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PubMed2005

Phagocytosis-mediated retroviral transduction: co-internalization of deactivated retrovirus and calcium-alginate microspheres by macrophages.

BACKGROUND: Efficiency of retrovirus-mediated gene transfer has been hampered by short retroviral half-life due to shedding of retroviral envelope proteins which is of utmost importance to the interaction between retrovirus and cell. METHODS: Calcium-alginate microspheres with average size of 780 nm were prepared by reverse emulsification and characterized by scanning electron microscopy and microelectrophoresis. To obtain deactivated retroviruses produced from 293 packaging cells, retrovirus-containing media were pre-incubated at 37 degrees C for 6, 12, 18, and 24 h, respectively. Murine J774A.1 macrophages were co-treated with Ca-alginate microspheres and deactivated retroviruses encoding the enhanced green fluorescent protein (eGFP) gene. Through phagocytosis, deactivated retroviruses and Ca-alginate microspheres were co-internalized into macrophages. After retroviral transduction for 24 and 48 h, the percentages of macrophages with eGFP expression were determined by bright-field and fluorescence microscopy. RESULTS: After 48-h incubation with Ca-alginate microspheres and deactivated retroviruses, phagosomes turned into large vacuoles occupied almost half of the cytoplasmic space. This was probably attributed to the erosion of Ca-alginate microspheres by destructive agents within vacuolar compartments and concomitant osmotic swelling. It was reasoned that deactivated retroviruses escaped such enlarged vesicles easily and underwent reverse transcription in the cytosol. The expression of eGFP in macrophages infected by retroviruses pre-incubated for 24 h in the presence of Ca-alginate microspheres was thereby augmented up to tenfold in comparison with the cells treated with 24-h deactivated retroviruses only. CONCLUSIONS: Ca-alginate microspheres performed as auxiliary agents for the enhancement of retrovirus-mediated gene transfer efficiency even though retroviruses had been deactivated due to the loss of envelope proteins.

Alginates↗

PubMed1995

Pseudomonas aeruginosa biofilms: role of the alginate exopolysaccharide.

Pseudomonas aeruginosa synthesizes an exopolysaccharide called alginate in response to environmental conditions. Alginate serves to protect the bacteria from adversity in its surroundings and also enhances adhesion to solid surfaces. Transcription of the alginate biosynthetic genes is induced upon attachment to the substratum and this leads to increased alginate production. As a result, biofilms develop which are advantageous to the survival and growth of the bacteria. In certain circumstances, P. aeruginosa produces an alginate lyase enzyme which cleaves the polymer into short oligosaccharides. This negates the anchoring properties of the alginate and results in increased detachment of the bacteria away from the surface, allowing them to spread and colonize new sites. Thus, both alginate biosynthetic and degradative enzymes are important for the development, maintenance and spread of P. aeruginosa biofilms.

Alginates↗

PubMed2005

In vitro stimulation of murine peritoneal monocytes induced by alginates.

In this trial we assessed the effect of soluble alginates on murine cells. Mouse peritoneal monocytes were stimulated in vitro with a solution of alginate. The production of TNF-alpha and nitric oxide (NO), the expression of surface molecules CD80 and CD86, and the ability of monocytes to phagocyte bacteria were assessed, in order to evaluate the effect of alginate on cell functionality. We showed that mouse peritoneal monocytes stimulated with alginate produce NO and TNF-alpha. In addition, alginate is able also to increase their phagocytic activity and to a lesser extent also to increase the expression of CD80. Even with different degrees, it implies that alginates per se act directly on immune response, being able to effectively stimulate proinflammatory activity. These findings corroborate the idea that alginates can represent interesting adjuvants to use to increase the efficacy of antigenic stimulation.

Adjuvants, Immunologic↗

PubMed2003

Azotobacter vinelandii mutants that overproduce poly-beta-hydroxybutyrate or alginate.

Azotobacter vinelandii produces two polymers of industrial importance, i.e. alginate and poly-beta-hydroxybutyrate (PHB). Alginate synthesis constitutes a waste of substrate when seeking to optimize PHB production and, conversely, synthesis of PHB is undesirable when optimizing alginate production. In this study we evaluated the effect of a mutation in algA, the gene encoding the enzyme that catalyzes the first step of the alginate biosynthetic pathway in the production of PHB. We also evaluated production of alginate in strain AT6 carrying a phbB mutation that impairs PHB synthesis. The algA mutation prevented alginate production and increased PHB accumulation up to 5-fold, determined in milligrams per milligram of protein. Similarly, the phbB mutation increased alginate production up to 4-fold.

Acyltransferases↗

PubMed2000

Biocompatible alginate from freshly collected Laminaria pallida for implantation.

A simple procedure is described for the extraction and purification of alginate from the inner stipes of the kelp Laminaria pallida. Alginate yield was about 10-15% of the dry mass, with a 70:30 mannuronic/guluronic acid ratio. Analysis of the purified alginate revealed a low polyphenol content while proteins were below detection level. The purified alginate was highly viscous, with 10-15 mPa s and 281 mPa s for a 0.1% and 0.5% solution, respectively, indicating a very high molecular mass (larger than 250 kDa). Bead formation occurred in the presence of divalent cations, but also in the presence of artificial serum (FCSIII) without added divalent cations. The biocompatibility of the alginate was tested with the in vitro mice lymphocyte test as well as by implantation of Ba2+ cross-linked beads beneath the kidney capsule of BB/OK rats. There was no evidence for significant mitogenic activity or fibrotic reaction. Biocompatibility of the alginate was also demonstrated by the encapsulation of human chondrocytes into Ca2+ cross-linked alginate beads. Immobilized chondrocytes grew and remained functional (i.e. they produced collagen).

Alginates↗

PubMed1997

Bacterial alginates: biosynthesis and applications.

Alginate is a copolymer of beta-D-mannuronic acid and alpha-L-guluronic acid (GulA), linked together by 1-4 linkages. The polymer is a well-established industrial product obtained commercially by harvesting brown seaweeds. Some bacteria, mostly derived from the genus Pseudomonas and belonging to the RNA superfamily I, are also capable of producing copious amounts of this polymer as an exopolysaccharide. The molecular genetics, regulation and biochemistry of alginate biosynthesis have been particularly well characterized in the opportunistic human pathogen Pseudomonas aeruginosa, although the biochemistry of the polymerization process is still poorly understood. In the last 3 years major aspects of the molecular genetics of alginate biosynthesis in Azotobacter vinelandii have also been reported. In both organisms the immediate precursor of polymerization is GDP-mannuronic acid, and the sugar residues in this compound are polymerized into mannuronan. This uniform polymer is then further modified by acetylation at positions O-2 and/or O-3 and by epimerization of some of the residues, leading to a variable content of acetyl groups and GulA residues. In contrast, seaweed alginates are not acetylated. The nature of the epimerization steps are more complex in A. vinelandii than in P. aeruginosa, while other aspects of the biochemistry and genetics of alginate biosynthesis appear to be similar. The GulA residue content and distribution strongly affect the physicochemical properties of alginates, and the epimerization process is therefore of great interest from an applied point of view. This article presents a survey of our current knowledge of the molecular genetics and biochemistry of bacterial alginate biosynthesis, as well as of the biotechnological potential of such polymers.

Alginates↗

PubMed2006

Bacterial alginates: from biosynthesis to applications.

Alginate is a polysaccharide belonging to the family of linear (unbranched), non-repeating copolymers, consisting of variable amounts of beta-D-mannuronic acid and its C5-epimer alpha- L-guluronic acid linked via beta-1,4-glycosidic bonds. Like DNA, alginate is a negatively charged polymer, imparting material properties ranging from viscous solutions to gel-like structures in the presence of divalent cations. Bacterial alginates are synthesized by only two bacterial genera, Pseudomonas and Azotobacter, and have been extensively studied over the last 40 years. While primarily synthesized in form of polymannuronic acid, alginate undergoes chemical modifications comprising acetylation and epimerization, which occurs during periplasmic transfer and before final export through the outer membrane. Alginate with its unique material properties and characteristics has been increasingly considered as biomaterial for medical applications. The genetic modification of alginate producing microorganisms could enable biotechnological production of new alginates with unique, tailor-made properties, suitable for medical and industrial applications.

Alginates↗

PubMed2005

Comparison of bone marrow cell growth on 2D and 3D alginate hydrogels.

Calcium cross-linked sodium alginate hydrogels have several advantageous features making them potentially suitable as tissue engineering scaffolds and this material has been previously used in many biomedical applications. 3D cell culture systems are often very different from 2D petri dish type cultures. in this study the effect of alginate hydrogel architecture was investigated by comparing rat bone marrow cell proliferation and differentiation on calcium cross linked sodium alginate discs and 1mm internal diameter tubes. It was found that bone marrow cell proliferation was diminished as the concentration of alginate in the 2D hydrogel substrates increased, yet proliferation was extensive on tubular alginate constructs with high alginate contents. Alginate gel thickness was found to be an important parameter in determining cell behaviour and the different geometries did not generate significant alterations in BMC differentiation profiles.

Absorbable Implants↗

PubMed1992

Alginate as immobilization matrix and stabilizing agent in a two-phase liquid system: application in lipase-catalysed reactions.

Alginate was evaluated as an immobilization matrix for enzyme-catalyzed reactions in organic solvents. In contrast to most hydrogels, calcium alginate was found to be stable in a range of organic solvents and to retain the enzyme inside the gel matrix. In hydrophobic solvents, the alginate gel (greater than 95% water) thus provided a stable, two-phase liquid system. The lipase from Candida cylindracea, after immobilization in alginate beads, catalysed esterification and transesterification in n-hexane under both batch and continuous-flow conditions. The operational stability of the lipase was markedly enhanced by alginate entrapment. In the esterification of butanoic acid with n-butanol, better results were obtained in the typical hydrophilic calcium alginate beads than in less hydrophilic matrices. The effects of substrate concentration, matrix area, and polarity of the substrate alcohols and of the organic solvent on the esterification activity were examined. The transesterification of octyl 2-bromopropanoate with ethanol was less efficient than that of ethyl 2-bromopropanoate with octanol. By using the hydrophilic alginate gel as an immobilization matrix in combination with a mobile hydrophobic phase, a two-phase liquid system was achieved with definite advantages for a continuous, enzyme-catalysed process.

Alcohols↗

PubMed1987

Graft copolymer emulsions of sodium alginate with hydroxyalkyl methacrylates for microencapsulation.

A hybrid material with interesting capsule forming properties has been prepared by gamma-initiated polymerization of hydroxyethyl methacrylate (HEMA) or other acrylic monomers in the presence of aqueous sodium alginate. For example, an aqueous solution of HEMA (1 wt%) and sodium alginate (1 wt%) was irradiated in a gamma-source at a dose of 0.15 MR, to produce a stable emulsion which coalesced in the form of a homogeneous precipitate when added to 0.1 M CaCl2. Polymerization of HEMA was presumed to be initiated by free radicals generated in the alginate by gamma-irradiation. The emulsion consisted largely of polyHEMA homopolymer stabilized by an alginate-polyacrylate graft (or block) copolymer of highly branched and ill-defined character, which acts as surfactant to prevent coagulation of the emulsion. Erythrocytes were encapsulated in such an emulsion by extrusion of cell/copolymer emulsion mixture into HEPES buffered CaCl2 (10 mM Ca). Cells appeared intact and functional after encapsulation. By SEM it appeared that the capsule wall consisted of microspheres of polyHEMA on the inner and outer surfaces held together by the alginate. Furthermore the capsule did not contain the internal meshwork, characteristic of calcium alginate homopolymer gels. Although much remains to be learned about these materials, the combination of the easy gelling characteristics of the alginate with the range of desirable properties (e.g. biocompatibility) afforded by the acrylic monomers may have a significant impact on the development of cell microencapsulation technology.

Alginates↗

PubMed2004

Effect of substrate mechanics on chondrocyte adhesion to modified alginate surfaces.

This study characterized the attachment of chondrocytes to RGD-functionalized alginate by examining the effect of substrate stiffness on cell attachment and morphology. Bovine chondrocytes were added to wells coated with 2% alginate or RGD-alginate. The alginate was crosslinked with divalent cations ranging from 1.25 to 62.5 mmol/g alginate. Attachment to RGD-alginate was 10-20 times higher than attachment to unmodified alginate and was significantly inhibited by antibodies to integrin subunits alpha3l and beta1, cytochalasin-D, and soluble RGD peptide. The equilibrium level and rate of attachment increased with crosslink density and substrate stiffness. Substrate stiffness also regulated chondrocyte morphology, which changed from a rounded shape with nebulous actin on weaker substrates to a predominantly flat morphology with actin stress fibers on stiffer substrates. The dependence of attachment on integrins and substrate stiffness suggests that chondrocyte integrins may play a role in sensing the mechanical properties of the matrices to which they are attached.

Alginates↗

PubMed2004

Studies on the nature of interaction of iron(III) with alginates.

The interactions between the polysaccharide alginate and iron(III) were investigated. The solution properties were studied through pH-metry, viscometry, zeta potential and particle size measurements. In the presence of alginate, iron(III) was stabilized and no precipitation was observed. Studies indicate that iron(III)-alginate system was more stable than iron(III) or alginate alone. The binding constant is of the order of 10(4) M(-1). A case for 'site binding model' for the interaction between alginate and Fe(III) has been made based on the studies using circular dichroism and zeta potential experiments. The number of binding sites per molecule of alginate has been estimated to be 66. This indicates that the alginate can bind more number of Fe(III) ions and thus provide a stable complex which can find wide industrial applications.

Alginates↗

PubMed2006

The promotion of oriented axonal regrowth in the injured spinal cord by alginate-based anisotropic capillary hydrogels.

Appropriate target reinnervation and functional recovery after spinal cord injury depend on longitudinally directed regrowth of transected axons. To assess the capacity to promote directed axon regeneration, alginate-based highly anisotropic capillary hydrogels (ACH) were introduced into an axon outgrowth assay in vitro and adult rat spinal cord lesions in vivo. In an entorhino-hippocampal slice culture model, alginate-based scaffolds elicit highly oriented linear axon regrowth and appropriate target neuron reinnervation. Coating of alginate-based ACH with the extracellular matrix components collagen, fibronectin, poly L-ornithine and laminin did not alter the axon regrowth response as compared to uncoated alginate-based ACH. After implantation into acute cervical spinal cord lesions in adult rats, alginate-based ACH integrate into the spinal cord parenchyma without major inflammatory responses, maintain their anisotropic structure and in parallel to findings in vitro induce directed axon regeneration across the artificial scaffold. Furthermore, adult neural progenitor cells (NPC), which have been shown to promote cell-contact-mediated axon regeneration, can be seeded into alginate-based ACH as a prerequisite to further improve the regenerative capacity of these artificial growth supportive matrices. Thus, alginate-based ACH represent a promising strategy to induce directed nerve regrowth following spinal cord injury.

Alginates↗

PubMed2006

A novel oligoalginate lyase from abalone, Haliotis discus hannai, that releases disaccharide from alginate polymer in an exolytic manner.

We previously reported the isolation and cDNA cloning of an endolytic alginate lyase, HdAly, from abalone Haliotis discus hannai [Carbohydr. Res.2003, 338, 2841-2852]. Although HdAly preferentially degraded mannuronate-rich substrates, it was incapable of degrading unsaturated oligomannuronates smaller than tetrasaccharide. In the present study, we used conventional chromatographic techniques to isolate a novel unsaturated-trisaccharide-degrading enzyme, named HdAlex, from the digestive fluid of the abalone. The HdAlex showed a molecular weight of 32,000 on SDS-PAGE and could degrade not only unsaturated trisaccharide but also alginate and mannuronate-rich polymers at an optimal pH and temperature of 7.1 and 42 degrees C, respectively. Upon digestion of alginate polymer, HdAlex decreased the viscosity of the alginate at a slower rate than did HdAly, producing only unsaturated disaccharide without any intermediate oligosaccharides. These results indicate that HdAlex degrades the alginate polymer in an exolytic manner. Because HdAlex split saturated trisaccharide producing unsaturated disaccharide, we considered that this enzyme cleaved the alginate at the second glycoside linkage from the reducing terminus. The primary structure of HdAlex was deduced with cDNAs amplified from an abalone hepatopancreas cDNA library by the polymerase chain reaction. The translational region of 822 bp in the total 887-bp sequence of HdAlex cDNA encoded an amino-acid sequence of 273 residues. The N-terminal sequence of 16 residues, excluding the initiation methionine, was regarded as the signal peptide of this enzyme. The amino-acid sequence of the remaining 256 residues shared 62-67% identities with those of the polysaccharide lyase family-14 (PL14) enzymes such as HdAly and turban-shell alginate lyase SP2. To our knowledge, HdAlex is the first exolytic oligoalginate lyase belonging to PL14.

Alginates↗

PubMed2004

Adsorption of Cu and Mn on covalently cross-linked alginate gel beads.

The covalently cross-linked alginate gel beads were prepared by the reactions of Ca(2+)-doped alginate gel beads, which were formed by spraying a viscous alginate solution into a calcium chloride solution, with cyanogen bromide and following 1,6-diaminohexane. The cross-linking of alginate matrix decreased the mean bead diameter by about 30% and made the beads durable in some extent under alkaline conditions. The adsorption of metal ions on the covalently cross-linked alginate gel beads was rapid and reached at equilibrium within 30 min at 25 degrees C. Adsorption isotherms of Cu(II), Mn(II), and Ca2+ on the beads possessed a stepwise shape, which was firstly determined by Rorrer et al. [Ind. Eng. Chem. Res. 32 (1993) 2170] for cross-linked chitosan gel beads and explained by a pore-blockage mechanism. Higher selectivity was determined against Cu(II) over Mn(II) and Ca2+, especially at a low concentration region. These metal adsorption profiles for the covalently cross-linked alginate gel beads was almost the same as those for the un-cross-linked beads, indicating that the cross-linking reactions were performed without interfering the adsorption characteristics of alginate gel beads.

Adsorption↗

PubMed2004

Preparation of alginate-chitosan hybrid gel beads and adsorption of divalent metal ions.

Naturally occurring polysaccharides such as alginic acid and chitosan have been recognized as one of the most effective adsorbents to eliminating low levels of heavy metal ions from waste water stream. The present study intended to use alginic acid and chitosan simultaneously, which are expected to form a rigid matrix structure of beads due to electrostatic interaction between carboxyl groups on alginic acid and amino groups on chitosan, and to prepare alginate-chitosan hybrid gel beads. This could be achieved for the first time by using water-soluble chitosan, which was obtained by deacetylating chitin to 36-39% degree. The water-soluble chitosan dissolved in water could remain in solution in the presence of sodium alginate, and the homogeneous solution of chitosan and alginate was dispensed into a CuCl2 solution to give gel bead particles. The resultant beads were then reinforced by a cross-linking reaction of aldehyde groups on glutaraldehyde with amine groups on the chitosan. The cross-linking reaction made the beads durable under acidic conditions. The adsorption of Cu(II), Co(II), and Cd(II) on the beads was significantly rapid and reached at equilibrium within 10 min at 25 degrees C. Adsorption isotherms of the metal ions on the beads exhibited Freundlich and/or Langmuir behavior, contrary to gel beads either of alginate or chitosan showing a step-wise shape of adsorption isotherm.

Adsorption↗

PubMed2004

In situ cross-linking of sodium alginate with calcium and aluminum ions to sustain the release of theophylline from polymeric matrices.

Small matrices of calcium alginate or aluminium alginate have been investigated as possible controlled release systems for drugs. The objective of the present study was to sustain the release of theophylline from alginate matrices using different concentrations of aluminium chloride and calcium chloride in presence and absence of HPMC. Tablets containing differing concentrations of aluminium and calcium chloride were produced and the release rate of theophylline was tested using the basket dissolution apparatus over 8 h. Increasing amounts of aluminium chloride from 0.0001 to 0.00068 moles decreased the release of theophylline from 95.1 +/- 0.27 to 29.5 +/- 1.5, indicating a significant effect of aluminium ions on a reduction in the release rate of theophylline from sodium alginate matrices. In the case of matrices containing different concentrations of calcium ions, as the concentration of calcium chloride increased, the release rate increased to an optimum then declined after this. This was due to insufficient calcium ions being available to cross-link with the sodium alginate to form an insoluble gel. The effect of aluminium ions, as this is a trivalent ion compared to calcium, which is a divalent ion, aluminium ions are able to decrease the release rate with a smaller concentration compared to calcium ions. The results also showed that the presence of HPMC caused a reduction in release rate of theophylline from alginate matrices containing calcium chloride. Whereas, in the case of alginate matrices containing aluminium chloride the release rate of theophylline increased in presence of HPMC. For comparing the dissolution data, dissolution efficiency (DE) was used. The values of DE are consistent with the dissolution data. The results show that within a formulation series, DE values generally decrease when the cation concentration increases and this criterion can be used to describe the effect of calcium and aluminium ions on the release behaviour of theophylline from polymeric matrices.

Alginates↗

PubMed2004

The immune stimulatory effect of sodium alginate on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus.

The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (9.4-11.3 g) were injected individually with sodium alginate at 10, 20 or 50 microg g(-1). No significant differences in THC, DHC and superoxide dismutase activity were observed among the shrimp injected with saline and those injected with sodium alginate at 10, 20 or 50 microg g(-1). However, L. vannamei injected with sodium alginate at 20 microg g(-1)increased its phenoloxidase activity and respiratory burst after 2 days and one day, respectively. L. vannamei injected with sodium alginate at 50 microg g(-1)maintained a higher phagocytic activity and clearance efficiency to V. alginolyticus after 4 days. In another experiment, L. vannamei which had been injected with sodium alginate, were challenged with V. alginolyticus at 2x10(5)colony-forming units (CFU) shrimp(-1)and then placed in seawater of 34 per thousand. The survival of shrimp that received sodium alginate at either dose was significantly higher than that of control shrimp at the termination of the experiment (6 days after the challenge). It is therefore concluded that L. vannamei received sodium alginate at 10 microg g(-1)or more and increased its immune ability and resistance from V. alginolyticus infection.

Alginates↗