphology that depends on the numerous parameters of the system. We discuss one particular case of spherical particles (containing the minority solvent) forming a hard gel with cubic structure and demonstrate using high-resolution synchrotron scattering experiments that the self-organized solution has a BCC structure. After fitting one- and two-dimensional form factors we extract from the data the one- and two-dimensional structure factors, S(q) and S(q,phi). The experimental S(q) corresponds almost quantitatively, up to the 9th order Bragg peak, to that calculated numerically for a randomly-oriented, finite-size BCC crystal. S(q,phi) contains a large number of reflections that allow the structure to be identified more exactly as a twin BCC morphology with some imperfections. Examination of the dependence of the structural parameters on polymer concentration reveals that the dilution law predicted theoretically for the center-to-center distance of the spheres is confirmed experimentally while the size of the spherical objects does not follow theoretical predictions due to chain extension with increasing concentration.

Interaction between phospholipids bilayer and chitosan in liposomes investigated by 31P NMR spectroscopy.

The interaction between chitosan and the polar head of phosphatidylcholine (PC) is discussed for a composite nanovesicle obtained by incorporating chitosan in the organic phase before PC self-assembling. Nanovesicles free of chitosan are studied in parallel to allow the comparison concerning modifications produced on the composite system. Zeta Potential increases in the presence of chitosan and with the increase in its concentration proving the localization of the polymer over the external surface of the vesicle as one interaction site. A (31)P resonance around 0 ppm, characteristic of the system, is reduced with addition of chitosan at 25 degrees C, indicating motional freedom reduction of the polar head phosphate group. The same resonance signal remains almost constant after increasing the temperature to 60 degrees C, suggesting that chitosan shields the phospholipids polar heads as a consequence of the electrostatic interactions leading to an increase in the thermodynamic stability of the composite.

Effect of nanovesicle-encapsulated nisin on growth of Listeria monocytogenes in milk.

Commercial nisin was encapsulated in nanovesicles (mean diameter 140 nm) prepared from partially purified soy lecithin. Nisin-loaded liposomes and unencapsulated (free) nisin were initially tested in BHI medium and skim milk inoculated with Listeria monocytogenes and incubated for 48 h at 30 degrees C. At such abuse temperature conditions, free nisin showed better inhibitory than the liposomal counterparts. Subsequently, the effect of encapsulated or free nisin was evaluated in combination with refrigeration (7 +/- 1 degrees C) in both whole (3.25% fat) and skim (0% fat) milk for up to 14 day. A decrease of 3-4 log cycles in L. monocytogenes counts was observed for free and encapsulated nisin at 0.5 mg/ml concentration. Liposome encapsulation of antimicrobial peptides may be important to overcome stability issues and interaction with food components. The utilization of nanovesicle-encapsulated nisin in combination with low temperatures appeared to be effective to control L. monocytogenes in milk, emphasizing the importance of hurdle technology to assure food safety.

Determining the simultaneous presence of drug nanocrystals in drug-loaded polymeric nanocapsule aqueous suspensions: a relation between light scattering and drug content.

The encapsulation of lipophilic drugs in polymeric nanoparticles can form simultaneously both polymeric nanoparticles and drug nanocrystals. The objective was to detect the presence of nanocrystals in the nanoparticle suspensions using a simple methodology, and to determine if the nanocrystals are formed during preparation or by drug leakage from the particles during storage. Indomethacin was chosen as drug model. Unloaded and drug-loaded (1mg/mL) nanocapsules showed diameters close to 280nm and polydispersity lower than 0.20, remaining constant after 120 days. Comparing indomethacin loaded (3mg/mL) and unloaded formulations, variations in the scattered light depolarization degree indicated the simultaneous presence of nanocrystals and nanocapsules in the suspensions. A relation between the scattered light intensities and the drug precipitation was established. As a function of time, when the decrease in the Rayleigh ratios occurred, the drug contents decreased due to precipitation. On the other hand, when the Rayleigh ratios slightly increase, the drug contents are constant. The nanocrystals formed in the oversaturated formulations, agglomerate and precipitate during storage. When the drug is adsorbed on the nanocapsules, but the system is not oversaturated, no nanocrystal was formed and the formulation is physico-chemically stable at least for 150 days of storage.

LUVs recovered with Chitosan: a new preparation for vaccine delivery.

Chitosan, alpha-(1-4)-amino-2-deoxy-beta-D-glucan, is a deacetylated form of chitin, an abundant natural polysaccharide present in crustacean shells. Its unique characteristics such as positive charge, biodegradability, biocompatibility, nontoxicity, and rigid linear molecular structure make this macromolecule ideal as drug carrier. The association between chitosan and liposomes was carefully described, where REVs (reverse phase evaporation vesicles) were sandwiched by chitosan. The usage of these particles in vaccine formulation is here proposed for the first time in the literature. The Chitosan-REVs now stabilized by polyvinilic alcohol were the vehicle for Diphtheria toxoid (Dtxd). Round chitosan-sandwiched REVs (REVs-Chi) particles of 373 +/- 17 nm containing 65% Dtxd were obtained. After 200 min of incubation in a simulated gastric fluid, 70% of the Dtxd was liberated from REVs-Chi in comparison to 100% of Dtxd liberated from pure REVs. In PBS, the Dtxd liberation from REVS-Chi was about 60%. Mice were immunized with Dtxd encapsulated within REVs-Chi and with other REVs/Dtxd formulations adsorbed onto Freund adjuvant or alumen [AIF and Al(OH)(3)]. The response patterns and the immune maturity were measured by IgG(1) and IgG(2a) titrations. REVs-Chi containing Dtxd elicited both antibodies production giving the animals higher immune response and selectivity. It was interesting that the memory of those mice immunized with REVs-Chi containing Dtxd enhanced, after booster, antibody production by 47% in contrast with 17 and 7% in mice immunized with the antigen vehiculated in REVs-AIF or REVs-Al(OH)(3), respectively.

ESIPT-exhibiting protein probes: a sensitive method for rice proteins detection during starch extraction.

The 2-(4'-isothiocyanate-2'-hydroxyphenyl)benzoxazole dye was successfully applied as label of rice proteins during the alkaline extraction of starch. Direct fluorescence measurements were used to observe the presence of proteins labelled in different steps of rice starch extraction. The results were compared to those obtained with the well-known biuret colorimetric test. Whereas the colorimetric test indicates the absence of protein after the third extraction step, the fluorescence emission of the conjugate could be observed in all extraction steps. The separation of different rice proteins could also be observed.

Structural evaluation of phospholipidic nanovesicles containing small amounts of chitosan.

In this study we present a full characterization of nanovesicles containing soybean phosphatidylcholine and polysaccharide chitosan. The nanovesicles were prepared by the reverse phase evaporation method, including the preparation of reverse micelles followed by the formation of an organogel, which is dispersed in water to yield the final liposomal particles. Structural changes as a function of the chitosan amount and the filter porosity used in the nanovesicles preparation were studied employing Static and Dynamic Light Scattering as well as Small Angle X-ray Scattering. The hydrodynamic radius of the nanovesicles ranged between 106 and 287 nm, depending on the chitosan contents and the filter porosity. A comparison with nanovesicles free of chitosan indicates the existence of higher contents of multilamellar structures that depends on the chitosan concentration in the vesicles containing chitosan. Typical spherical vesicles having nanometric diameters with polydispersity mostly desired in the biomedical area could only be achieved by filtration through a 0.45 microm porous filter.

Production of soybean phosphatidylcholine-chitosan nanovesicles by reverse phase evaporation: a step by step study.

In the present work, we describe the preparation of composite nanovesicles containing soybean phosphatidylcholine and polysaccharide chitosan by the reverse phase evaporation method. Nanovesicles free from chitosan prepared in the same way were studied as reference. The production method involves the preparation of reverse micelles followed by the formation of an organogel, which is dispersed in water to yield the final liposomal structures. Structural changes in each step of the nanovesicles preparation were studied by means of static and dynamic light scattering as well as small angle X-ray scattering. Chitosan was also fully characterized in solution. The hydrodynamic radius of the composite nanovesicles is in the range of 174-286 nm, depending on the chitosan contents. A comparison with nanovesicles free from chitosan indicates the existence of higher contents of multilamellae structures in the composites, as well as improved stability in water.

Alkaline hydrolysis as a tool to determine the association form of indomethacin in nanocapsules prepared with poly(eta-caprolactone).

To determine the association form of indomethacin in nanocapsules prepared with poly(eta-caprolactone) as polymer and a triglyceride as oil, two methods were studied. The indomethacin ethyl ester was prepared as control, which showed a higher affinity for the oil than the indomethacin. Two differently loaded nanocapsule formulations were prepared. For both formulations, a burst effect was detected using ethanol as release medium. Light scattering (PCS) and NMR analyses suggested the ethanol diffuses through the nanocapsule polymeric wall promoting the total release of indomethacin and its ester. The results showed the inability of this approach to determine the association form of indomethacin. On the other hand, the alkaline hydrolysis of indomethacin and its ester, followed by their disappearance (HPLC), were evaluated. The nanocapsule suspensions containing indomethacin or its ester were treated with 50 mM NaOH. The total disappearance of indomethacin associated with nanocapsules was determined after 2 min, whereas the ester associated with colloids was consumed during 24 h. The constant particle sizes (264 and 259 nm) during the hydrolysis reactions showed that neither the nanocapsules were dissolved nor the polymer sorbed water during the contact with NaOH aqueous solution. The ester rate hydrolysis was determined by its diffusion from the nanocapsules to the interface particle/water. Finally, the indomethacin association model considers the burst release of drug after the addition of NaOH by the formation of its carboxylate, followed by its hydrolysis in aqueous solution promoted by the excess of NaOH. The adsorption was the mechanism of indomethacin association with nanocapsules.