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PubMed2006

The efficacy of alginate encapsulated CHO-K1 single chain-TRAIL producer cells in the treatment of brain tumors.

OBJECT: Patients with astrocytic tumors in the central nervous system (CNS) have low survival rates despite surgery and radiotherapy. Innovative therapies and strategies must be developed to prolong survival of these patients. The alginate microencapsulation method, used to continuously release a certain cytotoxic agent in the vicinity of the tumor, is such a novel therapeutic strategy. The biological functionality of the apoptosis inducing scFv425:sTRAIL protein, which was released through the microencapsulation method, was studied in vitro. Analysis of the intracerebral biocompatibility of alginate capsules was performed by implantation of empty alginate capsules in the brain of mice. METHOD: Chinese Hamster Ovary cells (CHO-K1) were recombinantly engineered to produce the single chain anti-EGFR-sTRAIL protein (scFv425:sTRAIL). The CHO-K1 producer cells were encapsulated in an alginate capsule with a semi-permeable membrane through which the scFv425:sTRAIL protein could be released. RESULTS: In vitro studies show maintained biological functionality of the released scFv425:sTRAIL protein. There was no immunological tissue response detectable after intracerebral implantation of the alginate capsules in mice brains. CONCLUSION: Biological functionality of the produced scFv425:sTRAIL protein is maintained and intracerebral biocompatibility of the capsules is warranted. Alginate encapsulation of CHO-K1--scFv425:sTRAIL--producer cells and subsequently their intracerebral implantation is technically feasible. This study justifies further in vivo experiments.

Alginates↗

PubMed1991

Lipid-alginate interactions render changes in phospholipid bilayer permeability.

Lipid vesicles, e.g. liposomes, generally release their contents in a continuous manner. However, when these vesicles are entrapped in Ca-alginate and coated with poly(L-lysine), they release their contents in an unusual fashion, in 'bursts'. Molecular-level studies indicated that lipid-alginate interactions are responsible for changes in the barrier properties of lipid vesicles. Differential scanning calorimetry revealed that exposure of liposomes to alginate resulted in a 4-fold reduction in the phase transition enthalpy, with no change in the melting temperature. Size-exclusion chromatography of liposomes-in-alginate gave an additional liposomal peak with a smaller elution volume. These studies suggested that alginate is inserted into the lipid bilayer of vesicles. Lipid-alginate interactions were highly dependent on phospholipid head group charge and the phase transition temperature of the phospholipid. Based on these interactions, a mechanism to explain the 'burst' from these entrapped liposomes is suggested.

Alginates↗

PubMed1992

Analysis of Macrocystis pyrifera and Pseudomonas aeruginosa alginic acids by the reductive-cleavage method.

Permethylated alginic acids comprised of 4-linked D-mannopyranosyluronic acid and 4-linked L-gulopyranosyluronic acid residues undergo reductive cleavage to yield, after acetylation, methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-D-mannonate (2b) and methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-D-gluconate (3b) as major products. Small amounts (ca. 13%) of ring-contracted products, namely methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-D-mannonate (9) and methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-D-gluconate (10), were also observed in these experiments. These results are in marked contrast to previous results on the reductive cleavage of 4-linked D-glucopyranosyluronic acid residues, wherein the ring-contracted product was formed exclusively. Formation of the ring-contracted products could be completely eliminated by reduction (LiAlH4) of ester groups in the permethylated alginic acid prior to reductive cleavage. In the latter experiments, 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-D-mannitol (5b) and 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-L-gulitol (6b) were the sole products of reductive cleavage of the 4-linked ManA and 4-linked GulA residues, respectively. However, in the previous experiments it was noted that low yields of permethylated alginic acids were obtained and that extensive depolymerization occurred under methylation conditions. Depolymerization could be avoided and higher yields of permethylated polysaccharides could be obtained, by reduction of the carboxyl groups of the alginic acids prior to methylation. Reductive cleavage of the latter polysaccharides yielded the products expected from 4-linked D-mannopyranosyl and 4-linked L-gulopyranosyl residues, namely 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-D-mannitol (13b) and 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-L-gulitol (14b), respectively. Using the latter analytical strategy, it was established that the Macrocystis pyrifera alginate was comprised of 60% 4-linked ManA and 40% 4-linked GulA residues, whereas the Pseudomonas aeruginosa alginate was comprised of 80% 4-linked ManA and 20% 4-linked GulA residues.

Alginates↗

PubMed1992

Comparison of calcium sodium alginate (KALTOSTAT) and porcine xenograft (E-Z DERM) in the healing of split-thickness skin graft donor sites.

In a controlled, prospective study, calcium sodium alginate and porcine xenograft were compared in the treatment of split-thickness skin graft donor sites on 20 patients. Half of each donor site was dressed with each material. Time to complete healing, quality of regenerated skin and patient comfort were assessed. Time to healing was 8.1 days with alginate and 11.3 days with porcine xenograft (P less than 0.001). Quality of healed skin was consistently good with the alginate, and better than under the xenograft in 95 per cent of patients (P less than 0.001). Hypertrophic scarring was not observed under alginate dressings but occurred in 25 per cent of xenograft-dressed sites (P less than 0.01). In addition, evidence was found that allergic reactions to porcine xenograft could occur. Alginate was preferred by 75 per cent of patients and none preferred porcine xenograft (P less than 0.01); the remainder had no preference. It was concluded that porcine xenograft is inferior to calcium sodium alginate as a dressing for split-thickness skin donor sites.

Alginates↗

PubMed1995

Alginate regulatory and biosynthetic gene homologs in Pseudomonas putida WCS358: correlation with the siderophore regulatory gene pfrA.

A previous study [Venturi et al., Mol. Microbiol. 10 (1993) 63-73] demonstrated that the siderophore regulatory gene pfrA of Pseudomonas putida (Pp) WCS358 is highly similar and interchangeable with the alginate regulatory gene algQ (algR2) of P. aeruginosa (Pa). The algQ gene is physically linked to two other alginate regulators in the Pa chromosome, namely algR (algR1), a response regulator, and algP (algR3), a histone-like gene. In this study, we have identified the same genes and a similar genetic organization in the Pp chromosome. The two genes linked to pfrA, designated pprA and pprB, are similar to algR and algP, respectively. Chromosomal mutants of pprA and pprB were constructed showing that unlike pfrA, the two newly identified regulators are not involved in siderophore regulation. The pprA gene complemented a Pa algR mutant phenotype, suggesting that it could be involved in alginate gene regulation. The WCS358 strain is not producing alginate, but we demonstrated by Southern analysis that it also possesses, in addition to pprA and pprB, algD and algU (algT) gene homologs, two genes essential for alginate biosynthesis. Using an algD-xylE transcriptional fusion, we observed that the algD promoter is active in strain WCS358 and absolutely requires pfrA. The possibility that all five genes of Pp WCS358 are involved in alginate biosynthesis is discussed.

Alginates↗

PubMed1994

Magnetic resonance imaging (MRI) of calcium alginate gels.

The spatial heterogeneity of calcium alginate gels has been visualised by the variation of image intensity across the gel by one-, two-, and three-dimensional MRI. From the linear dependence of (T2)-1 on alginate concentration it is now possible to use MRI to map alginate concentration gradients within calcium alginate gels noninvasively. Since the concentration of alginate determines the pore size of the gel matrix, those alginate concentration maps will be useful in determining the pore size distribution within the gel, which is important in controlled release applications.

Alginates↗

PubMed2005

Size control of calcium alginate beads containing living cells using micro-nozzle array.

Size-controlled small (i.e. less than 300 microm) polyelectrolyte complex gel beads are urgently desired for wide-spread application, including use in medical, pharmaceutical, and bioengineering fields. However, it was impossible to obtain smaller beads less than 300 microm with conventional apparatuses. We developed a novel microfluidics device that utilizes silicon micro-nozzle (MN) array, enabling to produce 50-200 microm calcium alginate beads with a narrow size distribution. Alginate aqueous solution was extruded through a precisely fabricated thin (30 microm x 30 microm) and short (500 microm) MN and was sheared by the viscous drag force of oil flow to form alginate droplets. Alginate droplets were immediately reacted with CaCl2 droplets at the downstream of oil flow to form calcium alginate gel beads. This device enabled us to successfully encapsulate living cells into 162 microm calcium alginate beads with maintaining viability, which was confirmed by the expression of marker protein.

Alginates↗

PubMed2005

Release of angiogenic growth factors from cells encapsulated in alginate beads with bioactive glass.

Attempts to stimulate therapeutic angiogenesis using gene therapy or delivery of recombinant growth factors, such as vascular endothelial growth factor (VEGF), have failed to demonstrate unequivocal efficacy in human trials. Bioactive glass stimulates fibroblasts to secrete significantly increased amounts of angiogenic growth factors and therefore has a number of potential applications in therapeutic angiogenesis. The aim of this study was to assess whether it is possible to encapsulate specific quantities of bioactive glass and fibroblasts into alginate beads, which will secrete growth factors capable of stimulating angiogenesis. Human fibroblasts (CCD-18Co) were encapsulated in alginate beads with specific quantities of 45S5 bioactive glass and incubated in culture medium (0-17 days). The conditioned medium was collected and assayed for VEGF or used to assess its ability to stimulate angiogenesis by measuring the proliferation of human dermal microvascular endothelial cells. At 17 days the beads were lysed and the amount of VEGF retained by the beads measured. Fibroblasts encapsulated in alginate beads containing 0.01% and 0.1% (w/v) 45S5 bioactive glass particles secreted increased quantities of VEGF compared with cells encapsulated with 0% or 1% (w/v) 45S5 bioactive glass particles. Lysed alginate beads containing 0.01% and 0.1% (w/v) 45S5 bioactive glass contained significantly more VEGF (p<0.01) compared with beads containing no glass particles. Endothelial cell proliferation was significantly increased (p<0.01) by conditioned medium collected from alginate beads containing 0.1% (w/v) 45S5 bioactive glass particles. The results of this study demonstrate that bioactive glass and fibroblasts can be successfully incorporated into alginate beads for use in delivering angiogenic growth factors. With further optimization, this technique offers a novel delivery device for stimulating therapeutic angiogenesis.

Alginates↗

PubMed2005

Characteristics of Poly-L-Ornithine-coated alginate microcapsules.

Poly-L-Lysine (PLL) is the most widely used biomaterial for providing perm-selectivity in alginate microcapsules for islet transplantation. We had previously reported that Poly-L-Ornithine (PLO) is less immunogenic than PLL, and in the present study, we have compared the physical characteristics of PLO- and PLL-coated hollow alginate microcapsules. Microspheres made with 1.5% alginate were divided into 2 groups that were first coated with either 0.1% PLO or PLL, followed by a second coating with 0.25% alginate. After liquefaction of the inner alginate core with sodium citrate, the microcapsules were washed with saline and used for experiments. Pore size exclusion studies were performed with FITC-labeled lectins incubated with encapsulated pig islets followed by examination for fluorescence activity. Mechanical strength was assessed by an osmotic pressure test and by 36 h of mechanical agitation of microcapsules with inert soda lime beads. The pore size exclusion limit of microcapsules after 20 min of coating was significantly smaller with PLO. While the mean +/- SEM diameter of PLL-coated microcapsules increased from 718+/-17 to 821 +/- 17 microm (p < 0.05) during 14 days incubation at 37 degrees C, the PLO group did not change in size. Also, PLL group had a higher percentage of broken capsules (52.7 +/- 4.9%) compared to 3.1 +/- 2.05% for PLO capsules (p < 0.0001,n = 6). We conclude that PLO-coated alginate microcapsules are mechanically stronger and provide better perm-selectivity than PLL-coated microcapsules.

Alginates↗

PubMed2005

Utilization of an exopolysaccharide produced by Chryseomonas luteola TEM05 in alginate beads for adsorption of cadmium and cobalt ions.

Cadmium and cobalt adsorption from aqueous solution onto calcium alginate, sodium alginate with an extracellular polysaccharide (EPS) produced by the activated sludge bacterium Chryseomonas luteola TEM05 and immobilized C. luteola TEM05 was studied. In addition, solutions containing both of these ions were prepared and partial competitive adsorption of these mixtures was investigated. Metal adsorption onto gel beads was carried out at pH 6.0 and 25 degrees C. The maximum adsorption capacities determined by fitting Langmuir isotherms to the data for calcium alginate, calcium alginate+EPS, calcium alginate + C. luteola TEM05 and calcium alginate + EPS + C. luteola TEM05 were 45.87, 55.25, 49.26, 51.81 mg g(-1) for Co(II) and 52.91, 64.10, 62.5, 61.73 mg g(-1) for Cd(II), respectively. The biosorption capacity of the carrier for both metal ions together in competition was lower than those obtained when each was present alone.

Adsorption↗

PubMed2006

Cyclodextrin-linked alginate beads as supporting materials for Sphingomonas cloacae, a nonylphenol degrading bacteria.

Calcium alginate beads covalently linked with alpha-cyclodextrin (alpha-CD-alginate beads) were prepared and examined for their ability to serve as a supporting matrix for bacterial degradation of nonylphenol, an endocrine disruptor. Column chromatographic experiment using alpha-CD-alginate beads with diameter of 657+/-82 microm and with degree of CD substitution of 0.16 showed a strong affinity for nonylphenol adsorption. Although addition of alpha-CD (2.7-27 mM) to the culture broth of Sphingomonas cloacae retarded nonylphenol degradation, the immobilized bacteria on the CD-alginate beads were effective for the degradation. Batch degradation tests using the immobilized bacteria on alpha-CD-alginate-beads showed 46% nonylphenol recovery after 10-day incubation at 25+/-2 degrees C, and the recovery reached to about 17% when wide and shallow incubation tubes were used to facilitate uptake of the viscous liquid of nonylphenol on the surface of the medium. Scanning electron microscopic photographs revealed that multiplicated bacteria was present both on the surface and inside the beads and the matrix of CD-alginate was stable and suitable during 10-day incubation.

Adsorption↗

PubMed2006

Insulin encapsulation in reinforced alginate microspheres prepared by internal gelation.

Insulin-loaded alginate microspheres prepared by emulsification/internal gelation were reinforced by blending with polyanionic additive polymers and/or chitosan-coating in order to increase the protection of insulin at simulated gastric pH and obtain a sustained release at simulated intestinal pH. Polyanionic additive polymers blended with alginate were cellulose acetate phtalate (CAP), Eudragit L100 (EL100), sodium carboxymethylcellulose (CMC), polyphosphate (PP), dextran sulfate (DS) and cellulose sulfate (CS). Chitosan-coating was applied by using a one-stage procedure. The influence of additive polymers and chitosan-coating on the size distribution of microspheres, encapsulation efficiency and release profile of insulin in simulated gastrointestinal pH conditions was studied. The mean diameter of blended microspheres ranged from 65 to 106 microm and encapsulation efficiency of insulin varied from 14 to 100%, reaching a maximum value when CS and DS were incorporated in the alginate matrix. Insulin release, at pH 1.2, was almost prevented by the incorporation of PP, DS and CS. When uncoated microspheres were transferred to pH 6.8, a fast dissolution occurred, independently of the additive polymer blended with alginate, and insulin was completely released. Increasing the additive polymer concentration in the alginate matrix and/or chitosan-coating the blended alginate microspheres did not promote a sustained release of insulin from microspheres at pH 6.8.

Alginates↗

PubMed2005

Structure-activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells.

Guluronate and mannuronate oligomers with various degree of polymerization were prepared from polyguluronate (PG) and polymannuronate (PM) with an alginate lyase from a Pseudoalteromonas sp., and their activities to induce cytokine secretion from mouse macrophage cell line RAW264.7 cells were examined. Enzymatically depolymerized unsaturated alginate oligomers induced tumor necrosis factor (TNF)-alpha secretion from RAW264.7 cells in a structure-depending manner, while the activities of saturated alginate oligomers prepared by acid hydrolysis were fairly low or only trace levels. These results suggest that unsaturated end-structure of alginate oligomers was important for the TNF-alpha-inducing activity. Among the unsaturated guluronate (G3-G9) and mannuronate (M3-M9) oligomers, G8 and M7 showed the most potent activity, respectively. Bio-Plex assay revealed that interleukin (IL)-1alpha, IL-1beta, and IL-6 secretion from RAW264.7 cells were also induced by unsaturated alginate oligomers with similar structure-activity relationship profiles as seen in TNF-alpha, and the most potent activities were observed with G8 and M7. These results suggest that G8 and M7 may have the most suitable molecular size or entire structural conformation as stimulant for cytokine secretion. Since antibodies to Toll-like receptor (TLR)2 and TLR4 effectively inhibited the G8- and M7-induced production of TNF-alpha, these alginate oligomers may stimulate innate immunity through the pattern recognition receptors on macrophages similar to microbial products.

Alginates↗

PubMed2004

Characterization of the Azotobacter vinelandii algC gene involved in alginate and lipopolysaccharide production.

Azotobacter vinelandii is a soil gamma-proteobacteria that fixes nitrogen and forms desiccation-resistant cysts. The exopolysaccharide alginate is an integral part of the layers surrounding the cysts. Here, we reported the cloning of A. vinelandii algC, encoding the enzyme catalyzing the second step of alginate pathway. We showed that AlgC is involved not only in alginate production, but also in lipopolysaccharide (LPS) synthesis and that it seems to have both phosphomannomutase and phosphoglucomutase activities. The transcriptional analysis of the A. vinelandii algC gene showed that it contained two start sites, one of which was dependent on the alternative sigma factor AlgU/AlgT. This finding explains why alginate biosynthesis is dependent on AlgU activity, since all other alginate biosynthetic genes have been characterized previously and algC is the only alginate structural gene that is directly transcribed by this sigma factor.

Alginates↗

PubMed2006

The immunostimulatory effects of sodium alginate and iota-carrageenan on orange-spotted grouper Epinephelus coicoides and its resistance against Vibrio alginolyticus.

The lysozyme activity, alternative complement activity (ACH50), respiratory burst, SOD (superoxide dismutase) activity and phagocytic activity of orange-spotted grouper Epinephelus coicoides were examined when the fish were injected intraperitoneally with sodium alginate at 10, 20, 30mgkg-1 and iota-carrageenan at 10, 20, 30mgkg-1, respectively after 24, 72 and 120 h. Serum ACH50 increased directly with dose after 24 and 72 h for both sodium alginate and iota-carrageenan treatments. The fish that received sodium alginate at 20mgkg-1 after 24 and 72 h, and the fish that received iota-carrageenan after 72 and 120 h showed significantly increased respiratory burst, SOD activity and phagocytic activity, respectively. In another experiment, E. coicoides which had been injected individually with sodium alginate and iota-carrageenan at 10, 20, 30mgkg-1, were challenged with Vibrio alginolyticus at 1.8x10(9) colony-forming units (cfu)fish-1 and then placed in seawater of 33 per thousand. The survival of fish that received sodium alginate at 20mgkg-1, and the fish that received iota-carrageenan at 30mgkg-1 was significantly higher than that of fish which received saline and the control fish after 48 h as well as at the termination of the experiment (120 h after the challenge). It is therefore concluded that E. coicoides which received sodium alginate at 20mgkg-1 or iota-carrageenan at 30mgkg-1 increased the non-specific immune response and resistance from V. alginolyticus infection.

Adjuvants, Immunologic↗

PubMed2006

Pharmacokinetic and pharmacodynamic behaviour of antitubercular drugs encapsulated in alginate nanoparticles at two doses.

This study was designed to evaluate the pharmacokinetics and tissue distribution of free and alginate-encapsulated antitubercular drugs in mice at different doses. Alginate nanoparticles encapsulating isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB) were prepared by controlled cation-induced gelification of alginate. The formulation was orally administered to mice at two dose levels (D1 and D2). A comparison was made in mice receiving free drugs at equivalent doses. Drugs were analysed by high performance liquid chromatography (HPLC). The average size of alginate nanoparticles was found to be 235.5+/-0.0 nm with a polydispersity index of 0.44; drug encapsulation was 70-90% for INH and PZA, 80-90% for RIF and 88-95% for EMB. In the free drug groups, plasma levels of RIF and INH were higher and PZA and EMB levels were lower in the D1 group (per body surface area of mice) compared with the D2 group (recommended human dose). The plasma drug levels of all drugs were higher in the D1 encapsulated group compared with D2, resulting in higher values of area under the plasma drug concentration-time curve (AUC(0-infinity)). The relative bioavailabilities of all drugs encapsulated in alginate nanoparticles were significantly higher compared with free drugs. Drug levels were maintained at or above the minimum inhibitory concentration (MIC(90)) until Day 15 in organs after administration of encapsulated drugs, whilst free drugs stayed at or above the MIC(90) up to Day 1 only irrespective of dose. The levels of drugs in various organs remained above the MIC at both doses for equal periods, demonstrating their equiefficiency. Alginate nanoparticles hold great potential in reducing dosing frequency of antitubercular drugs.

Alginates↗

PubMed2005

Alginate microparticles prepared by spray-coagulation method: preparation, drug loading and release characterization.

A spray-coagulation method was developed for the preparation of large scale of porous alginate microparticles. The effect of three variables on porosity was evaluated: (1) alginate solution concentration (2) the concentration of CaCl2 in the coagulation medium and (3) the ratio of guluronic acid to manuronic acid of the alginate. Methylene blue (MB), a highly water-soluble compound and a practically water-insoluble compound, 4-phenylazoaniline (PAA) were used as the model drugs to study drug loading and release characteristics from alginate microparticles. The release of the model compounds from the microparticles was found to depend upon the release medium. Incomplete in vitro release of both model drugs in deionized (DI) water was observed. The release of MB in simulated gastrointestinal fluid (0.1N HCl) was fast and complete, while the release of PAA was slow in 0.1N HCl and fast in phosphate buffer solution (pH 6.8). Interactions between the model drugs and alginate microparticles were identified from scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR) analysis. The results indicated that (1) porous alginate microparticles can be produced by the spray-coagulation method; (2) drugs can be loaded by the adsorption method; (3) and the obtained microparticles may be used for delaying the release of drugs of low water solubility in acidic conditions.

Adsorption↗

PubMed2006

Rheological properties of aqueous Pluronic-alginate systems containing liposomes.

Rheological and erosion studies regarding a liposome-containing polymeric blend that is propaedeutic to its use in paving techniques in tubular organs, such as blood vessels, are reported. Attention is focused on an aqueous polymeric blend composed of Pluronic (PF127) and alginate (Protanal LF 10/60) because both polymers, when dissolved in water at a sufficiently high concentration, are subjected to different structural mechanisms, which are driven by temperature increase and addition of bivalent cations, respectively, and both result in marked viscoelastic and plastic properties. After proving the compatibility between PF127 and alginate, we show that the structural transition temperature of the blend, T(ST), can be properly modulated. In particular, we found that T(ST) for an aqueous solution of pure Pluronic 20% w/w is about 21 degrees C and that even slight reductions in polymer concentration result in considerable T(ST) decrease. The addition of salts or alginate (provided as Na-alginate) provokes a substantial decrease of T(ST) and thus the alginate concentration in the blend should not exceed 1% w/w. In addition, liposomes slow down the structural transition but do not substantially affect the rheological properties of the system in the final state at higher temperatures, thus showing that they can be added to the polymeric blend without significant effects. Finally, erosion tests show that after contact with a source of bivalent cations, the polymeric blend containing PF127 and alginate shows an erosion resistance neatly improved with respect to the simple structured Pluronic system having the same polymer concentration. As a whole, all these results constitute the basis for future potential applications of the considered polymeric blend in tubular organs such as blood vessels.

Alginates↗