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PubMed2000

Encapsulation of probiotic bacteria with alginate-starch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt.

A modified method using calcium alginate for the microencapsulation of probiotic bacteria is reported in this study. Incorporation of Hi-Maize starch (a prebiotic) improved encapsulation of viable bacteria as compared to when the bacteria were encapsulated without the starch. Inclusion of glycerol (a cryo-protectant) with alginate mix increased the survival of bacteria when frozen at -20 degrees C. The acidification kinetics of encapsulated bacteria showed that the rate of acid produced was lower than that of free cultures. The encapsulated bacteria, however, did not demonstrate a significant increase in survival when subjected to in vitro high acid and bile salt conditions. A preliminary study was carried out in order to monitor the effects of encapsulation on the survival of Lactobacillus acidophilus and Bifidobacterium spp. in yoghurt over a period of 8 weeks. This study showed that the survival of encapsulated cultures of L. acidophilus and Bifidobacterium spp. showed a decline in viable count of about 0.5 log over a period of 8 weeks while there was a decline of about 1 log in cultures which were incorporated as free cells in yoghurt. The encapsulation method used in this study did not result in uniform bead size, and hence additional experiments need to be designed using uniform bead size in order to assess the role of different encapsulation parameters, such as bead size and alginate concentration, in providing protection to the bacteria.

Alginates↗
PubMed2001

Factors affecting protein release from alginate-chitosan coacervate microcapsules during production and gastric/intestinal simulation.

A series of experiments was performed to evaluate the influence of a number of physico-chemical factors on the diffusion of a model protein, bovine serum albumin (BSA), from dried chitosan-coated alginate microcapsules. Diffusion of BSA was quantified during the microcapsule manufacture processes (gelation, washing, rinsing) and during incubation in conditions simulating the pH encountered during the gastric (0.1 N HCl; pH 1.5) and intestinal (200 mM Tris-HCl; pH 7.5) phases of digestion. Factors tested included alginate and chitosan concentration, calcium chloride (CaCl2) concentration in the gelation medium, loading rate, chitosan molecular mass and pH of the gelation medium. Microcapsule size and gelation time were altered in order to determine their effects on protein retention. Alginate and chitosan concentration significantly influenced BSA retention during microcapsule manufacture and acid incubation, as did calcium chloride concentration in the gelation medium (P<0.05). BSA retention during manufacture was not significantly altered by protein loading rate or pH of the encapsulation medium, however, protein retention during acid incubation decreased significantly with increasing protein loading rate and encapsulation medium pH (P<0.05). Microcapsules that were washed with acetone following manufacture demonstrated significantly increased protein retention during acid incubation (P<0.05). In microcapsules that had been acetone-dried to a point whereby their mass was reduced to 10% of that immediately following encapsulation, protein retention was over 80% following 24-h acid incubation vs. only 20% protein retention from non acetone-dried microcapsules. The presence of calcium in the neutral buffer medium significantly reduced BSA diffusion in a concentration-dependent manner (P<0.05).

Alginates↗
PubMed2002

Studies on alginate-chitosan microcapsules and renal arterial embolization in rabbits.

Spherical and well-dispersed alginate-chitosan microcapsules, with a mean diameter of 77.28+/-0.93 microm (n=3), were prepared by the emulsification-gelation method. Adriamycin hydrochloride (ADM) was used as a model drug to investigate the drug loading capacity and release characteristics of the microcapsules. The drug/carrier ratio and chitosan concentration influenced the encapsulation efficiency of adriamycin. The adriamycin release from microcapsules was obviously different in 0.1 mol/l HCl from that in phosphate-buffered saline (PBS, pH 7.4). The drug was completely and rapidly released in 0.1 mol/l HCl, while it showed a sustained release after a burst release in PBS. The increase in chitosan concentration had no effect on adriamycin release in PBS. Using sulforhodamin B (SRB)-staining survival assay, the inhibition of adriamycin alginate-chitosan microcapsules (ADM-ACM) to different cancer cell lines (human BGC-823 cells, Bel-7402 cells and Hela cells) in vitro was determined. The inhibitory rate of ADM-ACM suspension to the three cell lines significantly outran that of ADM solution, no matter at high or low concentration. The effects of blank alginate-chitosan microcapsules (BACM) on renal arterial embolization were examined with transcatheter arterial embolization in rabbits. The angiogram and histopathological results indicated the blank microcapsules had excellent short- and long-term effects on renal arterial embolization.

Alginates↗
PubMed2003

Numerical modelling of insulin and amyloglucosidase release from swelling Ca-alginate beads.

The release of insulin hexamer (39 kD) and amyloglucosidase (AMG, 97 kD), entrapped in spherical Ca-alginate beads, was investigated. While the release of insulin could be described solely by diffusion this was not the case for the 1.6 (rm/rm) larger AMG protein, where rm is the Stokes-Einstein effective molecular radius. Because the alginate bead size was not constant during the release experiments, it was hypothesised that in addition to the diffusional mass transfer, a non-negligible convective flow of liquid in or out of the beads was present due to swelling or shrinkage, respectively. Although it should be expected that the effective diffusion coefficient of AMG is only slightly lower than that of insulin, the results show that the effective diffusions coefficient of AMG was found to be much smaller. In the case of AMG, it was shown that including bead size changes and the resulting convective flow in the numerical model, release could be described more accurately. The numerical model was able to describe the release characteristics from both shrinking, swelling, and non-swelling alginate beads. To evaluate the effect of bead swelling on the protein release rate, a swelling modulus and a release index was defined, describing the different effects on release of smaller and larger proteins.

Alginates↗
PubMed2002

Electrophysiological and horseradish peroxidase-tracing studies of nerve regeneration through alginate-filled gap in adult rat spinal cord.

The spinal cord segments at T(9-10) were totally excised and the resulting gap was filled by implantation of alginate sponge in adult rats. A horseradish peroxidase-tracing study at 21 weeks after operation showed that numerous ascending and many but less numerous descending regenerating fibres traversed the alginate-filled gap, and that after re-entering the distal stump of the transected spinal cord, they extended randomly over a long distance away from the gap. Intracellular electrophysiological recording at the same postoperative time showed that both ascending and descending regenerating axons formed functional synapses with host neurons located beyond the gap. These findings suggest that alginate could be a promising material for the support of regenerating axons in the spinal cord.

Absorbable Implants↗
PubMed1999

Hyaluronate-alginate combination for the preparation of new biomaterials: investigation of the behaviour in aqueous solutions.

With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated. Crossed techniques were used to assess the existence of polymer interactions in aqueous solutions up to 20 mg/ml. Alginate was obtained from algae and hyaluronate was purified from rooster comb. Viscometry measurements using the capillary technique or the Couette flow, together with circular dichroism investigations, evidenced the moderate significance of interactions between the two polysaccharides in dilute solutions. In addition, the case of more concentrated solutions and containing 20 mg/ml alginate was approached by rheological measurements in the flow mode; the behaviour of the polymer associations appeared as a compromise between those of individual polysaccharides.

Alginates↗
PubMed1997

The Azotobacter vinelandii alg8 and alg44 genes are essential for alginate synthesis and can be transcribed from an algD-independent promoter.

A 2.8-kb DNA region, located immediately downstream of algD, contains the A. vinelandii alg8 and alg44 genes, whose sequences are highly homologous to those of the corresponding Pseudomonas aeruginosa genes. These genes occur on a transcript that does not include algD, and are transcribed from a promoter different from that transcribing algD; this is the fourth promoter described within the alginate biosynthetic gene cluster. alg8 and alg44 mutants were constructed and shown to be completely impaired in alginate production. Alg8 shares 28.20% identity and 38.09% similarity to Azorhizobium caulinodans NodC, a glycosyl transferase catalyzing the formation of beta-1,4 linkages. A topological model is predicted, which supports the idea of Alg8 being the polymerase responsible for alginate synthesis.

Alginates↗
PubMed2002

Preparation of alginate beads for floating drug delivery system: effects of CO(2) gas-forming agents.

Floating beads were prepared from a sodium alginate solution containing CaCO(3) or NaHCO(3) as gas-forming agents. The solution was dropped to 1% CaCl(2) solution containing 10% acetic acid for CO(2) gas and gel formation. The effects of gas-forming agents on bead size and floating properties were investigated. As gas-forming agents increased, the size and floating properties increased. Bead porosity and volume average pore size, as well as the surface and cross-sectional morphology of the beads were examined with Mercury porosimetry and Scanning Electron Microscopy. NaHCO(3) significantly increased porosity and pore diameter than CaCO(3). Incorporation of CaCO(3) into alginate solution resulted in smoother beads than those produced with NaHCO(3). Gel strength analysis indicated that bead strength decreased with increasing gas-forming agent from 9 to 4 N. Beads incorporating CaCO(3) exhibited significantly increased gel strength over control and NaHCO(3)-containing samples. Release characteristics of riboflavin as a model drug were studied in vitro. Release rate of riboflavin increased proportionally with addition of NaHCO(3). However, increasing weight ratios of CaCO(3) did not appreciably accelerate drug release. The results of these studies indicate that CaCO(3) is superior to NaHCO(3) as a gas forming agent in alginate bead preparations. The enhanced buoyancy and sustained release properties of CaCO(3)-containing beads make them an excellent candidate for floating drug dosage systems (FDDS).

Alginates↗
PubMed2002

Cross-linking mechanisms of calcium and zinc in production of alginate microspheres.

Calcium chloride and zinc sulphate were used to cross-link alginate microspheres prepared by an emulsification method. The microspheres cross-linked by a combination of these two salts showed different morphology and slower drug release compared with those cross-linked by the calcium salt alone. From viscosity study, it was found that zinc cations interacted with the alginate molecules to a greater extent than calcium cations. The varying effects of the salts on the properties of the microspheres were largely attributed to their ability to interact with the alginate molecules.

Alginates↗
PubMed2002

Alginate microparticles for enzyme peroral administration.

In order to protect protein and peptide drugs against inactivation by different barriers in the gastro-intestinal tract and to improve their absorption, alginate microparticles as a carrier of L-lactate dehydrogenase, were developed by spray-drying technique. However, alginate complexation and spray-drying conditions led to enzyme activity loss. Such a drawback was overcome by using protectant additives (carboxymethylcellulose sodium salt, polyacrylic acid sodium salt, lactose) preventing the enzyme inactivation by both interaction with alginate and experimental conditions, lactose having the most protective effect. Nevertheless, only polyacrylic acid sodium salt provided a microparticulate structure required for the target of the Peyer's patches.

Alginates↗
PubMed2003

The preparation and characterisation of drug-loaded alginate and chitosan sponges.

Sponges composed of sodium alginate and chitosan were prepared via a freeze drying process in order to assess the utility of mixed sponges as potential wound dressings or matrices for tissue engineering. Sponge preparation involved dissolving both polymers (either individually or mixed) in 1% acetic acid and freeze-drying the corresponding solutions. The mechanical properties of the sponges were assessed using texture analysis and the microstructure examined using scanning electron microscopy. The dissolution of a model drug (paracetamol) from the sponges was assessed as a function of polysaccharide composition. It was noted that the sponges had a flexible yet strong texture, as assessed macroscopically. Measurement of the resistance to compression ('hardness') indicated that the chitosan sponges were the 'hardest' while the alginate sponges showed the least resistance to compression, with all sponges showing a high degree of recovery. In contrast, the breaking force (tensile force) of the sponges were greatest for the single component systems, while the elongation prior to breaking was similar for each material. SEM studies indicated that the mixed systems had a less well-defined microstructure than the single component sponges. This was ascribed to the two polysaccharides interacting in aqueous solution via coulombic forces, leading to a more randomly ordered network being formed on freezing. Dissolution studies indicated that systems containing chitosan alone showed the slowest release profile, with the mixed systems showing a relatively rapid dissolution profile. The use of chitosan and alginates together, therefore, appears to allow the formulator to manipulate both the mechanical properties and the drug release properties of the sponges.

Alginates↗
PubMed2003

A novel two-step method for the formation of tissue-engineered cartilage by mature bovine chondrocytes: the alginate-recovered-chondrocyte (ARC) method.

Most attempts to tissue-engineer cartilage have involved seeding of cultured cells into a biological or synthetic scaffold. We have developed a novel two-step culture approach that makes possible the in vitro formation of cartilaginous-like tissue by mature adult bovine chondrocytes without the aid of a synthetic matrix. The first step consists of culturing chondrocytes under conditions that maintain their rounded shape and their molecular phenotype as assessed by type II collagen and aggrecan production. This step was accomplished by culturing the isolated chondrocytes in alginate beads until the cells have reestablished a proteoglycan-rich cell-associated matrix (CM). The second step consists of culturing the cells with their CM, after recovery from the beads, on a tissue culture insert with a porous membrane. In this study, young adult bovine articular chondrocytes were cultured in alginate beads in the presence of 10% or 20% fetal bovine serum (FBS). After 7 days of culture, the alginate beads were dissolved by incubating the beads for 20 min in sodium citrate buffer, a calcium chelator. Following a brief centrifugation, the cells with their CM were recovered, resuspended in medium containing 10% or 20% FBS and seeded onto a tissue culture insert. After 1 week of culture on the insert, the individual cells with their CM progressively became incorporated into a mass of cartilaginous tissue. Culture with 20% FBS resulted in the best formation of tissues. These tissues, easily recovered from the insert, were then subjected to biochemical and histological analyses. The biochemical results showed that the chondrocytes remain phenotypically stable in the tissues. The de novo tissue has a relatively high ratio of PG/collagen. Histological examination of the tissue revealed it contained a cartilage-like matrix strongly stained with toluidine blue. This scaffold-free system appears ideal to study, in vitro, the development of transplantable cartilaginous tissue.

Alginates↗
PubMed1999

In-vivo delivery of therapeutic proteins by genetically-modified cells: comparison of organoids and human serum albumin alginate-coated beads.

We have designed a self-assembling multimeric soluble CD4 molecule by inserting the C-terminal fragment of the alpha chain of human C4-binding protein (C4bp alpha) at the C-terminal end of human soluble CD4 genes. This CD4-C4bp alpha fusion protein (sMulti-CD4) and two other reference molecules, a fusion protein of human serum albumin (HSA) and the first two domains of CD4 (HSA-CD4) and monomeric soluble CD4 (sMono-CD4), were delivered in vivo by genetically modified 293 cells. These cells were implanted in mice as organoids and also encapsulated in HSA alginate-coated beads. sMulti-CD4 showed an apparent molecular weight of about 300-350 kDa, in accordance with a possible heptamer formula. sMulti-CD4 produced either in cell culture or in vivo in mice appeared to be a better invitro inhibitor of HIV infection than sMono-CD4. Plasma levels of sMulti-CD4, HSA-CD4, and sMono-CD4 reached approximately 2,300, 2,700, and 170 ng/mL, respectively, 13 weeks after in-vivo organoid implantation, which had formed tumours at that time. This suggests that the plasma half-life of sMulti-CD4 is much longer than that of sMono-CD4. The 293 xenogeneic cells encapsulated in HSA alginate-coated beads remained alive and kept secreting sMono-CD4 or HSA-CD4 continuously at significant levels for 18 weeks in nude mice, without tumour formation. When implanted in immunocompetent Balb/c mice, they were rejected two to three weeks after implantation. In contrast, encapsulated BL4 hybridoma cells remained alive and kept secreting BL4 anti-CD4 mAb for at least four weeks in Balb/c mice. These results suggest the clinical potential of the C4bp-multimerizing system, which could improve both the biological activity and the poor in-vivo pharmacokinetic performance of a monomeric functional protein like soluble CD4. These data also show that a systemic delivery of therapeutic proteins, including immunoglobulins, can be obtained by the in-vivo implantation of engineered allogeneic cells encapsulated in HSA alginate-coated beads.

Alginates↗
PubMed2000

Growth and survival of phosphate-solubilizing bacteria in calcium alginate.

Calcium alginate was superior to conventional charcoal-soil (3:1) carrier for phosphate-solubilizing bacteria (PSB). High populations of Pseudomonas striata (27) and Bacillus polymyxa (H-5) could be maintained in this polymer during storage. Incorporation of charcoal-soil (3:1) adversely affected the initial loading of these organisms in alginate gel. Alginate alone supported maximum survival of these organisms at elevated storage temperature (40 degrees C).

Alginates↗
PubMed2003

Macroaffinity ligand-facilitated three-phase partitioning for purification of glucoamylase and pullulanase using alginate.

Starch-degrading enzymes glucoamylase (from Aspergillus niger), and pullulanase (from Bacillus acidopullulyticus) were purified using alginates (polysaccharides consisting of mannuronic acids and guluronic acids) by a recently developed technique called macroaffinity ligand-facilitated three-phase partitioning (MLFTPP). In this process, a crude preparation of the enzyme was mixed with alginate. On addition of appropriate amounts of ammonium sulfate and t-butanol, the alginate bound enzyme appeared as an interfacial precipitate between the lower aqueous and the upper t-butanol phase. Enzyme activity from this interfacial precipitate was recovered using 1M maltose. Glucoamylase and pullulanase were purified 20- and 38-fold with 83% and 89% activity recovery, respectively. Both the purified preparations showed a single band on SDS-PAGE.

Alginates↗
PubMed2003

Method for monitoring alginate released in biological fluids by high-performance anion-exchange chromatography with pulsed amperometric detection.

The use of alginate-entrapped cells in cell therapy requires a method for monitoring possible released compound within biological fluids following either their implantation or inoculation in artificial organs. Oligomannuronic and oligoguluronic acids were prepared by enzymatic depolymerization with alginate lyase from Pseudomonas alginovora, characterized by high-performance anion-exchange chromatography with pulsed amperometric detection and quantitated in human, pig and rabbit blood, urine and tissue samples. The method was tested for linearity and detection limit, accuracy, intra- and inter-day precision. The limit of detection was 3 microgram/ml in both urine and plasma and 5 mg/g of tissues. The relative standard deviations (RSDs) of intra-day precision were 6.0-16.6% and 4.8-8.7% in plasma and urine, respectively; the RSDs of inter-day precision were 5.1-14.4% and 5.0-11.6% in plasma and urine, respectively. Thus, this method appears suitable for the measurement of released alginate from entrapped cells used in cell therapy.

Alginates↗
PubMed1999

Assessment of alginic acid molecular weight and chemical composition through capillary electrophoresis.

The polydispersity of alginic acids regarding distributions in molecular weight and chemical composition is analyzed by capillary electrophoresis. Low concentrations of linear polyacrylamide in the electrolyte provide the means for separation of alginate fractions into the megadalton range, while high-resolution polysaccharide mapping of structural variants could be achieved at higher LPAA concentrations. The fraction and block length of the structurally important guluronic acid constituent is determined through the selective complexation with calcium ions. The methodology presented here might serve as a general approach for the assessment of alginate composition and size and the relation to technical properties desired in medical and pharmaceutical applications.

Alginates↗
PubMed1996

Carbohydrate fluxes into alginate biosynthesis in Azotobacter vinelandii NCIB 8789: NMR investigations of the triose pools.

The metabolite flux of carbohydrates through primary catabolism and into hexose resynthesis has been investigated for alginic acid biosynthesis in Azotobacter vinelandii. To do these studies, we fed the microorganism a variety of 13C-labeled glucose precursors, including [U-13C6]glucose. The incorporations of the precursors were determined by 1D 13C-NMR, 2D 13C-DQF-COSY, and inverse triple-quantum correlation experiments. The results clearly show that the entire catabolism of hexose is through the Entner-Doudoroff (E-D) pathway and that the triose pools are in equilibrium. Reentry into gluconeogenesis prior to alginate synthesis occurs totally from the glyceraldehyde 3-phosphate generated by the E-D pathway. The obligatory intermediacy of triose intermediates in alginate biosynthesis was proved. The experiments and results presented in this paper constitute a new method for distinguishing the E-D pathway from glycolysis in bacteria.

Alginates↗