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PubMed1994

Pseudomonas aeruginosa: genes and enzymes of alginate synthesis.

Alginate is an important virulence factor of Pseudomonas aeruginosa, a bacterium that colonizes the pulmonary tracts of cystic fibrosis patients. Alginate is also widely used in the food, pharmaceutical and chemical industries, and consequently there is considerable interest in the molecular biology and biochemistry of alginate synthesis. As well as its therapeutic potential, research on mucoid P. aeruginosa may provide a lead to an alternative source of alginate for industrial use.

Alginates↗
PubMed2005

Cell density alters matrix accumulation in two distinct fractions and the mechanical integrity of alginate-chondrocyte constructs.

Chondrocyte density in articular cartilage is known to change with the development and growth of the tissue and may play an important role in the formation of a functional extracellular matrix (ECM). The objective of this study was to determine how initial chondrocyte density in an alginate hydrogel affects the matrix composition, its distribution between the cell-associated (CM) and further removed matrix (FRM) fractions, and the tensile mechanical properties of the developing engineered cartilage. Alginate constructs containing primary bovine chondrocytes at densities of 0, 4, 16, and 64 million cells/ml were fabricated and cultured for 1 or 2 weeks, at which time structural, biochemical, and mechanical properties were analyzed. Both matrix content and distribution varied with the initial cell density. Increasing cell density resulted in an increasing content of collagen and sulfated-glycosaminoglycan (GAG) and an increasing proportion of these molecules localized in the CM. While the equilibrium tensile modulus of cell-free alginate did not change with time in culture, the constructs with highest cell density were 116% stiffer than cell-free controls after 2 weeks of culture. The equilibrium tensile modulus was positively correlated with total collagen (r2=0.47, p<0.001) and GAG content (r2=0.68, p<0.001), and these relationships were enhanced when analyzing only those matrix molecules in the CM fraction (r2=0.60 and 0.72 for collagen and GAG, respectively, each p<0.001). Overall, the results of this study indicate that initial cell density has a considerable effect on the developing composition, structure, and function of alginate-chondrocyte constructs.

Alginates↗
PubMed2005

Controlling alginate gel degradation utilizing partial oxidation and bimodal molecular weight distribution.

Degradability is often a critical property of materials utilized in tissue engineering. Although alginate, a naturally derived polysaccharide, is an attractive material due to its biocompatibility and ability to form hydrogels, its slow and uncontrollable degradation can be an undesirable feature. In this study, we characterized gels formed using a combination of partial oxidation of polymer chains and a bimodal molecular weight distribution of polymer. Specifically, alginates were partially oxidized to a theoretical extent of 1% with sodium periodate, which created acetal groups susceptible to hydrolysis. The ratio of low MW to high MW alginates used to form gels was also varied, while maintaining the gel forming ability of the polymer. The rate of degradation was found to be controlled by both the oxidation and the ratio of high to low MW alginates, as monitored by the reduction of mechanical properties and corresponding number of crosslinks, dry weight loss, and molecular weight decrease. It was subsequently examined whether these modifications would lead to reduced biocompatibility by culturing C2C12 myoblast on these gels. Myoblasts adhered, proliferated, and differentiated on the modified gels at a comparable rate as those cultured on the unmodified gels. Altogether, this data indicates these hydrogels exhibit tunable degradation rates and provide a powerful material system for tissue engineering.

Alginates↗
PubMed2005

Real-time 3-D dark-field microscopy for the validation of the cross-linking process of alginate microcapsules.

Alginate-based microencapsulation is a promising method for long-term maintenance of cellular and membrane function of the cells and tissue fragments required for in vitro and in vivo biosensors, for tissue engineering and particularly for immunoisolation of non-autologous transplants. Microcapsules of high mechanical strength and optimum permeability can be produced by injection of BaCl2 crystals into alginate droplets before they come into contact with external Ba2+. A key requirement is that the system parameters (number of crystals, speed of the crystal stream etc.) are properly adjusted according to the mannuronic and guluronic acid ratio and the average molecular mass of the alginate as well as to the diameter of the microcapsules. Robust, reliable, rapid and low-cost validation tools are, therefore, needed for assurance of the microcapsule quality. Here, we describe a novel three-dimensional (3-D) dark-field microscopy that allows the real-time measurement of the number and spatial distribution of the injected Ba2+ ions throughout the microcapsules after treatment with sulphate. This novel method requires only a conventional microscope equipped with three polarising filters and a double aperture stop. In contrast to confocal laser scanning microscopy images, peripherally attached BaSO4 precipitates can clearly be distinguished from internal ones. The data also demonstrate that several steps of the alginate gelling process must be improved before such immunoisolation can be used in patients.

Alginates↗
PubMed2005

Physicochemical model of alginate-poly-L-lysine microcapsules defined at the micrometric/nanometric scale using ATR-FTIR, XPS, and ToF-SIMS.

Alginate-poly-L-lysine-alginate (APA) microcapsules are currently being investigated as a means to immuno-isolate transplanted cells, but their biocompatibility is limited. In this study, we verified the hypothesis that poly-L-lysine (PLL), which is immunogenic when unbound, is exposed at the APA microcapsule surface. To do so, we analysed the microcapsule membrane at the micrometric/nanometric scale using attenuated total reflectance Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry. The results indicate that PLL and alginate molecules interact within the membrane. PLL exists in considerable amounts near the surface, contributing to the majority of the carbon within the outermost 100 Angstroms of the membrane. PLL was also detected at the true surface (the outermost monolayer) of the microcapsules. The exposure of PLL does not appear to result from defects in the outer alginate coating. This physicochemical model of APA microcapsules could explain their immunogenicity and will play an important role in the optimization of the microcapsule design.

Alginates↗
PubMed2006

Intraperitoneal stability of alginate-polyornithine microcapsules in rats: an FTIR and SEM analysis.

Alginate-polycation microcapsule systems have been used over decades as delivery vehicles for cell and protein therapy. These systems have been unpredictable across a range of indications with questions resulting around the inherent stability of the alginate polysaccharide and failure mode of the delivery system. The current study focuses on such a system using 5 different alginates, 2 of which are commercially purified, which are crosslinked by polyornithine. Capsules formed by frequency-generated droplet formation were studied in the peritoneal cavity of Long-Evans rats over the course of 3 months by morphometry, Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy of the surface. Individual capsule components were also investigated on FTIR and a relative stability index was generated by titration for comparison to explanted samples over time. Using these techniques, a distinct degradation pattern was noted and is compared between the 5 alginate sources.

Alginates↗
PubMed2006

Biocompatibility of alginate-poly-L-lysine microcapsules for cell therapy.

Cell microencapsulation holds promise for the treatment of many diseases by the continuous delivery of therapeutic products. The biocompatibility of the microcapsules and their biomaterials components is a critical issue for the long-term efficacy of this technology. The objective of this paper is to provide detailed information about the principal factors affecting the biocompatibility of alginates and alginate-poly-l-lysine microcapsules, which are the most frequently employed biomaterials and encapsulation devices for cell immobilization, respectively. Some of these factors include the alginate composition and purification, the selection of the polycation, the interactions between the alginates and the polycation, the microcapsule fabrication process, the uniformity of the devices and the implantation procedure. Improved knowledge will lead to the production of standardized transplantation-grade biomaterials and biocompatible microcapsules.

Alginates↗
PubMed2005

Degradation of 4-aminobenzenesulfonate by alginate encapsulated cells of Agrobacterium sp. PNS-1.

Studies were carried out on 4-aminobenzenesulfonate (4-ABS) degradation by free and alginate entrapped cells of Agrobacterium sp. PNS-1. Degradation rate in batch reactors with free cells was marginally higher than Ca-encapsulated cells. Comparison of Ca2+ and Ba2+ as gelling agents showed that 4-ABS removal rate was significantly less with Ba-alginate entrapped cells. Specific degradation rates, using linear regression analysis and based on the initial biomass in the beads, varied from 49.7 mg/mg biomass/h to 92.0 mg/mg biomass/h for Ca-alginate encapsulated cells for different initial 4-ABS concentrations ranging from 200 to 800 mg/L. UV spectra of the aliquots drawn at different time intervals from batch reactors did not show accumulation of any intermediate during degradation. Ca-alginate immobilized cells could be repeatedly reused upto five cycles without any loss of activity. Studies with packed bed reactors, operated in a semi-continuous mode, showed that this could be used for 4-ABS degradation.

Alginates↗
PubMed2003

Sustained release of water-soluble drug from directly compressed alginate tablets.

The release of acetyl salicylic acid from directly compressed alginate tablets was investigated. The effect of the amount and type of alginate on the drug release rate was evaluated in different formulations. Four different grades of alginate were used. The in vitro release studies were carried out using the apparatus II (paddle) equipment as described in the USP 23/NF dissolution monograph. Dissolution medium was 0.1 M HCl for 2 h followed by phosphate buffer pH 6.8; both at 37 degrees C. Sustained drug release up to 16 h was achieved using sodium alginate in combination with dibasic calcium phosphate.

Alginates↗
PubMed2004

Oesophageal bioadhesion of sodium alginate suspensions: particle swelling and mucosal retention.

This paper describes a prospective bioadhesive liquid dosage form designed to specifically adhere to the oesophageal mucosa. It contains a swelling polymer, sodium alginate, suspended in a water-miscible vehicle and is activated by dilution with saliva to form an adherent layer of polymer on the mucosal surface. The swelling of alginate particles and the bioadhesion of 40% (w/w) sodium alginate suspensions were investigated in a range of vehicles: glycerol, propylene glycol, PEG 200 and PEG 400. Swelling of particles as a function of vehicle dilution with artificial saliva was quantified microscopically using 1,9-dimethyl methylene blue (DMMB) as a visualising agent. The minimum vehicle dilution to initiate swelling varied between vehicles: glycerol required 30% (w/w) dilution whereas PEG 400 required nearly 60% (w/w). Swelling commenced when the Hildebrand solubility parameter of the diluted vehicle was raised to 37 MPa(1/2). The bioadhesive properties of suspensions were examined by quantifying the amount of sodium alginate retained on oesophageal mucosa after washing in artificial saliva. Suspensions exhibited considerable mucoretention and strong correlations were obtained between mucosal retention, the minimum dilution to initiate swelling, and the vehicle Hildebrand solubility parameter. These relationships may allow predictive design of suspensions with specific mucoretentive properties, through judicious choice of vehicle characteristics.

Adhesives↗
PubMed2005

Oesophageal bioadhesion of sodium alginate suspensions 2. Suspension behaviour on oesophageal mucosa.

Sodium alginate suspensions in a range of water miscible vehicles were investigated as novel bioadhesive liquids for targeting the oesophageal mucosa. Such a dosage form might be utilised to coat the oesophageal surface and provide a protective barrier against gastric reflux, or to deliver therapeutic agents site-specifically. Alginate suspensions swelled and formed an adherent viscous layer on contact with the mucosa. The swelling kinetics of alginate particles on the oesophageal surface was examined with respect to vehicle composition and related to the extent, duration and location of bioadhesion within the oesophagus. Mucosal retention was evaluated in two in vitro models utilising tissue immersion and a peristaltic tube. By varying the vehicle composition it was possible to modulate the rate of swelling of alginate particles on the mucosa and the mucosal retention of suspensions. Suspensions containing predominantly glycerol exhibited superior retention and were preferentially retained within the lower oesophagus. The propensity of these suspensions to rapidly swell on the mucosa and establish adhesive/cohesive bonds may explain their enhanced retention. The potential to control, through vehicle composition, the extent, duration and location of oesophageal retention could provide a useful tool for site targeting of viscous polymers to the oesophagus.

Adhesives↗
PubMed2006

The effect of sodium alginate on the immune response of tiger shrimp via dietary administration: activity and gene transcription.

The total haemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (release of superoxide anions), and superoxide dismutase (SOD) activity, as well as expressions of beta-1,3-glucan-binding protein (betaGBP), prophenoloxidase (proPO), peroxinectin (PE), cytosolic SOD (cyt-SOD), penaeidin-5 (PA-5), and a single whey acidic protein (WAP) domain protein (SWDP) gene were determined in the tiger shrimp Penaeus monodon (15.6-19.5g) which had individually been fed diets containing sodium alginate at 0, 1.0, or 2.0gkg(-1) for 5months. Results showed that shrimp fed a diet containing 1.0 and 2.0gkg(-1) sodium alginate had significantly increased SOD activity but decreased respiratory bursts. The expressions of betaGBP, PE, cyt-SOD, PA-5, and SWDP were significantly elevated in shrimp fed the <or=2.0gkg(-1) sodium alginate-containing diet for 5months. However, no significant differences in THC, PO activity, or proPO mRNA transcription in shrimp were observed among the three treatments. It was concluded that sodium alginate can be used as an immunomodulator for shrimp through dietary administration to modify immune genes expression of shrimp.

Alginates↗
PubMed2005

Release of a macromolecular drug from alginate-impregnated microspheres.

Macroporous microspheres were impregnated with calcium alginate to encapsulate fluorescein isothiocyanate-labeled dextran (FITC-dextran) and control its release. The detailed study of the impregnation process lead to its optimization: the quantity of alginate in the impregnated microspheres and the FITC-dextran encapsulation efficiency were increased. FITC-dextran diffused out of the impregnated microspheres in a slow rate in deionised water, while in presence of sodium ions, its release rate was increased as a consequence of the progressive swelling and erosion of calcium alginate. Release studies from different formulations of impregnated microspheres were performed in a continuous flow apparatus. The release profiles were composed of a slow release phase explained by the progressive erosion of calcium alginate and a faster release phase related to eroded impregnated microspheres. Therefore, the delayed release by microspheres induced by impregnation would permit the delivery of their payload at the vascular occlusion site, limit the amount of drug lost in the systemic circulation and improve the therapy.

Alginates↗
PubMed2006

Alginate microspheres prepared by internal gelation: development and effect on insulin stability.

Recombinant human insulin was encapsulated within alginate microspheres by the emulsification/internal gelation technique with the objective of preserving protein stability during encapsulation procedure. The influence of process and formulation parameters was evaluated on the morphology and encapsulation efficiency of insulin. The in vitro release of insulin from microspheres was studied under simulated gastrointestinal conditions and the in vivo activity of protein after processing was assessed by subcutaneous administration of extracted insulin from microspheres to streptozotocin-induced diabetic rats. Microspheres mean diameter, ranging from 21 to 287 microm, decreased with the internal phase ratio, emulsifier concentration, mixer rotational speed and increased with alginate concentration. Insulin encapsulation efficiency, near 75%, was not affected by emulsifier concentration, mixer rotational speed and zinc/insulin hexamer molar ratio but decreased either by increasing internal phase ratio and calcium/alginate mass ratio or by decreasing acid/calcium molar ratio and alginate concentration. A high insulin release, above 75%, was obtained at pH 1.2 and under simulated intestinal pH a complete dissolution of microspheres occurred. Extracted insulin from microspheres decreased hyperglycemia of diabetic rats proving to be bioactive and showing that encapsulation in alginate microspheres using the emulsification/internal gelation is an appropriate method for protein encapsulation.

Alginates↗
PubMed2006

Modulation of drug release from glyceryl palmitostearate-alginate beads via heat treatment.

Diclofenac calcium alginate (DCA) beads containing glyceryl palmitostearate (GPS) were prepared by ionotropic gelation method. The effect of GPS amount and heat treatment on characteristics of the DCA beads was investigated. Incorporation of GPS into the DCA beads increased particle size and entrapment efficiency of diclofenac sodium (DS), but decreased water uptake in distilled water, and DS release rate. The heat treatment caused the DCA beads to be irregular shape particles and to possess higher water uptake. A slower release rate of DS in distilled water was found because of interaction of DS and alginate polymer matrix, and a restriction of water sorption into the inside region of the beads, which caused by the shrinkage of the beads after heating. However, the heat treatment did not affect particle shape and water uptake in distilled water of the 3%GPS-DCA beads. Differential scanning calorimetric study showed that GPS in the DCA beads was resolidified to different polymorph after cooling. Furthermore, the micro-Raman spectra indicated the existence of DS in the GPS matrix particles in the beads due to the partition of DS into the melted GPS during heat treatment. This led to a decrease in release rate of DS in pH 6.8 phosphate buffer and a change in DS release pattern in distilled water. Thus, not only the calcium alginate matrix, but also the resolidified GPS matrix in the alginate beads controlled the DS release from the 3%GPS-DCA beads with heat treatment.

Alginates↗
PubMed2006

Induction of systemic and mucosal immune responses by intranasal administration of alginate microspheres encapsulated with tetanus toxoid and CpG-ODN.

In the induction of systemic and mucosal immunity, particulate antigens are more effective than soluble antigens; possibly because they are more efficiently endocytosed by mucosal-associated lymphoid tissue (MALT) M cells. In this study, we determined the systemic and mucosal immune responses in rabbits following intranasal immunization with encapsulated tetanus toxoid (TT) and CpG-ODN in alginate microspheres. The microspheres were less than 4 microm in diameter. Encapsulation efficiency of TT and CpG-ODN was determined as 47.7+/-6.6 and 34.2+/-7.4, respectively. Release of TT and CpG-ODN in a simulated model with nasal cavity was 14.2+/-3.06 and 36.7+/-2.4% after 4 h. Encapsulated TT preserved its intact structure, but its immunoreactivity was decreased to about 91+/-5%. The highest serum IgG and antitoxin, and nasal lavage IgA titers were observed in groups immunized with microsphere formulations. CpG-ODN as an adjuvant could increase the serum IgG and antitoxin titers when co-administered with TT solution, but its co-encapsulation with TT in alginate microspheres failed to potentiate the systemic immune response while induced high IgA titers in nasal lavages. No hemolysis was occurred on incubation of alginate microspheres and human RBCs. Also after nasal administration of plain microspheres to human volunteers, no local irritation was observed. Intranasal administration of microspheres encapsulated with vaccines showed to be an effective way for inducing a variety of immune responses and that a strong systemic IgG and mucosal IgA responses can be induced in rabbits with intranasal administration of alginate microspheres encapsulated with TT.

Adjuvants, Immunologic↗
PubMed2006

Sequence analysis of alginate-derived oligosaccharides by negative-ion electrospray tandem mass spectrometry.

Negative-ion electrospray tandem mass spectrometry (ES-MS/MS) with collision-induced dissociation (CID) is attempted for sequence determination of alginate oligosaccharides, derived from polyanionic alginic acid, polymannuronate, and polyguluronate by partial depolymerization using either alginate lyase or mild acid hydrolysis. Sixteen homo- and hetero-oligomeric fragments were obtained after fractionation by gel-filtration and strong anion exchange high performance liquid chromatography. The product-ion spectra of these alginate oligosaccharides were dominated by intense B-, C-, Y-, and Z-type ions together with (0,2)A- and (2,5)A-ions of lower intensities. Internal mannuronate residues (M) produce weak but specific decarboxylated Z(int)-ions (Z(int) - 44 Da; int: denotes internal), which can be used for distinction of M and a guluronate residue (G) at an internal position. A reducing terminal M or G, although neither gives rise to a specific ion, can be identified by differences in the intensity ratio of fragment ions of the reducing terminal residue [(2,5)A(red)]/[(0,4)A(red)] (red: denotes reducing terminal).

Alginates↗
PubMed2006

Determination of alginate copolymer in pharmaceutical formulations by micellar electrokinetic chromatography.

A micellar electrokinetic chromatography method was developed for the determination and quantification of sodium alginate. The alginate peak migrated in the very short time of 1.33 min and calibrated easily though the polydisperse properties of alginates. The minimum detection limit (LOD) of the method was calculated as 0.393 mg/ml. This analysis method was successfully applied to the alginate quantification in an antacid pharmaceutical formulation. Precise and reproducible analysis results were obtained, with liquid formulations injected directly without any pre-separation process.

Alginates↗