high-G Alginate Research Articles

MS-proteomics provides insight into the host responses towards alginate microspheres.

Protein adsorption to biomaterial surfaces is considered a determining factor for the host response. Here we detail the protein adsorption profiles of alginate hydrogel microspheres relevant for cell therapy using mass spectrometry (MS)-based proteomics. The investigated microspheres include sulfated alginate (SA), high G alginate (HiG), and poly-l-lysine coated alginate (AP), which previously have been shown to exhibit different inflammatory and fibrotic responses. The biological significance was assessed in lepirudin-anticoagulated human whole blood (hWB) by functional analysis of the acute-phase responses (complement and coagulation). Proteomic profiling revealed distinct signatures for the microspheres, wherein Ingenuity Pathway Analysis identified complement and coagulation as the top enriched canonical pathways. The levels of complement and coagulation activators and inhibitors were distinctly different, which was reflected in the functional hWB analyses: SA was highly enriched with inhibitory factors of complement and coagulation (e.g. C1 inhibitor, factor H, antithrombin-III, heparin cofactor 2), other heparin-binding proteins and factors promoting fibrinolysis (factor XII, plasma kallikrein), conforming to an anti-inflammatory and anti-fibrotic profile. HiG enriched moderate levels of complement inhibitors, conforming to a low-inflammatory and pro-fibrotic profile. AP showed the most prominent enrichment of complement activators (e.g. C3, properdin, C-reactive protein) and low levels of inhibitors, conforming to a pro-inflammatory and highly pro-fibrotic profile. In conclusion, the extensive enrichment of inhibitory acute-phase proteins on SA could be a determining factor for its reduced host response. The interactions between the plasma proteins and hydrogel surfaces shown herein point to proteomics as an important supplement to existing in vitro and in vivo methods for designing biocompatible alginate-based hydrogels.

Coaxial Alginate Hydrogels: From Self-Assembled 3D Cellular Constructs to Long-Term Storage.

Alginate as a versatile naturally occurring biomaterial has found widespread use in the biomedical field due to its unique features such as biocompatibility and biodegradability. The ability of its semipermeable hydrogels to provide a favourable microenvironment for clinically relevant cells made alginate encapsulation a leading technology for immunoisolation, 3D culture, cryopreservation as well as cell and drug delivery. The aim of this work is the evaluation of structural properties and swelling behaviour of the core-shell capsules for the encapsulation of multipotent stromal cells (MSCs), their 3D culture and cryopreservation using slow freezing. The cells were encapsulated in core-shell capsules using coaxial electrospraying, cultured for 35 days and cryopreserved. Cell viability, metabolic activity and cell–cell interactions were analysed. Cryopreservation of MSCs-laden core-shell capsules was performed according to parameters pre-selected on cell-free capsules. The results suggest that core-shell capsules produced from the low viscosity high-G alginate are superior to high-M ones in terms of stability during in vitro culture, as well as to solid beads in terms of promoting formation of viable self-assembled cellular structures and maintenance of MSCs functionality on a long-term basis. The application of 0.3 M sucrose demonstrated a beneficial effect on the integrity of capsules and viability of formed 3D cell assemblies, as compared to 10% dimethyl sulfoxide (DMSO) alone. The proposed workflow from the preparation of core-shell capsules with self-assembled cellular structures to the cryopreservation appears to be a promising strategy for their off-the-shelf availability.

Effects of polyols on gelation kinetics, gel hardness, and drying properties of alginates subjected to internal gelation

This study investigates the influence of polyols (glycerol, sorbitol, mannitol and isomalt) on thickening of sodium alginate solutions, hardness and drying kinetics of calcium alginate gels and their glass transition temperature (Tg) after drying. Two alginate samples with 30% and 63% guluronate were used to prepare gels by internal gelation based on release of calcium ions from CaCO3 by hydrolysis of glucono-delta-lactone. The lag period, determined from viscosity-time plots, for the low-G alginate was about 13 min less than that for the high-G alginate. Presence of polyols did not influence the lag period values. The hardness value for high-G gels was about 100 g greater than that of low-G gels. Addition of isomalt resulted in gels with higher hardness values compared to other polyols. Presence of polyols had a significant effect on residual moisture and Tg of alginate films. Initial drying rates were the same for all samples but gels containing glycerol had high residual moisture (∼3.5 g/100 g) and produced very flexible films, whereas mannitol containing samples had low moisture (∼0.2 g/100 g) and produced brittle films. Tg of the films varied from −88 °C to 35 °C and decreased upon polyol addition.

A novel multilayer immunoisolating encapsulation system overcoming protrusion of cells

Application of alginate-microencapsulated therapeutic cells is a promising approach for diseases that require a local and constant supply of therapeutic molecules. However most conventional alginate microencapsulation systems are associated with low mechanical stability and protrusion of cells which is associated with higher surface roughness and limits their clinical application. Here we have developed a novel multilayer encapsulation system that prevents cells from protruding from capsules. The system was tested using a therapeutic protein with anti-tumor activity overexpressed in mammalian cells. The cell containing core of the multilayer capsule was formed by flexible alginate, creating a cell sustaining environment. Surrounded by a poly-L-lysine layer the flexible core was enveloped in a high-G alginate matrix that is less flexible and has higher mechanical stability, which does not support cell survival. The cells in the core of the multilayer capsule did not show growth impairment and protein production was normal for periods up to 70 days in vitro. The additional alginate layer also lowered the surface roughness compared to conventional cell containing alginate-PLL capsules. Our system provides a solution for two important, often overlooked phenomena in cell encapsulation: preventing cell protrusion and improving surface roughness.

The modulation of pancreatic lipase activity by alginates

Alginates are comprised of mannuronic (M) and guluronic acid (G) and have been shown to inhibit enzyme activity. Pancreatic lipase is important in dietary triacylglycerol breakdown; reducing pancreatic lipase activity would reduce triacylglycerol breakdown resulting in lower amounts being absorbed by the body.Lipase activity in the presence of biopolymers was assessed by enzymatic assay using natural and synthetic substrates. Alginate inhibited pancreatic lipase by a maximum of 72.2% (±4.1) with synthetic substrate (DGGR) and 58.0% (±9.7) with natural substrate. High-G alginates from Laminaria hyperborea seaweed inhibited pancreatic lipase to a significantly higher degree than High-M alginates from Lessonia nigrescens, showing that inhibition was related to alginate structure.High-G alginates are effective inhibitors of pancreatic lipase and are used in the food industry at low levels. They could be included at higher levels in foods without altering organoleptic qualities, potentially reduce the uptake of dietary triacylglycerol aiding in weight management.

The role of pathogen-associated molecular patterns in inflammatory responses against alginate based microcapsules

Alginate-based microcapsules are used for immunoisolation of cells to release therapeutics on a minute-to-minute basis. Unfortunately, alginate-based microcapsules are suffering from varying degrees of success, which is usually attributed to differences in tissue responses. This results in failure of the therapeutic cells. In the present study we show that commercial, crude alginates may contain pathogen-associated molecular patterns (PAMPs), which are recognized by the sensors of the innate immune system. Known sensors are Toll-like receptors (TLRs), NOD receptors, and C-type lectins. By using cell-lines with a non-functional adaptor molecule essential in Toll-like receptor signaling, i.e. MyD88, we were able to show that alginates signal mainly via MyD88. This was found for low-G, intermediate-G, and high-G alginates applied in calcium-beads, barium-beads as well as in alginate–PLL–alginate capsules. These alginates did stimulate TLRs 2, 5, 8, and 9 but not TLR4 (LPS receptor). Upon implantation in rats these alginates provoked a strong inflammatory response resulting in fibrosis of the capsules. Analysis demonstrated that commercial alginates contain the PAMPs peptidoglycan, lipoteichoic acid, and flagellin. By applying purification procedures, these PAMPs were largely removed. This was associated with deletion of the inflammatory tissue responses as confirmed by an implantation experiment in rats. Our data also show that alginate itself does not provoke TLR mediated responses. We were able to unravel the sensor mechanism by which contaminants in alginates may provoke inflammatory responses.

Binding and leakage of barium in alginate microbeads.

Microbeads of alginate crosslinked with Ca(2+) and/or Ba(2+) are popular matrices in cell-based therapy. The aim of this study was to quantify the binding of barium in alginate microbeads and its leakage under in vitro and accumulation under in vivo conditions. Low concentrations of barium (1 mM) in combination with calcium (50 mM) and high concentrations of barium (20 mM) in gelling solutions were used for preparation of microbeads made of high-G and high-M alginates. High-G microbeads accumulated barium from gelling solution and contained higher concentrations of divalent ions for both low- and high-Ba exposure compared with high-G microbeads exposed to calcium solely and to high-M microbeads for all gelling conditions. Although most of the unbound divalent ions were removed during the wash and culture steps, leakage of barium was still detected during storage. Barium accumulation in blood and femur bone of mice implanted with high-G beads was found to be dose-dependent. Estimated barium leakage relevant to transplantation to diabetic patients with islets in alginate microbeads showed that the leakage was 2.5 times lower than the tolerable intake value given by WHO for high-G microbeads made using low barium concentration. The similar estimate gave 1.5 times higher than is the tolerable intake value for the high-G microbeads made using high barium concentration. To reduce the risk of barium accumulation that may be of safety concern, the microbeads made of high-G alginate gelled with a combination of calcium and low concentration of barium ions is recommended for islet transplantation.

Growth and Nucleation of Calcium Carbonate Vaterite Crystals in Presence of Alginate

Structural biocomposites found in nature often have a well-defined organization on the nanometer scale. For mineralized materials, interactions between organic and inorganic phases are important for controlling crystal size, morphology, and spatial arrangement, which is a requirement when structural biomaterials are designed. In this paper, we studied influence of low concentrations of alginate on calcium carbonate crystallization by seeded and unseeded experiments, at controlled activity-based supersaturations. Crystal growth and nucleation were characterized by scanning electron microscopy (SEM), calcium concentration measurements, and crystal volume distribution measurements through the crystallization experiments. Alginate concentrations as low as 10 ppm were found to have a significant effect on growth of vaterite seeds, resulting in decreased growth rates and extensive agglomeration, compared to the case without alginate. For increased alginate concentrations (100 and 200 ppm), vaterite seed growth rates were decreased further. The decreased growth rates were probably caused by adsorption of alginate onto the active growth sites of the crystal surface. Alginate with 65% G-units (HighG) reduced the growth rate more than alginate with 43% G-units (LowG), which may be accounted for by the greater G-block length, and thus higher affinity to calcium, in HighG alginate. The unseeded experiments showed that mainly small vaterite crystals nucleated with 100 ppm alginate present, after an induction time of 50−80 min, while large calcite crystals were formed after some time by transformation from vaterite. The decreased crystal growth rates and higher nucleation rates caused by increased concentrations of alginate explain how small size mineral particles can be formed in alginate gel networks to form nanostructured composite materials.

Chemistry and the biological response against immunoisolating alginate–polycation capsules of different composition

Implantation of microencapsulated cells has been proposed as a therapy for a wide variety of diseases. An absolute requirement is that the applied microcapsules have an optimal biocompatibility. The alginate-poly-L-lysine system is the most commonly applied system but is still suffering from tissue responses provoked by the capsule materials. In the present study, we investigate the biocompatibility of microcapsules elaborated with two commonly applied alginates, i.e. an intermediate-G alginate and a high-G alginate. These alginates were coated with poly-L-lysine (PLL), poly-D-lysine (PDL) and poly-L-ornithine (PLO). The main objective of this study is to determine the interaction of each alginate matrix with the different polycations and the potential impact of these interactions in the modulation of the host's immune response. To address these issues the different types of microcapsules were implanted into the peritoneal cavity of rats for I month. After this period the microcapsules were recovered and they were evaluated by different techniques. Monochromatised X-ray photoelectron spectroscopy (XPS) was performance and the degree of capsular recovery, overgrowth on each capsule, and the cellular composition of the overgrowth were evaluated by histology. Our results illustrate that the different observed immune responses are the consequence of the variations in the interactions between the polycations and alginates rather than to the alginates themselves. Our results suggest that PLL is the best option available and that we should avoid using PLO and PDL in its present form since it is our goals to produce capsules that lack overgrowth and do not induce an immunological response as such.

Granulation of subtilisin by internal gelation of alginate microspheres for application in detergent formulation

Subtilisin was encapsulated within impact-resistant alginate granules produced by emulsification, internal gelation, and acetone extractive drying. The mechanical and controlled release properties of the granules were modified by adding to the alginate varying levels of formulation excipients, including titanium dioxide, polyvinyl alcohol, microcrystalline cellulose, starch and sucrose. Optimum protease activity and mass yields of 83 and 88%, respectively (mg active subtilisin/g granules), occurred for granules formulated with 3% alginate, 10% starch, 10% titanium dioxide, and 3% subtilisin. Mass losses occurred primarily during the gelation step. Maximum encapsulation efficiency is achieved by using higher molecular weight alginate, increasing the alginate concentration, and carefully controlling process temperature and pH. The strongest granules were obtained at the higher concentrations of medium-G or high-G alginate, while fastest granule dissolution was achieved when a lower concentration of alginate was used in combination with polyvinyl alcohol or microcrystalline cellulose as dispersants. Mechanical properties of alginate granules were found to be unaffected by the different cations employed in matrix gel formation.

MIN6 cells-enclosing aminopropyl-silicate membrane templated by alginate gels differences in guluronic acid content

Mouse insulinoma (MIN6) cells were encapsulated into aminopropyl-silicate membrane deposited on calcium alginate gel beads via the sol–gel synthesis. Two alginates with different guluronic acid (G) contents, high and intermediate, but with the same molecular weights were used. Viability of the cells in the membrane templated by the alginate with an intermediate content of guluronic acid (intermediate-G) was approximately 10% higher than those in the membrane templated by the alginate with a high content of guluronic acid immediately after encapsulation. Growth of cells in vitro was hindered in case of encapsulation in the aminopropyl-silicate membrane deposited on the high-G alginate gel. The MIN6 cells in the microcapsule made from high-G alginate needed a longer period to establish a normoglycemic in recipients than those in the microcapsule made from intermediate-G alginate despite the same number of viable cells implantation. Recipients of the microcapsule with the core made from the intermediate-G alginate maintained their blood glucose values less than 300 mg/dl for a longer period.

Small-angle X-ray scattering and rheological characterization of alginate gels. 3. Alginic acid gels.

Alginic acid gels were studied by small-angle X-ray scattering and rheology to elucidate the influence of alginate chemical composition and molecular weight on the gel elasticity and molecular structure. The alginic acid gels were prepared by homogeneous pH reduction throughout the sample. Three alginates with different chemical composition and sequence, and two to three different molecular weights of each sample were examined. Three alginate samples with fractions of guluronic acid residues of 0.39 (LoG), 0.50 (InG), and 0.68 (HiG), covering the range of commercially available alginates, were employed. The excess scattering intensity I of the alginic acid gels was about 1 order of magnitude larger and exhibited a stronger curvature toward low q compared to ionically cross-linked alginate. The I(q) were decomposed into two components by assuming that the alginic acid gel is composed of aggregated multiple junctions and single chains. Time-resolved experiments showed a large increase in the average size of aggregates and their weight fraction within the first 2 h after onset of gelling, which also coincides with the most pronounced rheological changes. At equilibrium, little or no effect of molecular weight was observed, whereas at comparable molecular weights, an increased scattering intensity with increasing content of guluronic acid residues was recorded, probably because of a larger apparent molecular mass of domains. The results suggest a quasi-ordered junction zone is formed in the initial stage, followed by subsequent assembling of such zones, forming domains in the order of 50 A. The average length of the initial junction zones, being governed by the relative fraction of stabilizing G-blocks and destabilizing alternating (MG) blocks, determines the density of the final random aggregates. Hence, high-G alginates give alginic acid gels of a higher aggregate density compared to domains composed of loosely packed shorter junction zones in InG or LoG system.

Fourier transform infrared spectroscopy studies of alginate-PLL capsules with varying compositions.

Microencapsulation of cells is a promising approach to prevention of rejection in the absence of immunosuppression. Clinical application, however, is hampered by insufficient insight into the factors that influence the biocompatibility of the capsules. Capsules prepared of alginates with a high guluronic (G) acid content proved to be more adequate for clinical application since they are more stable, but, unfortunately, they are less biocompatible than capsules prepared of intermediate-G alginate. In order to get some insight into the physicochemical factors that influence the biocompatibility of capsules for the encapsulation of living cells, the chemical compositions of alginate[bond]Ca beads and alginate[bond]PLL capsules were studied by Fourier transform infrared spectroscopy. We found that during the transition of the alginate[bond]Ca beads to alginate[bond]PLL capsules, Ca connecting the alginate molecules, disappeared at the surface of both high-G and intermediate-G alginate[bond]PLL capsules. At the same time, it turned out that high-G alginate[bond]PLL capsules contained more hydrogen bonding than did intermediate[bond]G alginate capsules. Thus the well-known higher stability of high-G alginate[bond]PLL compared to intermediate-G alginate[bond]PLL capsules is not caused by a higher degree of binding to Ca of the alginate molecules but rather by the presence of more hydrogen bonds. Another observation was that after the transition from bead to capsule, high-G alginate[bond]PLL capsules contained 20% more PLL than the intermediate-G alginate[bond]PLL capsules. Finally, we show that in both high-G and intermediate-G alginate[bond]PLL capsules, the PLL exists in the alpha-helix, in the antiparallel beta-sheet, and in the random coil conformation. This study shows that FT-IR allows for successful analyses of the chemical factors essential for understanding differences in the biocompatibility of alginate[bond]PLL capsules.

Biocompatibility of microcapsules for cell immobilization elaborated with different type of alginates

The biocompatibility of alginate-PLL-alginate (APA) microcapsules has been evaluated with respect to impurity levels. The impurity content of three different alginates (a raw high M-alginate, a raw high G-alginate and a purified high G-alginate) has been determined and the in vivo antigenic response of APA beads made with each alginate assessed. Results show that purification of the alginate not only reduces the total amount of impurities (63% less in polyphenols, 91.45% less in endotoxins and 68.5% less in protein in relation to raw high M-alginate), but also avoids an antibody response when microcapsules of this material are implanted in mice. In contrast, raw alginates produced a detectable antibody response though the differences in their impurity content. Consequently, this work revealed that purity of the alginate rather than their chemical composition, is probably of greater importance in determining microcapsule biocompatibility.

Chemistry and biocompatibility of alginate-PLL capsules for immunoprotection of mammalian cells.

Transplantation of encapsulated living cells is a promising approach for the treatment of a wide variety of diseases. Large-scale application of the technique, however, is hampered by insufficient biocompatibility of the capsules. In order to get means to study factors influencing the biocompatibility of capsule for encapsulation of living cells, we have correlated the chemical composition of the surface of commonly applied alginate-PLL capsules with the biological response in rats. Capsules prepared of alginates with an intermediate guluronic (G) acid content proved to be biocompatible, whereas capsules prepared of high-G alginates were overgrown by inflammatory cells. We applied X-ray photoelectron spectroscopy to correlate the biological responses with the chemical compositions of the capsule surfaces. High-G alginate capsules proved to have a higher PLL content but less surface binding sites for PLL than low-G alginates. This study, shows for the first time that biological responses against capsules can be successfully correlated to its chemical characteristics.

Biosynthetic implications of NMR analyses of alginate homo- and heteropolymers from New Zealand brown seaweeds

Homopolymeric (M- and G-) and heteropolymeric (MG-) blocks have been prepared from alginates that have been isolated from seven Southern Hemisphere brown seaweeds and from three commercial algal alginates. The blocks have been analysed by 1H and 13C NMR spectroscopy, and the analyses show that well-defined M- and G-homopolymers are obtained only from the five polysaccharides that have been previously designated as either high-M or high-G alginates. However, the five intermediate algal alginates (which have F M values between 0.6 and 0.7) appear to contain mainly MG or heteropolymeric blocks. It is concluded that the results of NMR spectroscopic analysis of whole alginates can give a misleading picture of the block structure of some alginates. The NMR analyses of the homopolymers isolated from both the high-M and high-G alginates also show that the order of the d-mannuronosyl and l-guluronosyl residues in these blocks fits a first-order Markov distribution pattern. This suggests that 5-epimerization of some of the GDP- d-mannuronosyl residues at the monomer level, followed by addition copolymerization catalyzed by a GDP-guluronic acid transferase system, as originally suggested by Lin and Hassid [1,2], may contribute to the biosynthesis of the homopolymeric block structures in these high-M and high-G alginates isolated from brown algae. The NMR analysis of homopolymeric blocks from several algal alginates are shown to have first-order Markov distributions of d-mannuronosyl and l-guluronosyl residues. This is consistent with a biosynthetic pathway that involves a C-5 epimerisation at the monomer level.

Effect of the alginate composition on the biocompatibility of alginate-polylysine microcapsules

Alginate-polylysine (PLL) capsules are commonly applied for immunoprotection of endocrine tissues. Alginate is composed of mannuronic acid (M) and guluronic acid (G). Different types of alginate have different ratios of G to M, but little is known of the influence of these differences on biocompatibility. Therefore, we have investigated in vivo the effect of the G-content of the alginate on the biocompatibility of the capsules. Capsules prepared of commercially available alginates with either a high or an intermediate G-content were implanted in the peritoneal cavity of rats and retrieved one month later for histological evaluation. The fibrotic reaction was more severe against high-G alginate capsules than to intermediate-G alginate capsules. The majority of the high-G capsules proved to be overgrown and adherent to the abdominal organs whereas with intermediate-G alginate most capsules were found freely floating in the peritoneal cavity and free of any adhesion of cells. This was not caused by the alginate as such but rather by inadequate binding of high-G alginate to PLL since in the absence of PLL, i.e. with beads instead of capsules, no fibrotic reaction was observed. As high-G alginates have beneficial effects for islet encapsulation, efforts should be made to apply polycations which more effectively interact with high-G alginate than PLL.