DNA protection from extracapsular nucleases, within chitosan- or poly-L-lysine-coated alginate beads

DNA was immobilized within alginate matrix using an external or an internal calcium source, and then membrane coated with chitosan or poly-L-lysine. Membrane thickness increased with decreasing polymer molecular weight and increasing degree of deacetylation (chitosan). Beads were exposed to a 31,000 molecular weight nuclease to determine the levels of DNA protection offered by different membrane and matrix combinations. Almost total hydrolysis of DNA was observed in alginate beads following nuclease exposure. Less than 1% of total double-stranded DNA remained unhydrolyzed within chitosan- or poly-L-lysine-coated beads, corresponding with an increase in DNA residuals (i.e. double- and single-stranded DNA, polynucleotides, bases). Chitosan membranes did not offer sufficient DNA protection from DNase diffusion since all of the double-stranded DNA was hydrolyzed after 40 min of exposure. Both chitosan and poly-L-lysine membranes reduced the permeability of alginate beads, shown by enhanced retention of DNA residuals after DNase exposure. The highest level of DNA protection within freshly prepared beads was obtained with high molecular weight (197,100) poly-L-lysine membranes coated on beads formed using an external calcium source, where over 80% of the double-stranded DNA remained after 40 min of DNase exposure. Lyophilization and rehydration of DNA beads also reduced permeability to nucleases, resulted in DS-DNA recoveries of 60% for chitosan-coated, 90% for poly-L-lysine-coated, and 95% for uncoated alginate beads.     

Influence of oligoguluronates on alginate gelation, kinetics, and polymer organization

Structural polysaccharides of the alginate family form gels in aqueous Ca2+-containing solutions by lateral association of chain segments. The effect of adding oligomers of alpha-l-guluronic acid (G blocks) to gelling solutions of alginate was investigated using rheology and atomic force microscopy (AFM). Ca-alginate gels were prepared by in situ release of Ca2+. The gel strength increased with increasing level of calcium saturation of the alginate and decreased with increasing amount of free G blocks. The presence of free G blocks also led to an increased gelation time. The gel point and fractal dimensionalities of the gels were determined based on the rheological characterization. Without added free G blocks the fractal dimension of the gels increased from df = 2.14 to df = 2.46 when increasing [Ca2+] from 10 to 20 mM. This increase was suggested to arise from an increased junction zone multiplicity induced by the increased concentration of calcium ions. In the presence of free G blocks (G block/alginate = 1/1) the fractal dimension increased from 2.14 to 2.29 at 10 mM Ca2+, whereas there was no significant change associated with addition of G blocks at 20 mM Ca2+. These observations indicate that free G blocks are involved in calcium-mediated bonds formed between guluronic acid sequences within the polymeric alginates. Thus, the added oligoguluronate competes with the alginate chains for the calcium ions. The gels and pregel situations close to the gel point were also studied using AFM. The AFM topographs indicated that in situations of low calcium saturation microgels a few hundred nanometers in diameter develop in solution. In situations of higher calcium saturation lateral association of a number of alginate chains are occurring, giving ordered fiber-like structures. These results show that G blocks can be used as modulators of gelation kinetics as well as local network structure formation and equilibrium properties in alginate gels.     

Conjugative plasmid transfer between Pseudomonas strains within alginate bead microcosms: effect of the internal gel structure.

Because microorganisms frequently live in an immobilized state in natural habitats, a cell-confined system was used to study bacterial conjugation. Two Pseudomonas putida strains were introduced together within calcium alginate gels. Different alginate beads were designed by varying the polysaccharide and the gelation solution concentrations. Microscopic examinations showed that 2% gels were quite homogeneous, but that 1.5% and 1% gels were rather heterogeneous. In these two last cases, shaft-shaped macrostructures were present. They were colonized during the culture by great densities of highly motile bacteria. Gene transfers due to conjugation were investigated in such alginate gel bead microcosms, in batch and continuous cultures. High-initial transfer frequencies were detected whatever the gel, but no conjugation events seemed to occur with further growth in the beads. Transfer frequency values were roughly similar in the different tested systems. Alginate gels used as artificial microcosms may be valuable to study the effect of cell microenvironment on genetic transfers in complex systems.     

Calcium Alginate Gels as Stem Cell Matrix – Making Paracrine Stem Cell Activity Available for Enhanced Healing after Surgery

Regeneration after surgery can be improved by the administration of anabolic growth factors. However, to locally maintain these factors at the site of regeneration is problematic. The aim of this study was to develop a matrix system containing human mesenchymal stem cells (MSCs) which can be applied to the surgical site and allows the secretion of endogenous healing factors from the cells. Calcium alginate gels were prepared by a combination of internal and external gelation. The gelling behaviour, mechanical stability, surface adhesive properties and injectability of the gels were investigated. The permeability of the gels for growth factors was analysed using bovine serum albumin and lysozyme as model proteins. Human MSCs were isolated, cultivated and seeded into the alginate gels. Cell viability was determined by AlamarBlue assay and fluorescence microscopy. The release of human VEGF and bFGF from the cells was determined using an enzyme-linked immunoassay. Gels with sufficient mechanical properties were prepared which remained injectable through a syringe and solidified in a sufficient time frame after application. Surface adhesion was improved by the addition of polyethylene glycol 300,000 and hyaluronic acid. Humans MSCs remained viable for the duration of 6 weeks within the gels. Human VEGF and bFGF was found in quantifiable concentrations in cell culture supernatants of gels loaded with MSCs and incubated for a period of 6 weeks. This work shows that calcium alginate gels can function as immobilization matrices for human MSCs.     

Swelling behaviour of alginate–chitosan microcapsules prepared by external gelation or internal gelation technology

Swelling behaviour is one of the important properties for microcapsules made by hydrogels, which always affects the diffusion and release of drugs when the microcapsules are applied in drug delivery systems. In this paper, alginate–chitosan microcapsules were prepared by different technologies called external or internal gelation process respectively. With the volume swelling degree (S_w) as an index, the effect of properties of chitosan on the swelling behaviour of both microcapsules was investigated. It was demonstrated that the microcapsules with low molecular weight and high concentration of chitosan gave rise to low S_w. Considering the need of maintaining drug activity and drug loading, neutral pH and short gelation time were favorable. It was also noticed that S_w of internal gelation microcapsules was lower than that of external gelation microcapsules, which was interpreted by the structure analysis of internal or external gelation Ca–alginate beads with the aid of confocal laser scanning microscope.     

Stomach-specific drug delivery of 5-fluorouracil using floating alginate beads

A multiple-unit-type oral floating dosage form (FDF) of 5-fluorouracil (5-FU) was developed to prolong gastric residence time, target stomach cancer, and increase drug bioavailability. The floating bead formulations were prepared by dispersing 5-FU together with calcium carbonate into a mixture of sodium alginate and hydroxypropyl methylcellulose solution and then dripping the dispersion into an acidified solution of calcium chloride. Calcium alginate beads were formed, as alginate undergoes ionotropic gelation by calcium ions and carbon dioxide develops from the reaction of carbonate salts with acid. The evolving gas permeated through the alginate matrix, leaving gas bubbles or pores, which provided the beads buoyancy. The prepared beads were evaluated for percent drug loading, drug entrapment efficiency, image, surface topography, buoyancy, and in vitro release. The formulations were optimized for different weight ratios of gas-forming agent and sodium alginate. The beads containing higher amounts of calcium carbonate demonstrated instantaneous, complete, and excellent floating ability over a period of 24 hours. The optimized formulation was subjected to in vivo antitumor studies to check the therapeutic efficacy of the floating dosage forms containing 5-FU against benzo(a)pyrene-induced stomach tumors in albino female mice (Balb/C strain). The multiple-bead FDF was found to reduce the tumor incidence in mice by 74%, while the conventional tablet dosage form reduced this incidence by only 25%. Results indicate that FDF performed significantly better than the simple tablet dosage form. Published: June 22, 2007     

Diffusion characteristics of calcium alginate gels.

The diffusivity of a protein solute (bovine serum albumin) within calcium alginate gels made from sodium alginate of different guluronic acid content was determined. It was found that protein diffusion within alginate gels, prepared to be isotropic in structure, was greatest for gels prepared from sodium alginate of low guluronic acid content as opposed to those prepared from sodium alginate of high guluronic acid content. This finding was explained in terms of the difference in flexibility of the polymer backbone of the two alginates. The greater the polymer backbone flexibility, the greater the solute diffusivity within the gel.     

Production of alginate beads by emulsification/internal gelation. II. Physicochemistry

Alginate microspheres were produced by emulsification/internal gelation of alginate sol dispersed within vegetable oil. Gelification was initiated within the alginate sol by a reduction in pH (7.5 to 6.5), releasing calcium from an insoluble complex. Smooth, spherical beads with the narrowest size dispersion were obtained when using low-guluronic-acid and low-viscosity alginate and a carbonate complex as the calcium vector. A more finely dispersed form of the complexed calcium within the alginate sol promotes a more homogeneous gelification. Microsphere mean diameters ranging from 50 m to 1000 m were obtained with standard deviations ranging from 35% to 45% of the mean.     

Structural changes during heat-induced gelation of globular protein dispersions

Macroscopic and molecular structural changes during heat-induced gelation of beta-lactoglobulin, bovine serum albumin, ovalbumin, and alpha-lactalbumin aqueous dispersions were probed by the mechanical and CD spectroscopy, respectively. Aqueous solutions of the native globular proteins, except for alpha-lactalbumin, exhibited solid-like mechanical spectra-namely, the predominant storage modulus G' over the loss modulus G" in the entire frequency range examined (0.1-100 rad/s), suggesting that these protein solutions were highly structured even before gelation, possibly due to strong repulsions among protein molecules. Such solid-like structures were susceptible to nonlinearly large shear but recovered almost immediately at rest. During gelation by isothermal heating, major changes in the secondary structure of the globular proteins completed within a few minutes, while values of the modulus continued to develop for hours with maintaining values of tandelta (= G"/G') less than unity. As a result, a conventional criterion for mechanically defining the gelation point, such as a crossover between G' and G", was inapplicable to these globular protein systems. beta-Lactoglobulin gels that had passed the gelation point satisfied power laws (G' approximately G" approximately omega(n)) believed to be valid only at the gelation point, suggesting that fractal gel networks, similar to those of critical gels (i.e., gels at the gelation point), were formed.     

Effect of Sodium Alginate Addition on Physical Properties of Rennet Milk Gels

Effect of addition of sodium alginate (alginate) to milk on the storage modulus (G′), water holding capacity (WHC) and hardness of rennet gels was evaluated as a function of alginate (0–0.25 g/100 g) and fat (0.5–3.0 g/100 g) concentrations. There was a significant effect of alginate addition on ionic calcium in milk and whey (Ca²⁺), and particle size distribution in alginate added milk. Results showed a positive correlation of alginate with WHC; negative correlation of alginate and positive correlation of fat with G′; and negative correlation of interaction of fat and alginate with gel hardness of rennet gels. Hence, the rennet gels with lower fat content and higher added alginate tended to be softer due to the high water holding capacity of the alginate particles.     

Improved Properties of Bacillus coagulans β‐Galactosidase through Immobilization

Thermostable β‐galactosidase from Bacillus coagulans RCS3 was purified by successive column chromatography using DEAE‐cellulose and Sephadex G‐50. Immobilization of the purified enzyme was studied with DEAE‐cellulose and calcium alginate. The efficiency of β‐galactosidase retention was 87 % with DEAE‐cellulose (17 mg protein/mL of matrix) and 80 % with calcium alginate (2.2 mg protein/g bead). Comparative studies of immobilization displayed a shift in the optimum temperature from 65 °C to 70 °C provoked by DEAE‐cellulose, although no effect was observed with calcium alginate. The heat inactivation curve revealed an improvement in the stability (t_1/2 of 14.5 h for the immobilized enzyme as compared to 2 h for the free enzyme at 65 °C) in a calcium alginate system. This immobilized enzyme has a wide pH stability range (6.5–11). β‐Galactosidase immobilized by DEAE‐cellulose and calcium alginate allowed a 57 and 70 % lactose hydrolysis, respectively, to be achieved within 48 h after repeated use for twenty times.     

Water status and thermal analysis of alginate beads used in cryopreservation of plant germplasm

Encapsulation and dehydration techniques using alginate beads are used increasingly for the pre-treatment of various plant materials for cryopreservation to improve survival post-cryogenic storage. This study reports the effects of the water content of beads (formed with 3% (w/v) alginic acid in liquid S-RIB), polymerisation time (in 100 mM calcium chloride solution), osmotic dehydration (in 0.75 M sucrose solution), and evaporative air desiccation on the thermal properties of alginate beads used in cryopreservation protocols. Experimental beads were assayed using a differential scanning calorimeter (DSC) with a cooling programme to -150 degrees C, followed by re-warming. Resultant thermograms were evaluated with particular reference to the onset temperature and enthalpy of the melt endotherm from which the quantities of frozen and unfrozen water were calculated. Treatments were applied sequentially to samples of beads and their thermal features evaluated at each stage of the protocol. Using 'standard' beads (40-55 mg fresh weight), formed using plastic disposable pipettes, the degree of polymerisation (>10 min) proportionally reduced their dry weight and increased their water content. Thermal characteristics of the beads were unaffected by polymerisation times >10 min, but the maximum level (23%) of unfrozen (osmotically inactive) water was achieved after 15 min polymerisation. Osmotic dehydration using 0.75 M sucrose significantly lowered bead water content and mean dry weight approximately doubled with 20-24 h immersion time. Bead desiccation in still air reduced their water content by 83% of fresh weight, whilst dry weight remained constant. After 8 h desiccation in air between 27 and 37% of the water in the bead was osmotically inactive (unfrozen) in DSC scans. Desiccation >18 h reduced this fraction to zero. The melt onset temperature and the enthalpy of melting were directly related to bead water content. The unfrozen water fraction increased substantially with reduced water content of the beads (from 23 to 37% of total water content), concomitant with a reduction in the ratio of unfrozen to frozen water from 1:3 to 1:2. For successful vitrification and the production of a glass that did not destabilise on rewarming, a bead water content of ca. 26% of fresh weight (0.4 g waterg(-1) dry weight) was required, much of which was osmotically inactive water. These data are discussed in relation to optimal pre-treatments for alginate bead encapsulation techniques used in the cryopreservation of a range of plant germplasm. It is proposed that increased standardisation of alginate beads, in terms of volume, fresh weight, and water content, is required to reduce the variability in physical and thermal features, which in turn will improve survival of plant samples post-cryopreservation.     

Production of alginate beads by emulsification/internal gelation. I. Methodology

Summary Small diameter alginate beads (microspheres) were formed via internal gelation of alginate solution emulsified within vegetable oil. Gelation was initiated by addition of an oil-soluble acid thereby reducing the pH of the alginate solution and releasing soluble Ca²⁺ from the citrate complex. Smooth, spherical, micron-sized beads were formed. The mean diameter ranged from 200 to 1000 m, controlled by the reactor impeller design and rotational speed. The technique has potential for large-scale and continuous applications in immobilization.Correspondence to: R. J. Neufeld     

Pancreatic cell immobilization in alginate beads produced by emulsion and internal gelation

Alginate has been used to protect transplanted pancreatic islets from immune rejection and as a matrix to increase the insulin content of islet progenitor cells. The throughput of alginate bead generation by the standard extrusion and external gelation method is limited by the rate of droplet formation from nozzles. Alginate bead generation by emulsion and internal gelation is a scaleable alternative that has been used with biological molecules and microbial cells, but not mammalian cells. We describe the novel adaptation of this process to mammalian cell immobilization. After optimization, the emulsion process yielded 90 ± 2% mouse insulinoma 6 (MIN6) cell survival, similar to the extrusion process. The MIN6 cells expanded at the same rate in both bead types to form pseudo‐islets with increased glucose stimulation index compared to cells in suspension. The emulsion process was suitable for primary pancreatic exocrine cell immobilization, leading to 67 ± 32 fold increased insulin expression after 10 days of immobilized culture. Due to the scaleability and broad availability of stirred mixers, the emulsion process represents an attractive option for laboratories that are not equipped with extrusion‐based cell encapsulators, as well as for the production of immobilized or encapsulated cellular therapeutics on a clinical scale. Biotechnol. Bioeng. 2011;108: 424–434. © 2010 Wiley Periodicals, Inc.     

Microbial and algal alginate gelation characterized by magnetic resonance

Advanced magnetic resonance (MR) relaxation and diffusion correlation measurements and imaging provide a means to non-invasively monitor gelation for biotechnology applications. In this study, MR is used to characterize physical gelation of three alginates with distinct chemical structures; an algal alginate, which is not O-acetylated but contains poly guluronate (G) blocks, bacterial alginate from Pseudomonas aeruginosa, which does not have poly-G blocks, but is O-acetylated at the C2 and/or C3 of the mannuronate residues, and alginate from a P. aeruginosa mutant that lacks O-acetyl groups. The MR data indicate that diffusion-reaction front gelation with Ca(2+) ions generates gels of different bulk homogeneities dependent on the alginate structure. Shorter spin-spin T(2) magnetic relaxation times in the alginate gels that lack O-acetyl groups indicate stronger molecular interaction between the water and biopolymer. The data characterize gel differences over a hierarchy of scales from molecular to system size.     

DNA encapsulation by an air-agitated, liquid-liquid mixer

Smooth and spherical alginate microspheres and nylon-membrane bound microcapsules were formed in an air-agitated, liquid-liquid mixer by emulsification/internal gelation and interfacial polymerization respectively. The mean diameter of the alginate microspheres ranged from 100 to 800 mum, and was controlled by process modifications. Increase in emulsifier concentration, gas flowrate, and emulsification time resulted in smaller microsphere size as did a decrease in liquid height. Increase in the dispersed phase viscosity resulted in a longer emulsification time required for approaching a minimum microsphere size. Microspheres could be formed with the proportion of dispersed phase approaching 30%. The yield of alginate microspheres was 70%, with losses attributed to incomplete recovery during washing and filtration operations. The yield of DNA encapsulation within the fraction of recovered microspheres, was 94%. The small loss was thought to occur by surface release during the washing of the microspheres. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 464-470, 1997.     

Granulation of subtilisin by internal gelation of alginate microspheres for application in detergent formulation

Subtilisin was encapsulated within impact-resistant alginate granules produced by emulsification, internal gelation, and acetone extractive drying. The mechanical and controlled release properties of the granules were modified by adding to the alginate varying levels of formulation excipients, including titanium dioxide, polyvinyl alcohol, microcrystalline cellulose, starch and sucrose. Optimum protease activity and mass yields of 83 and 88%, respectively (mg active subtilisin/g granules), occurred for granules formulated with 3% alginate, 10% starch, 10% titanium dioxide, and 3% subtilisin. Mass losses occurred primarily during the gelation step. Maximum encapsulation efficiency is achieved by using higher molecular weight alginate, increasing the alginate concentration, and carefully controlling process temperature and pH. The strongest granules were obtained at the higher concentrations of medium-G or high-G alginate, while fastest granule dissolution was achieved when a lower concentration of alginate was used in combination with polyvinyl alcohol or microcrystalline cellulose as dispersants. Mechanical properties of alginate granules were found to be unaffected by the different cations employed in matrix gel formation.     

Phase separation in calcium alginate gels

Alginates are polysaccharides consisting of beta-D-mannuronate and alpha-L-guluronate units. In the presence of bivalent cations like calcium the guluronate blocks form physically cross-linked gels. The gelation properties of alginates play an important role in the stability of extracellular polymer substances and in the food industry. When stock solutions of Ca2+ ions and alginate are mixed, the gelation starts before the Ca2+ ions are evenly distributed, which leads to non-uniform gels. In this contribution, Ca alginate gels were prepared by in situ gelation using glucono-delta-lactone and CaCO3. In this way, uniform gels could be prepared directly in the measuring cell. Below a critical concentration, highly viscous solutions were obtained, which were below the critical point of gel formation. In these solutions at low rotational speeds a Schlieren peak arose, which became smaller and steeper with increasing time until a new meniscus could be detected. This behaviour is in contrast to the peak broadening due to diffusion after a synthetic boundary was formed. Evaluation of the data leads to negative diffusion coefficients. It has been shown by others that the mutual diffusion coefficient must be negative in the spinodal region. This phenomena is known as uphill diffusion and leads to phase separation of a binary system. The formation of the gel phase in this case is therefore discussed as uphill diffusion.     

Spherical alginate granules formulated for quick-release active subtilisin

Novel attrition-resistant and spherical enzyme granules encapsulating active subtilisin were formed by emulsification of 2% alginate sol loaded with active enzyme, instantaneous gelation triggered through in situ release of Ca(2+) (internal gelation), particle separation, and finally acetone extractive drying. Granular subtilisin was highly active, readily dispersible, and mechanically robust. This technique serves as a new and attractive alternative to established enzyme granulation processes, such as fluid bed coating, extrusion followed by marumerization, drum granulation, or prilling, for use in industrial enzyme applications such as detergents, textile manufacturing, and food processing. The formulation and encapsulation conditions were optimized to maximize the resistance of the granule to compression and impact forces, consistent with enzyme release and particle dispersion in detergent solutions. Well characterized alginates, with specified guluronic/mannuronic acid (G/M) content and molecular weight, were used in the formulation. The characteristics of the resulting microspheres, including their size and distribution, morphology, shrinkage, compression resistance, impact strength, solubility and encapsulation yield, were examined. Spherical dry granules were formulated with a mean diameter of 500 microm with particle sizes ranging from 300 to 800 microm. Dry alginate granules were discrete, spherical, and glossy white and exhibited impact strength, compression resistance, and solubility difference dependent on composition. Reduced starch levels, high alginate concentration, low alginate molecular weight, and use of high guluronate alginates resulted in the lowest dust level and highest compression resistance. Subtilisin mass yields were approximately 50%, and specific activity yields ranged from 60% to 100%. A formulation consisting of 3% SG150 alginate, 10% starch, 10% TiO(2), and 1% CaCO(3) provided granules appropriate for use in detergent application.     

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane-based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2-4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5-3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44-155 kD-lowering their diffusion rates from 10- to 100-fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 A for 1.5 and 3% alginate gels, respectively, and 480 and 360 A for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different-size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration. (c) 1996 John Wiley & Sons, Inc.