Continuous Formation and Separation of Calcium-Alginate Capsules Containing Viable Mammalian Cells in Microfluidic Devices

Microfluidic devices can be used for many applications, including the formation of well-controlled emulsions. In this study, the capability to continuously create monodisperse droplets in a microfluidic device was used to form calcium-alginate capsules.Calcium-alginate capsules have many potential uses, such as immunoisolation of cells and microencapsulation of active drug ingredients or bitter agents in food or beverage products. The gelation of calcium-alginate capsules is achieved by crosslinking sodiumalginate with calcium ions. Calcium ions dissociated from calcium carbonate due to diffusion of acetic acid from a sunflower oil phase into an aqueous droplet containing sodium-alginate and calcium carbonate. After gelation, the capsules were separated from the continuous oil phase into an aqueous solution for use in biological applications. Typically, capsules are separated bycentrifugation, which can damage both the capsules and the encapsulated material. A passive method achieves separation without exposing the encapsulated material or the capsules to large mechanical forces, thereby preventing damage. To achieve passiveseparation, the use of a microfluidic device with opposing channel wa hydrophobicity was used to stabilize co-laminar flow of im of hydrophobicity is accomplished by defining one length of the channel with a hydrogel. The chosen hydrogel was poly (ethylene glycol) diacrylate, which adheres to the glass surface through the use of self-assembled monolayer of 3-(trichlorosilyl)-propyl methacrylate. Due to the difference in surface energy within the channel, the aqueous stream is stabilized near a hydrogel and the oil stream is stabilized near the thiolene based optical adhesive defining the opposing length of the channel. Passive separation with co-laminar flow has shown success in continuously separating calcium-alginatecapsules from an oil phase into an aqueous phase. In addition to successful formation and separation of calcium alginate capsules,encapsulation of Latex micro-beads and viable mammalian cells has been achieved. The viability of encapsulated mammalian cells was determined using a live/dead stain. The co-laminar flow device has also been demonstrated as a means of separating liquid-liquidemulsions.

Chitosan-based scaffolds for tissue regeneration: preparation and microstructure characterization

Scaffolds are porous three-dimensional supports, designed to mimic the extracellular environment and remain temporarily integrated into the host tissue while stimulating, at the molecular level, specific cellular responses to each type of body tissues. The major goal of the research work entertained herein was to study the microstructure of scaffolds made from chitosan (Ch), blends of chitosan and sodium alginate (Ch/NaAlg), blends of chitosan, sodium alginate and calcium chloride (Ch/NaAlg/CaCl2) and blends of chitosan, sodium alginate and hydroxyapatite (Ch/NaAlg/HA). Scaffolds possessing ideal physicochemical properties facilitate cell proliferation and greatly increase the rate of recovery of a damaged organ tissue. Using CT three-dimensional images of the scaffolds, it was observed that all scaffolds had a porosity in the range 64%-92%, a radius of maximum pore occurrence in the range 95m-260m and a permeability in the range 1×10-10-18×10-10 m2. From the results obtained, the scaffolds based on Ch, Ch/NaAlg and Ch/NaAlg/CaCl2 would be most appropriate both for the growth of osteoid and for bone tissue regeneration, while the scaffold made with a blend of Ch/NaAlg/HA, by possessing larger pores size, might be used as a support for fibrovascular tissue.

Quantifying diffusion in biofilms : from model hydrogels to living biofilms

Les biofilms sont des communautés de microorganismes incorporés dans une matrice exo-polymérique complexe. Ils sont reconnus pour jouer un rôle important comme barrière de diffusion dans les systèmes environnementaux et la santé humaine, donnant lieu à une résistance accrue aux antibiotiques et aux désinfectants. Comme le transfert de masse dans un biofilm est principalement dû à la diffusion moléculaire, il est primordial de comprendre les principaux paramètres influençant les flux de diffusion. Dans ce travail, nous avons étudié un biofilm de Pseudomonas fluorescens et deux hydrogels modèles (agarose et alginate) pour lesquels l’autodiffusion (mouvement Brownien) et les coefficients de diffusion mutuels ont été quantifiés. La spectroscopie par corrélation de fluorescence a été utilisée pour mesurer les coefficients d'autodiffusion dans une volume confocal de ca. 1 m3 dans les gels ou les biofilms, tandis que les mesures de diffusion mutuelle ont été faites par cellule de diffusion. En outre, la voltamétrie sur microélectrode a été utilisée pour évaluer le potentiel de Donnan des gels afin de déterminer son impact sur la diffusion. Pour l'hydrogel d'agarose, les observations combinées d'une diminution du coefficient d’autodiffusion et de l’augmentation de la diffusion mutuelle pour une force ionique décroissante ont été attribuées au potentiel de Donnan du gel. Des mesures de l'effet Donnan (différence de -30 mV entre des forces ioniques de 10-4 et 10-1 M) et l'accumulation correspondante d’ions dans l'hydrogel (augmentation d’un facteur de 13 par rapport à la solution) ont indiqué que les interactions électrostatiques peuvent fortement influencer le flux de diffusion de cations, même dans un hydrogel faiblement chargé tel que l'agarose. Curieusement, pour un gel plus chargé comme l'alginate de calcium, la variation de la force ionique et du pH n'a donné lieu qu'à de légères variations de la diffusion de sondes chargées dans l'hydrogel. Ces résultats suggèrent qu’en influençant la diffusion du soluté, l'effet direct des cations sur la structure du gel (compression et/ou gonflement induits) était beaucoup plus efficace que l'effet Donnan. De même, pour un biofilm bactérien, les coefficients d'autodiffusion étaient pratiquement constants sur toute une gamme de force ionique (10-4-10-1 M), aussi bien pour des petits solutés chargés négativement ou positivement (le rapport du coefficient d’autodiffusion dans biofilm sur celui dans la solution, Db/Dw ≈ 85 %) que pour des nanoparticules (Db/Dw≈ 50 %), suggérant que l'effet d'obstruction des biofilms l’emporte sur l'effet de charge. Les résultats de cette étude ont montré que parmi les divers facteurs majeurs qui affectent la diffusion dans un biofilm environnemental oligotrophe (exclusion stérique, interactions électrostatiques et hydrophobes), les effets d'obstruction semblent être les plus importants lorsque l'on tente de comprendre la diffusion du soluté. Alors que les effets de charge ne semblaient pas être importants pour l'autodiffusion de substrats chargés dans l'hydrogel d'alginate ou dans le biofilm bactérien, ils ont joué un rôle clé dans la compréhension de la diffusion à travers l’agarose. L’ensemble de ces résultats devraient être très utiles pour l'évaluation de la biodisponibilité des contaminants traces et des nanoparticules dans l'environnement.

Produção de lipase pelo fungo Myceliophthora sp. F. 2.1.4, caracterização e imobilização da solução enzimática bruta

Pós-graduação em Microbiologia - IBILCE

Precursores funcionalizantes de poros à base de alginato para obtenção de cerâmicas bioativas

A complexidade de desenvolver novas tecnologias para aplicações em reconstituição óssea se deve à necessidade de combinar várias propriedades químicas e físicas para que o material proporcione o desempenho almejado. Particularmente, em aplicações que visam osteogênese, os enxertos sintéticos devem ser bioativos, possuir porosidade com volume, geometria e interconectividade de poros controlados, além de ter boas propriedades mecânicas, dentro de limites relativamente rígidos. Por essa razão, o recobrimento de materiais bioinertes com cerâmicas bioativas se tornou o foco da presente pesquisa. O objetivo desse estudo foi desenvolver um novo método de produção de enxertos cerâmicos com macroporosidade funcionalizada, onde a formação e o revestimento dos poros são realizados em uma única etapa. Foi realizado o estudo de recobrimento com vidro bioativo e fosfato de cálcio. Para isso, agentes porogênicos na forma de grânulos (de 600 μm a 2 mm de diâmetro) foram sintetizados pelo método da gelificação de uma solução aquosa de alginato de sódio gotejada em solução de nitrato de cálcio (0,5 M), com incorporação de outros elementos para a formação de biovidro ou fosfato de cálcio. Esses grânulos foram conglomerados a um vidro ou alumina em pó, formando um compósito, que foi tratado termicamente para sinterização e formação de poros. No caso da matriz vítrea, a sinterização ocorreu com cristalização simultânea e concorrente. As cerâmicas resultantes foram caracterizadas por microscopia óptica e eletrônica de varredura, sendo possível observar a formação de macroporos aproximadamente esféricos (de 600 μm a 2 mm de diâmetro) revestidos internamente por uma camada de material com possível composição bioativa.

Encapsulation and release characteristics of glibenclamide loaded calcium-alginate beads

The aims of this study were to formulate calcium-alginate beads containing glibenclamide, characterize the resulting microparticles, evaluate the release characteristics of this type of delivery system in an in vitro dissolution test, and compare it with two commercially available trademarks (Daonil® and Glibetab®). We obtained glibenclamide loaded calcium-alginate beads with a rough surface and a particle size between 150-200 µm. For the in vitro dissolution test Daonil® at 45 min showed a Q > 70%, whereas Glibetab® and glibenclamide calcium-alginate beads a Q < 70%; in spite of that glibenclamide calcium-alginate beads showed significant release properties.

Application of Response Surface Methodology to study the effect of different calcium sources in fish muscle-alginate restructured products

Sodium alginate needs the presence of calcium ions to gelify. For this reason, the contribution of the calcium source in a fish muscle mince added by sodium alginate, makes gelification possible, resulting a restructured fish product. The three different calcium sources considered were: Calcium Chloride (CC); Calcium Caseinate (CCa); and Calcium lactate (CLa). Several physical properties were analyzed, including mechanical properties, colour and cooking loss. Response Surface Methodology (RSM) was used to determine the contribution of different calcium sources to a restructured fish muscle. The calcium source that modifies the system the most is CC. A combination of CC and sodium alginate weakened mechanical properties as reflected in the negative linear contribution of sodium alginate. Moreover, CC by itself increased lightness and cooking loss. The mechanical properties of restructured fish muscle elaborated were enhanced by using CCa and sodium alginate, as reflected in the negative linear contribution of sodium alginate. Also, CCa increased cooking loss. The role of CLa combined with sodium alginate was not so pronounced in the system discussed here.

Production of isomaltulose obtained by Erwinia sp. cells submitted to different treatments and immobilized in calcium alginate

In recent decades, there has been an increase in the studies of isomaltulose obtainment, due to its physicochemical properties and physiological health benefits. These properties, which include low cariogenicity, low glycemic index and greater stability, allow the use of this sweetener as a substitute for sucrose in foods; besides the fact that it can be converted to isomalt, a dietary non-cariogenic sugar alcohol used in pharmaceuticals as well as in the food industry. Isomaltulose (6-O-α-D-glucopyronosyl-1-6-D-fructofuranose) is a disaccharide reducer obtained by the enzymatic conversion of sucrose - the α-glucosyltransferase enzyme. Different treatments were performed for the preparation of whole cells; lysed cells; and crude enzyme extract of Erwinia sp. D12 strain immobilized in calcium alginate. The packed bed column of granules, containing Erwinia sp. cells sonicated and immobilized in calcium alginate (CSI), reached a maximum conversion of 53-59% sucrose into isomaltulose and it presented activity for 480 hours. The converted syrup was purified and the isomaltulose crystallization was performed through the lowering of temperature. The isomaltulose crystals presented purity of 96.5%.

Enhanced viability of corneal epithelial cells for efficient transport/storage using a structurally-modified calcium alginate hydrogel

Aims: Therapeutic limbal epithelial stem cells could be managed more efficiently if clinically validated batches were transported for ‘on-demand’ use. Materials & methods: In this study, corneal epithelial cell viability in calcium alginate hydrogels was examined under cell culture, ambient and chilled conditions for up to 7 days. Results: Cell viability improved as gel internal pore size increased, and was further enhanced with modification of the gel from a mass to a thin disc. Ambient storage conditions were optimal for supporting cell viability in gel discs. Cell viability in gel discs was significantly enhanced with increases in pore size mediated by hydroxyethyl cellulose. Conclusion: Our novel methodology of controlling alginate gel shape and pore size together provides a more practical and economical alternative to established corneal tissue/cell storage methods.

Isomaltulose production from Sucrose by Protaminobacter rubrum immobilized in calcium alginate

Different culture conditions for Protaminobacter rubrum and enzymatic reaction parameters were evaluated with the goal of improving isomaltulose production. P. rubrum was grown in a medium with 1% (w/v) cane molasses and 0.5% yeast extract and achieved a maximum cell yield Y(x/s) of 0.295 g of cells/g sucrose and a specific growth rate (mu) of 0.192 h(-1). The immobilization of P. rubrum cells was carried out with calcium alginate, glutaraldehyde and polyethyleneimine. Stabile immobilized cell pellets were obtained and used 24 times in batch processes. Enzymatic conversion was carried out at different sucrose concentrations and in pH 6 medium with 70% (w/v) sucrose at 30 degrees C an isomaltulose yield of 89-94% (w/v) was obtained. The specific activity of the P. rubrum immobilized pellets in calcium alginate at 30 degrees C ranged from 1.6 to 4.0 g isomaltulose g(-1) pellet h(-1), respectively with 70% and 65% sucrose solution, while in lower sucrose concentration had higher specific activities presumably due to substrate inhibition of the isomaltulose synthase in higher sucrose concentrations. (C) 2009 Elsevier Ltd. All rights reserved.

Production of fructooligosaccharides by the mycelia of Aspergillus japonicus immobilized in calcium alginate

This work describes fructose oligosaccharide (FOS) production by the immobilized mycelia (IM) of a strain of Aspergillus japonicus, isolated from soil. The microorganism was inoculated into 50 mi of medium composed of sugar cane molasses (5.0% of total sugars); yeast powder; 2.0%; K2HPO4, 0.5%; NaNO3, 0.2%; MgSO4. 7H(2)O, 0.05%; KCl, 0.05%, final pH 5.0, and the flasks were agitated in an orbital shaker at 200 rpm for 60 h, at 30 degrees C. The beta-fructofuranosidase activity (Uf), transfructosylating activity (Ut), hydrolyzing activity (Uh), and FOS production were analyzed by high performance liquid chromatography. FOS production was performed in a batch process in a 2-l jar fermenter by IM in calcium alginate beads. The optimum pH and temperature were 5.0-5.6 and 55 degrees C, respectively No loss of activity was observed when the mycelium was maintaned at 60 degrees C for 60 min. Maximum production was obtained using 5.75% (cellular weight/volume) of mycelia (122.4 Ut g(-1)) and 65% sucrose solution (w:v) for 4 h of reaction when the final product reached 61.28% of fetal FOS containing GF(2) (30.56%), GF(3) (26.45%), GF(4) (4.27%), sucrose (9.6%) and glucose (29.10%). In the assay conditions, 23 batches were performed without loss of activity of the IM, showing that the microorganism and the process utilized have potential for industrial applications. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Determination of calcium by atomic absorption spectrophotometry applied to the preparation of alginate:chitosan complexes

Polysaccharicles, as alginate and chitosan, have been used to obtain modified release dosage forms. Alginate, due to its property of building gels during the complex formation with calcium ions, allows the building of capsules containing a core constituted by calcium alginate. This work had for objective to determine the appropriate calcium concentration for the preparation of alginate-chitosan capsules, by means of calcium quantification using atomic absorption spectrophotometry. The methodology of calcium quantification was validated through analysis of the limit of detection, precision, accuracy and recovery of the method. The capsules, containing or not the drug, were prepared by the complex coacervation/ionotropic gelification method. Calcium was quantified after samples mineralization and dilution in lantanium solution. The results showed that the amount of calcium incorporated into the capsules depends on the amount of calcium added to the medium, and this ratio increases until the concentration of 1.5% of initial calcium chloride and above this concentration there is a decrease in the proportion of calcium bonded. It was observed that the proportion of calcium that links to the polymer is inversely proportional to the amount of calcium added. The calcium amount incorporated depends on the concentration of the polymeric dispersions used as well as on the ratio between the two polymers.

Evaluation of dry protonated calcium alginate beads for biosorption applications and studies of lead uptake

Alginate polysaccharide is a promising biosorbent for metal uptake. Dry protonated calcium alginate beads for biosorption applications were prepared, briefly characterized and tested for lead uptake. Several advantages of this biosorbent are reported and discussed in comparison with other alginate-based sorbents. The alginate beads contained 4.7 mmol/g of COOH groups, which suffered hydrolysis near pH 4. The Weber and Morris model, applied to kinetic results of lead uptake, showed that intraparticle diffusion was the rate-controlling step in lead sorption by dry alginate beads. Equilibrium experiments were performed and the data were fitted with different isotherm models. The Langmuir equation was the most adequate to model lead sorption. The maximum uptake capacity (qmax) was estimated as 339 mg/g and the Langmuir constant (b) as 0.84 l/mg. These values were compared with that of other sorbents found in the literature, indicating that dry protonated calcium alginate beads are among the best biosorbents for the treatment and recovery of heavy metals from aqueous streams.

Evaluation of dry protonated calcium alginate beads for biosorption applications and studies of lead uptake

Alginate polysaccharide is a promising biosorbent for metal uptake. Dry protonated calcium alginate beads for biosorption applications were prepared, briefly characterized and tested for lead uptake. Several advantages of this biosorbent are reported and discussed in comparison with other alginate-based sorbents. The alginate beads contained 4.7 mmol/g of COOH groups, which suffered hydrolysis near pH 4. The Weber and Morris model, applied to kinetic results of lead uptake, showed that intraparticle diffusion was the rate-controlling step in lead sorption by dry alginate beads. Equilibrium experiments were performed and the data were fitted with different isotherm models. The Langmuir equation was the most adequate to model lead sorption. The maximum uptake capacity (qmax) was estimated as 339 mg/g and the Langmuir constant (b) as 0.84 l/mg. These values were compared with that of other sorbents found in the literature, indicating that dry protonated calcium alginate beads are among the best biosorbents for the treatment and recovery of heavy metals from aqueous streams.

Layer-by-layer coating of alginate matrices with chitosan–alginate for the improved survival and targeted delivery of probiotic bacteria after oral administration

The oral administration of probiotic bacteria has shown potential in clinical trials for the alleviation of specific disorders of the gastrointestinal tract. However, cells must be alive in order to exert these benefits. The low pH of the stomach can greatly reduce the number of viable microorganisms that reach the intestine, thereby reducing the efficacy of the administration. Herein, a model probiotic, Bifidobacterium breve, has been encapsulated into an alginate matrix before coating in multilayers of alternating alginate and chitosan. The intention of this formulation was to improve the survival of B. breve during exposure to low pH and to target the delivery of the cells to the intestine. The material properties were first characterized before in vitro testing. Biacore™ experiments allowed for the polymer interactions to be confirmed; additionally, the stability of these multilayers to buffers simulating the pH of the gastrointestinal tract was demonstrated. Texture analysis was used to monitor changes in the gel strength during preparation, showing a weakening of the matrices during coating as a result of calcium ion sequestration. The build-up of multilayers was confirmed by confocal laser-scanning microscopy, which also showed the increase in the thickness of coat over time. During exposure to in vitro gastric conditions, an increase in viability from <3 log(CFU) per mL, seen in free cells, up to a maximum of 8.84 ± 0.17 log(CFU) per mL was noted in a 3-layer coated matrix. Multilayer-coated alginate matrices also showed a targeting of delivery to the intestine, with a gradual release of their loads over 240 min.