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Entrapment efficiency and release characteristics of polyethyleneimine-treated or -untreated calcium alginate beads loaded with propranolol-resin complex.

Propranolol-HCl-loaded calcium alginate (ALG) beads, propranolol-resin complex (resinate)-loaded calcium alginate (RALG) beads and polyethyleneimine (PEI)-treated RALG (RALG-PEI) beads were prepared by ionotropic gelation/polyelectrolyte complexation method. The beads were evaluated and compared in respect of drug entrapment efficiency (DEE) and release characteristics in simulated gastric fluid (SGF, 0.1(N) HCl, pH 1.2) and simulated intestinal fluid (SIF, phosphate buffer, pH 6.8). DEE of RALG beads was considerably higher than that of ALG beads containing unresinated drug. However, DEE of RALG beads decreased with increase in both gelation time and concentration of the gel forming Ca2+ ions due to drug displacement from resinate. PEI treatment of RALG beads further decreased DEE as the polycation also displaced the drug from the resinate. The release of drug from all the beads was slow and incomplete in SGF owing to considerably less swelling of the beads and the decrease in drug release from the beads followed the order: RALG-PEI<RALG<ALG. In contrast to rapid discharge of the drug by ALG beads in SIF, RALG beads provided marginal prolongation in drug release as both ALG and RALG beads swelled and eroded rapidly although at different rates. On the other hand, drug release from RALG-PEI beads in SIF was considerably prolonged for different periods of time depending upon the conditions of PEI treatment. Interaction of the polycation with alginate resulted in the formation of polyelectrolyte complex membrane as evident from scanning electron microscopy (SEM), differential scanning calorimetry (DSC) and fourier-transform infrared spectroscopy (FTIR) studies. The membrane having reduced swelling and erosion properties behaved as a physical barrier to drug release. Kinetics of the drug release also confirmed the formation of physical barrier as anomalous transport type of release associated with. RALG beads tended to shift towards Fickian transport in case of RALG-PEI beads.

Alginates↗

Xanthan-alginate composite gel beads: molecular interaction and in vitro characterization.

Xanthan gum (XG), a trisaccharide branched polymer, was applied to reinforce calcium alginate beads in this study. Composite beads consisting of XG and sodium alginate (SA) were prepared using ionotropic gelation method. Diclofenac calcium-alginate (DCA) beads incorporated with different amounts of XG were produced as well. Molecular interaction between SA and XG in the composite beads and the XG-DCA beads was investigated using FTIR spectroscopy. Physical properties of the XG-DCA beads such as entrapment efficiency of diclofenac sodium (DS), thermal property, water uptake, swelling and DS release in various media were examined. XG could form intermolecular hydrogen bonding with SA in the composite beads with or without DS. Differential scanning calorimetric study indicated that XG did not affect thermal property of the DCA beads. The DS entrapment efficiency of the DCA beads increased with increasing amount of XG added. The XG-DCA beads showed higher water uptake and swelling in pH 6.8 phosphate buffer and distilled water than the DCA beads. A longer lag time and a higher DS release rate of the XG-DCA beads in pH 6.8 phosphate buffer were found. In contrast, the 0.3%XG-DCA beads could retard the drug release in distilled water because interaction between XG and SA gave higher tortuosity of the bead matrix. However, higher content of XG in the DCA beads increased the release rate of DS. This can be attributed to erosion of small aggregates of XG on the surface of the DCA beads. This finding suggested that XG could modulate physicochemical properties and drug release of the DCA beads, which based on the existence of molecular interaction between XG and SA.

Alginates↗

Preparation of dual crosslinked alginate-chitosan blend gel beads and in vitro controlled release in oral site-specific drug delivery system.

Alginate-chitosan (ALG-CS) blend gel beads were prepared based on Ca2+ or dual crosslinking with various proportions of alginate and chitosan. The homogeneous solution of alginate and chitosan was dripped into the solution of calcium chloride; the resultant Ca2+ single crosslinked beads were dipped in the solution of sodium sulfate sequentially to prepare dual crosslinked beads. The dual crosslinkage effectively promoted the stability of beads under gastrointestinal tract conditions. The sustained release profiles of single and dual crosslinked gel beads loaded bovine serum albumin (BSA), a model protein drug, were investigated in simulated gastric fluid (SGF), simulated intestinal fluid (SIF) and simulated colonic fluid (SCF). In SGF, compared to Ca2+ single crosslinked beads, from which BSA released fast and the cumulative drug release percentages were about 80% of all formations in 4 h, the BSA total release from dual crosslinked gel beads was no more than 3% in 8 h. In SIF and SCF, Ca2+ single crosslinked beads were disrupted soon associating with the fast drug release. As to the dual crosslinked beads, the BSA total release from the ALG-CS mass ratio 9:1 (81.24%) was higher than that of 7:3 and 5:5 (less than 60%) in 8 h in SIF; the BSA release from all beads was much faster in SCF than in SIF. The dual crosslinked beads incubated in gastrointestinal tract conditions, the BSA cumulative release of ALG-CS mass ratios 9:1, 7:3 and 5:5 were respectively 2.35, 1.96, 1.76% (in SGF 4 h), 82.86, 78.83, 52.91% (in SIF 3 h) and 97.84, 96.81, 87.26% (in SCF 3 h), which suggested that the dual crosslinked beads have potential small intestine or colon site-specific drug delivery property.

Alginates↗

Quantitative determination of alginic acid in pharmaceutical formulations using capillary electrophoresis.

A capillary electrophoresis (CE) method has been developed and validated for the quantitative determination of alginic acid, which is used as a rafting agent in complex antacid formulations. The method involves a preliminary separation of the alginic acid from the formulation by washing the sample matrix with methanol, diluted HCl and water. This is followed by electrophoresis within a fused silica capillary using borate/boric acid buffer as the electrolyte, and the quantification is performed by a UV detector monitoring at 200 nm, where the intrinsic absorption of alginic acid is measured. An assay precision of better than 3% was achieved in intra- and interday determinations. No interference was found from the matrix of the antacid formulations.

Alginates↗

Osteogenic protein-1 promotes the formation of tissue-engineered cartilage using the alginate-recovered-chondrocyte method.

OBJECTIVE: This study examined the effects of a growth factor, recombinant human osteogenic protein-1 (rhOP-1), on the formation of tissue-engineered cartilaginous tissue by adult bovine articular chondrocytes using the alginate-recovered-chondrocyte (ARC) method. DESIGN: To ascertain if rhOP-1 enhances the formation of the cell-associated matrix (CM) and the characteristics of CM formation, bovine articular chondrocytes were first cultured for up to 14 days in alginate beads in medium supplemented with serum, with or without rhOP-1. Then, the recovered chondrocytes and their associated CM were resuspended in medium, with or without OP-1, seeded onto culture inserts, and incubated for an additional 14 days. The fabricated ARC tissues were subjected to biochemical and histological analyses. RESULTS: The addition of rhOP-1 to the medium in the alginate bead culture step resulted in an increased accumulation of both proteoglycan (PG) and collagen, with a ratio of PG to collagen that was higher than that found in native adult cartilage. The addition of rhOP-1 in the second step had a similar stimulatory effect during 14 days of culture. Histological examination of the tissue formed under all conditions revealed a cartilage-like matrix, stained strongly by toluidine blue. The thickness of the tissues obtained from culture conditions that included the addition of rhOP-1 was four times greater than that of the tissues cultured without rhOP-1. CONCLUSIONS: Using the ARC method, rhOP-1 enhanced the formation of matrix and generated a voluminous tissue-engineered cartilaginous construct. These characteristics may be beneficial in generating constructs that can cover large defects.

Alginates↗

Calcium alginate immobilized marine microalgae: experiments on growth and short-term heavy metal accumulation.

Growth of 11 calcium alginate immobilized marine microalgal species belonging to eight taxonomical groups has been checked in the present work. Cellular densities inside the calcium alginate beads were monitored during 17 days. Good growth and maintenance of the structure of the beads were both found for some of the assayed species. One of those species (Tetraselmis chui, Prasinophyceae) was selected in order to perform a short term (up to 24 h) heavy metal accumulation experiment. Beads of calcium alginate containing (or not) cells of T. chui were exposed to 820 microg L(-1) Cu and 870 microg L(-1) Cd separately during a 24 h period, and accumulation of heavy metals in the beads was measured after this time and compared. Concentration of each metal in the supernatants was monitored at 5, 10, 60 min and 24 h from the beginning of the experiment. After 24 h, practically all Cu was removed by the beads. Beads with immobilized algae removed around 20% of total Cd, while beads without algae removed half of that percentage.

Alginates↗

Cloning, high yield overexpression, purification, and characterization of AlgH, a regulator of alginate biosynthesis in Pseudomonas aeruginosa.

The most common cause of mortality among cystic fibrosis sufferers is infection by antibiotic resistant strains of Pseudomonas aeruginosa. Means to control these strains continue to be an important goal. An integral component of the ability of many of these strains to defy antibiotic therapies is the protection afforded by the mucoexopolysaccharide alginate. Production of alginate by P. aeruginosa is tightly regulated at the transcriptional level. AlgH, a putative transcriptional regulator, is involved in regulating alginate biosynthesis as well as nucleoside diphosphate kinase activity and succinyl coenzyme A synthetase activity in P. aeruginosa. Sequence homologues are found in many bacterial species. Here, we describe a method for high level overexpression and high yield/high purity production of AlgH for biophysical and functional studies. The algH gene was cloned and AlgH was overexpressed in Escherichia coli using a commercially available vector with an inducible T7 promoter. We purified the recombinantly produced protein using a rapid classical purification scheme. The yield of purified protein, either isotopically labeled for NMR studies or unlabeled, is excellent (30-37 mg of purified protein per liter of minimal media culture), as is the purity (>95% pure). Analysis of the secondary structure using circular dichroism and NMR indicates that the protein is comprised of both beta-sheet and alpha-helical secondary structural elements. Heteronuclear NMR spectra indicate that AlgH is a monodisperse, folded globular protein. This rapid, high yield, and high purity method for AlgH production will permit further biophysical characterization of this protein including high resolution structural studies.

Alginates↗

Short-term immunosuppression reduces fibrotic cellular infiltration around barium-M-alginate microbeads injected intraportally.

INTRODUCTION: One of the major barriers affecting the viability of encapsulated islets is pericapsular fibrotic infiltration (PFI). This study aimed to design strategies to reduce PFI around intraportally injected alginate microbeads. METHODS: Empty, highly purified, barium-M-alginate microbeads (400 microm) were injected intraportally into Lewis rats (3000 beads/rat). Rats (n = 9/group) were treated daily with either rapamycine (RAPA; 1 mg/kg/d p.o.), tacrolimus (TAC; 2 mg/kg/d p.o.), a combination of both, or gadolinium-chloride (GdC13, 20 mg/kg/d i.v., at day -1 and day +4). Treatment was discontinued at 10 days. Three rats/group were sacrificed at 3, 7, and 42 days after transplantation. Cellular composition of PFI was evaluated by immunohistochemistry. Severity of the reaction to the beads was determined by measuring the thickness of PFI on histology. RESULTS: The main cellular components of PFI in the liver were macrophages and myofibroblasts. There was a significant (P <.05) reduction in the thickness of PFI in all treated groups, even 6 weeks after transplantation. Encapsulated rat islets showed excellent insulin response to glucose in vitro, with a stimulation index of 3.6 +/- 2.0. CONCLUSION: Combination of highly purified alginate with short-term immunosuppression reduces fibrotic overgrowth around microbeads injected intraportally.

Alginates↗

Determination of the diadic composition of alginate by means of circular dichroism: a fast and accurate improved method.

Circular dichroism (CD) is presented as a reliable and sensitive method of determining the diadic frequency composition of alginate (F(GG), F(MM) and F(GM+MG)). The availability of samples, very largely or even completely conforming to the limiting structures of polymannuronate (MM)(n), polyguluronate (GG)(n) and polyalternating MG (MG)(n), respectively, allowed the limiting CD spectra for each alginate diad to be obtained. These showed very different CD behaviour, thus pointing out the crucial importance of the neighbouring residue in chiroptical properties. Using an iterative best-fit procedure, the diadic composition of commercial alginates could be obtained from their respective CD spectra by means of a linear combination of the spectra of the three limiting diads. The results were found in excellent agreement with the composition parameters obtained by 1H NMR spectroscopy.

Alginates↗

First pregnancies and livebirths from transfer of sodium alginate encapsulated embryos in a rodent model.

OBJECTIVE: To determine the effect of sodium alginate encapsulation of rodent embryos on in vitro embryonic cleavage rates, implantation rates, and livebirth rates, and to find the in vivo degradation time for the capsules. DESIGN: Studies were conducted using both CB6F1 mice and Golden Syrian hamsters. RESULTS: Capsules made with 3.0% sodium alginate degraded in vivo within 24 to 48 hours after transfer. In vitro embryonic cleavage of encapsulated embryos was not impaired, nor were implantation rates in CB6F1 mice. Finally, 8.6% of transferred encapsulated embryos resulted in livebirths. CONCLUSIONS: Encapsulation of rodent embryos in 3.0% sodium alginate is not detrimental to embryonic development, implantation rates, or fetal development. Because the capsule degrades within 48 hours after transfer, encapsulating embryos may be beneficial for human in vitro fertilization and embryo transfer.

Alginates↗

Effect of media composition on long-term in vitro stability of barium alginate and polyacrylic acid multilayer microcapsules.

For a number of applications stability of microcapsules is a critical factor. Since the maintenance of polyelectrolyte complexes depends considerably on the ion composition we tested the physical properties of barium alginate capsules and searched for conditions to improve stability by a multilayer coating with polyethylenimine (PEI) and polyacrylic acid (PAA). Mechanical stability and diameters were determined in barium alginate capsules and compared with multilayer capsules. Multilayer coating resulted in smaller capsules than barium complexing alone. The difference was more pronounced when CaCl2 was used instead of NaCl during coating. Barium alginate capsules and application of CaCl2 during coating led to continuous pressure profiles, whereas NaCl resulted in bursting at a defined pressure, indicating the additional contribution to mechanical stability by the outer layers. After 7 d culture, mechanical stability of coated capsules decreased in RPMI and NaCl but was most pronounced in sodium citrate. The capsule diameter increased in sodium citrate, less pronounced in NaCl and was significantly different to RPMI and double distilled water. During long-term culture in RPMI, the diameter increased and mechanical stability decreased significantly. Multilayer coating improved mechanical stability which was impeded most in sodium citrate, to a lesser extent by NaCl and RPMI even after long-term exposure.

Acrylic Resins↗

Designing alginate hydrogels to maintain viability of immobilized cells.

Hydrogel-forming materials have been widely utilized as an immobilization matrix and transport vehicle for cells. Success in these applications is dependent upon maintaining cell viability through the gel preparation process. We hypothesized that the high viscosity of pre-gelled solutions typically used in these applications may decrease cell viability due to the high shear forces required to mix cells with these solutions. Further, we proposed this harmful effect could be mediated by decreasing the molecular weight (Mw) of the polymer used to form the gel, while maintaining its gel-forming ability. To investigate this hypothesis, alginate was used as model system, as this copolymer consists of cross-linkable guluronic acid (G) blocks and non-cross-linkable blocks. Decreasing the Mw of alginate using irradiation (e.g., irradiating at dose of 2 Mrad) decreased the low shear viscosity of 2% (w/w) pre-gelled solutions from 1000 to 4 cP, while maintaining high elastic moduli, once cross-linked to form a gel. Importantly, the immobilization of cells with these polymer hydrogels increased cell viability from 40% to 70%, as compared to using high Mw polymer chains to form the gels. Furthermore, the solids concentration of gels formed with the low Mw alginate could be raised to further increase the moduli of gels without significantly deteriorating the viability of immobilized cells. This was likely due to the limited increase in the viscosity of these solutions. This material design approach may be useful with a variety of synthetic or naturally occurring block copolymers used to immobilize cells.

Alginates↗

Physico-chemical characterization of Ca-alginate microparticles produced with different methods.

In the present paper the physico-chemical characterization of Ca-alginate microparticles produced with different methods is presented. Ca-alginate microparticles were obtained either by emulsification method or by dripping an aqueous alginate solution into a solution of calcium salt. Inverse Size Exclusion Chromatography (ISEC) was used for the determination of dimensions of the pores and porous volume of microparticles having a mean diameter of 220 microm when obtained by emulsification method. The same technique was used to study the variation of the pore size and porous volume with pH. The results were related with the content of calcium and sodium in the microparticles, before and after their treatment with different HCl solutions. For the particles with a mean diameter of 1.2 mm (obtained by dripping method) we adopted an other approach based on the steric exclusion of solute at equilibrium. For a convenient interpretation of the obtained data, determination of water regain, swelling degree, and scanning electron microscopy (SEM) were performed. Finally, a comparison of the characteristics of microparticles produced by ionic and epichlorohydrin crosslinkings was made. The maximum dimensions of the pores of the microparticles obtained by emulsification were found smaller than those obtained by other technics. The variation of the dimensions of the pores and porous volume with pH is not significant. The structure of the chemically crosslinked beads with epichlorohydrin is more elastic and the swelling is reversible; after drying and reswelling process, the dimensions of the pores and porous volume of these microparticles remain unchanged. On the opposite, for the microparticles obtained by emulsification or dripping method in the presence of calcium ions, these characteristics are changed after a first drying process.

Alginates↗

Tissue-engineered cartilage using serially passaged articular chondrocytes. Chondrocytes in alginate, combined in vivo with a synthetic (E210) or biologic biodegradable carrier (DBM).

In vitro multiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generate neo-cartilage, but is known to induce cell-dedifferentiation. The aim of this study was to investigate whether multiplied chondrocytes can be used to generate neo-cartilage in vivo. Adult bovine articular chondrocytes, of various differentiation stages, were suspended in alginate at densities of 10 or 50 million/ml, either directly after isolation (P0) or after multiplication in monolayer for one (P1) or three passages (P3). Alginate with cells was seeded in demineralized bovine bone matrix (DBM) or a fleece of polylactic/polyglycolic acid (E210) and implanted in nude mice for 8 weeks. The newly formed tissue was evaluated by Alcian Blue and immunohistochemical staining for collagen type-II and type-I. Structural homogeneity of the tissue, composed of freshly isolated as well as serially passaged cells, was found to be enhanced by high-density seeding (50 million/ml) and the use of E210 as a carrier. The percentage of collagen type-II positive staining P3-cells was generally higher when E210 was used as a carrier. Furthermore, seeding P3-chondrocytes at the highest density (50 million/ml) enhanced collagen type-II expression. This study shows promising possibilities to generate structurally regular neo-cartilage using multiplied chondrocytes in alginate in combination with a fleece of polylactic/polyglycolic acid.

Alcian Blue↗

Articular cartilage repair in rabbits by using suspensions of allogenic chondrocytes in alginate.

The feasibility of allogenic implants of chondrocytes in alginate gels was tested for the reconstruction in vivo of artificially full-thickness-damaged articular rabbit cartilage. The suspensions of chondrocytes in alginate were gelled by the addition of calcium chloride solution directly into the defects giving in situ a construct perfectly inserted and adherent to the subchondral bone and to the walls of intact cartilage. The tissue repair was controlled at 1, 2, 4 and 6 months after the implant by NMR microscopy, synchrotron radiation induced X-ray emission to map the sulfur of glycosaminoglycans and by histochemistry. Practically a complete repair of the defect was observed 4-6 months from the implant of the chondrocytes with the recovery of a normal tissue structure. Controls in which Ca-alginate alone was implanted developed only a fibrous cartilage.

Alginates↗

Cadmium biosorption by cells of Spirulina platensis TISTR 8217 immobilized in alginate and silica gel.

The biosorption of cadmium by immobilized Spirulina platensis on alginate gel and silica gel was studied. The maximum biosorption capacities for alginate immobilized cells and silica immobilized cells were 70.92 and 36.63 mg Cd/g biomass, respectively. Temperature did not have an influence on metal sorption, whereas an initial pH solution did. Sorption occurred in a wide pH range (pH 3-8). The highest adsorption of alginate immobilized cells was at pH 6, while silica immobilized cell adsorption was not affected at pH between 4 and 7. The immobilized cells were reused in consecutive adsorption-desorption. The results showed that immobilized cells could be repeatedly used in the sorption process up to five times.

Adsorption↗

Stabilization of alginate beads using radiation polymerized polyacrylamide.

A technique has been described for the stabilization of calcium alginate beads using radiation polymerized acrylamide. The technique involved dropping a mixture containing the cells (20%), sodium alginate (2%), acrylamide (2.5%) and N-N'-methylene-bis-acrylamide (0.1%) through a syringe needle into cold (-75 degrees C) toluene. The frozen beads obtained were exposed to 60Co gamma-rays (0.5 KGy) and were then thawed in 0.1 M CaCl2 solution. Unlike the calcium alginate beads the conjugate beads were not found to be dissolved when incubated in 3% trisodium citrate solution. Stabilized beads containing entrapped yeast cells could be reused for over 15 batches for the inversion of sucrose without loss in activity or chemical integrity of the beads.

Acrylic Resins↗

Induction of mucosal immune responses following enteric immunization with antigen delivered in alginate microspheres.

Oral immunization is the most effective way of inducing immune responses in the intestinal tract. Biodegradable microspheres have been used extensively for the delivery of antigens to the Peyer's patches (PPs) within the gut-associated lymphoid tissue (GALT). We evaluated various formulations of alginate microspheres for their capacity to induce mucosal immune responses in vivo. Multiple intestinal "loops" each containing a single PP, were surgically prepared in lambs. We have previously showed that PP in individual intestinal loops function as independent sites for the induction of immune responses. This animal model provides a system for directly comparing different antigen formulations within the same animal. Individual intestinal loops were injected with a model antigen, porcine serum albumin (PSA) encapsulated in three different formulations of alginate micropsheres. Three weeks after immunization, PSA-specific immune responses were assayed with antibody secreting cell (ASC) ELISPOT, lymphocyte proliferative responses (LPRs), IFN-gamma production and antibody secreted into intestinal loops. PSA encapsulated in alginate micropsheres or in saline induced humoral immune responses as indicated by the presence of numerous ASC. However, PSA-specific T-cell responses (LPR and IFN-gamma production) were not induced.

Alginates↗