Water in agarose gels studied by nuclear magnetic resonance relaxation in the rotating frame.

The dependence of the spin-lattice relaxation time in the rotating frame (T1rho) on radio frequency (RF) field strength and temperature has been studied for agarose gels in order to investigate molecular motion. The results indicate the presence of slow motions with a correlation time of ca. 5-10(-6) s at room temperature. This interaction is responsible for the short spin-spin relaxation times (T2) for water protons in agarose gels and is ascribed to firmly bound water. The fraction of bound water is estimated to about 0.003 for a 7.3% agarose gel. The motion of the more mobile protons in agarose-water systems can not be characterized by single correlation time. This fraction is presumably composed of water in different motional states and some of the agarose hydroxyl protons. Higher mobilities are the most common.     

Effect of locust bean gum and konjac glucomannan oh the conformation and rheology of agarose and κ-carrageenan

Mixing with locust bean gum (LBG) induces obvious gel-like character in very dilute solutions of K⁺ κ-carrageenan (< 0.01% w/w in 100 mM KCl). At higher concentration (0.085%), addition of LBG (0.036%) gives a shoulder on the high-temperature side of the DSC (differential scanning calorimetry) exotherm associated with the carrageenan disorder-order transition, with an accompanying increase in gelation temperature and enhancement in gel strength (storage modulus, G′). On substitution of LBG by konjac glucomannan (KM) the shoulder in DSC cornverts to a discernable peak. Van't Hoff analysis of optical rotation data indicates that the high-temperature thermal processes could arise from association of LBG or KM chains to the carrageenan double helix as it forms, with the main transition at lower temperature corresponding to ordering of surplus carrageenan. With K -carrageenan in the nongelling tetra-methylaminonium salt form, addition of LBG causes no delectable change in DSC; rheological enhancement at high concentration (1% w/w) is limited to development of a very tenuous network, and in dilute solution a decrease in viscosity is observed. Agarose shows only a very slight increase in the disorder-order transition temperature on addition of KM, and it shows no detectable change with LBG. These observations are interpreted as showing that efficient binding of mannan or glucomannan chains requires some aggregation of the algal polysaccharide helices, but that extensive aggregation restricts synergistic interaction by competition with heterotypic association. © 1995 John Wiley & Sons, Inc.     

Interpenetrating network formation in agarose–sodium gellan gel composites

Aqueous cold-set gels from mixtures of agarose and sodium gellan have been characterised structurally and mechanically using optical and electron microscopy, turbidity measurements, differential scanning calorimetry, mechanical spectroscopy and compression testing. Consistent with expectations for charged–uncharged polymer combinations at low ionic strength there is no liquid–liquid demixing in sols prior to gelation, and although transmission electron microscopy reveals heterogeneities in gel microstructures at the higher polymer concentrations, these are small in extent, and are unlikely to arise from normal segregative demixing. Overall, ‘molecularly’ interpenetrating networks (IPNs) are indicated, in which the gellan and agarose architectures pass through one another on a distance scale comparable to their pore sizes. At concentrations greater than 2% w/w gellan, where gellan is the first gelling species, and when the agarose concentration is greater than 0.5% w/w, the composite modulus falls below that expected for the agarose alone. At 0.5% w/w agarose, on the other hand, modulus contributions from the components are much closer to additive. These findings are reflected in the results of large deformation compression testing where breaking stresses show similar trends.     

Stability of hydrogels used in cell encapsulation: An in vitro comparison of alginate and agarose

The present studies were undertaken to evaluate the in vitro gel stability of the hydrogels alginate and agarose. Gel strength (of alginate and agarose) and protein diffusion (of alginate only) were shown to correlate with gel stability and to be useful techniques to monitor gel stability over time. The gel strengths of alginate and agarose were followed for a 90-day period using gel strength as a measure of gel stability. The gel strength of agarose diminished in the presence of cells because the cells likely interfered with the hydrogen bond formation required for agarose gelation. In the presence of cells, the gel strength of agarose decreased by an average of 25% from time 0 to 60 days, thereafter maintaining that value to 90 days. The gel strength of calcium- or barium-crosslinked alginate decreased over 90 days, with an equilibrium gel strength being achieved after 30 days. The presence of cells did not further decrease alginate gel strength. The gel strengths of calcium- and barium-crosslinked alginates were similar at 60 days-350 +/- 20 g and 300 +/- 60 g, respectively-indicating equivalence in their stability. The stability of calcium-crosslinked sodium alginate gels over a 60-day time period was monitored by diffusion of proteins ranging in molecular weight from 14.5 to 155 kD. From these diffusion measurements, the average pore size of the calcium-crosslinked alginate gels was estimated, using a semi-empirical model, to increase from approximately 176 to 289 A over a period of 60 days. (c) 1996 John Wiley & Sons, Inc.     

A solid state NMR study of locust bean gum galactomannan and Konjac glucomannan gels

This paper describes a ¹³C solid state NMR study of hydrated powders and gels of locust bean gum galactomannan-LBG and Konjac glucomannan-KGM. Changes in relative spectral intensities, cross-polarization dynamics (T_CH, T_1ρH) and relaxation times (T_1C, T_1H, T_2H) show that hydration (0–90%) of LBG powders increases the 10⁸ Hz frequency molecular motions, probably reflecting the enhanced motion of non-aggregating segments and chain ends. Slower motions (10⁴–10⁵ Hz) are enhanced only slightly at 90% hydration. LBG gel shows higher spatial distinction between aggregated and non-aggregated segments than the hydrated powder and relaxation times indicate higher mobility for galactose-ramified segments, compared to linear mannose segments. While the dynamics of KGM hydration is similar to that of LBG, i.e. mainly affecting fast 10⁸ Hz motions, the gel is significantly more rigid. Both spectra and relaxation times show that glucose residues in KGM gel are particularly hindered, probably due to their preferential involvement in chain aggregation.     

Impact of incorporations of various polysaccharides on rheological and microstructural characteristics of heat-induced quinoa protein isolate gels

This study aimed to investigate the properties of heat-induced gels (85 °C for 30 min) of quinoa protein isolate (QPI) in the presence and absence of various polysaccharides including guar gum (GG), locust bean gum (LBG), and xanthan gum (XG) at pH 7. For this purpose, samples with three gum concentrations (0.05, 0.1, and 0.2 wt%) at a fixed QPI concentration (10 wt%) and a fixed ionic strength (50 mM NaCl) were studied in terms of their gelation behaviour, small and large deformation rheological properties, water holding capabilities, and microstructural characteristics. Rheological measurements revealed that all polysaccharides incorporation could improve gel strength (complex modulus, G*) and breaking stress, accelerate gel formations, and more stiffer gels were obtained at greater polysaccharide concentrations. The XG exhibited the most gel strengthening effect followed by LBG and GG. Incorporation of 0.2 wt% XG led to a 15 folds increase in G* compared to the control. Confocal laser scanning microscopy observation revealed that the polysaccharides also altered gel microstructures, with the gels containing XG showing the most compact gel structures. The findings of this study may provide useful information for the fabrication of novel QPI based food gel products with improved texture.

     

Transient electric birefringence of agarose gels. II. Reversing electric fields and comparison with other polymer gels

The transient electric birefringence of low electroendosmosis (LE) agarose gels oriented by pulsed unidirectional electric fields was described in detail in Part I [J. Stellwagen and N. C. Stellwagen (1994), Biopolymers, Vol. 34, p. 187]. Here, the birefringence of LE agarose gels in rapidly reversing electric fields, similar in amplitude and duration to those used for field inversion gel electrophoresis, is reported. Symmetric reversing electric fields cause the sign of the birefringence of LE agarose gels, and hence the direction of orientation of the agarose fibers, to Oscillate in phase with the applied electric field. Because of long-lasting memory effects, the alternating sign of the birefringence appears to be due to metastable changes in gel structure induced by the electric field. If the reversing field pulses are equal in amplitude but different in duration, the orientation behavior depends critically on the applied voltage. If E < 7 V/cm, the amplitude of the birefringence gradually decreases with increasing pulse number and becomes unmeasurably small. However, if E > 7 V/cm, the amplitude of the birefringence increase more than 10-fold after ～ 20 pulses have been applied to the gel, suggesting that a cooperative change in gel structure has occurred. Because there is no concomitant change birefringence must be due to an increase in the number of agarose fibers and /or fiber bundles orienting in the lectric field, which in turn indicates a cooperatice breakdown of the noncovalent “junction zones” that corss-link the fibers in to the fgel matrix. The sign of the birefringence of LE agarose gels is always positive after extensive junction zone breakdown, indicating that the agarose fibers and fiber bundles preferentially orient parallel to the lectric field when they are freed from the constraints of the gel matrix. Three other gel-forming polymers, high electroendosmosis (HEEO) agarose (a more highly changed agarose), β-carrageenan (a stereoisomer of agarose), and polyacrylamide (a chemically corss-linked polymer) were alos studied in unidirectional and rapidly reversing electric fields. The birefringence of HEEO agarose backbone chain. The β-carrageenan gels exhibit variable orientation behavior in reversing electric fields, suggesting that its internal gel structure is not as tightly interconnected as that of agaroise gels. Both HEEO agarose and β-carrageenan gels exhibit a large increase in the amplitude of the birefringence with increasing pulse number when asymmetric reversing pulses > 7 V/cm are applied to the gels, suggesting that junction zone breakdown in a common feature of polysaccharide gels. Chemically cross-linked polyacrylamide gels exhibit very small birefringence signals, indicating that very little orientation occurs in pulsed lectric fields. The sign of the birefringence is independent of the polarity of the lectric field, as expected from the Kerr law, and normal orientation behavior is observed in reversing electric fields. Hence, the anomalous change in sign of the birefringence observed for agarose gels in reversing electric fields must be due to the metastable junction zones in the agarose gel matrix, which allow gel fiber rearrangements to occur. © 1994 John Wiley & Sons, Inc.     

Rheological properties of mixtures of kappa-carrageenan from Hypnea musciformis and galactomannan from Cassia javanica

Mixed gels of κ-carrageenan (κ-car) from Hypnea musciformis and galactomannans (Gal) from Cassia javanica (CJ) and locust bean gum (LBG) were compared using dynamic viscoelastic measurements and compression tests. Mixed gels at 5 g/l of total polymer concentration in 0.1 M KCl showed a synergistic maximum in viscoelastic measurements for κ-car/CJ and κ-car/LBG at 2:1 and 4:1 ratios, respectively. The synergistic maximum obtained from compression tests carried out for mixed gels at 10 g/l of total polymer concentration in 0.25 M KCl was the same for both κ-car/CJ and κ-car/LBG gels. An enhancement in the storage modulus (G′) and the loss modulus (G″) was observed in the mechanical spectra for the mixtures in relation to κ-car. The proportionally higher increase in G″ compared with G′, as indicated by the values of the loss tangent (tan δ), suggests that the Gal adhere non-specifically to the κ-car network.     

Molecular dynamics study of the influence of solvents on the structure and mechanical properties of poly(vinyl alcohol) gels

Molecular dynamics (MD) simulations were employed to study the influence of solvents on the structure and mechanical properties of physically crosslinked poly(vinyl alcohol) (PVA) gels. Firstly, three kinds of PVA precursor gels were made by adding water, dimethyl sulfoxide (DMSO) and a mixture of DMSO and water (4:1 by weight), respectively. The solvents in the precursor gels were then exchanged with water to obtain three kinds of PVA hydrogels. Solvent in the precursor gel with a mixture of DMSO and water was also exchanged with ethanol and DMSO, respectively. It was found that the tensile strength and failure strain of the PVA hydrogel prepared from precursor gel with a mixture of DMSO and water was the highest, and the polymer network was more homogeneous than the other two PVA hydrogels. The polymer network of PVA gel with ethanol or with DMSO was more heterogenous than with water, and the tensile strength and failure strain were much lower. The torsional activity of polymer chains of PVA gel with ethanol was much stronger than PVA gel with water and DMSO.     

The gel matrix of gastric mucus is maintained by a complex interplay of transient and nontransient associations

The gel nature of mucus is fundamental to its physiological functions; however, the structure of the mucus gel matrix is unclear. Here, small and large deformation rheology has been used to investigate the physical nature of the gel matrix and the forces that maintain this matrix in pig gastric mucus. The gelation process in mucus has been shown to be comparable with that of other polymer gel systems. Nongelling portions of mucin have been identified within the gel network, and the importance of transient, relaxable interactions to the maintenance of the mucus gel matrix has been demonstrated. The structure of the mucus gel matrix is considered in relation to the functional properties of mucus gels.     

Affinity electrophoresis: New simple and general methods of preparation of affinity gels

Two simple and generally applicable methods of preparation of affinity gels for affinity electrophoresis in agarose and polyacrylamide gels are described. In the first method, amino ligands are coupled to periodate-oxidized agarose gel beads (Sepharose 4B), and homogeneous affinity gels are obtained after mixing the melted substituted beads with either melted agarose solution or with the polymerization mixture used for the preparation of polyacrylamide gels. This type of affinity gel was used for affinity electrophoresis of lectins (immobilized p-aminophenyl glycosides), ribonuclease (immobilized uridine 3′,5′-diphosphate 5′-p-aminophenyl ester), trypsin (immobilized p-aminobenzamidine), and double-stranded phage DNA fragments (immobilized acriflavine). Alternatively, heterogeneous affinity gels are prepared from the suspension of ligand-substituted agarose, dextran, or polyacrylamide gel beads in the polymerization solution normally used for preparation of polyacrylamide electrophoretic gels. This technique was used for affinity electrophoresis of lectins, ribonuclease, and trypsin on affinity gels containing appropriate ligands coupled to the gel beads “activated” by various methods. Applicability of affinity gels prepared by the two methods described above for affinity isoelectric focusing is demonstrated.     

Rheological Study of the Gelation Process of Agarose-Based Solutions

The gelation of agarose is investigated by rheological methods and electron microscopy, as well as the thickening properties of xanthan. The gelling and thickening agents have been investigated in pure water to compare the results with theoretical models. The gelation of agarose was shown to follow two steps upon cooling, which could be addressed to the formation of helices and their aggregation. In addition to the rheology, transmission electron micrographs of freeze-dried samples have been taken to underline the date by corresponding structures at different stages of the gelling process. The xanthan molecules, which have been approximated by rigid highly charged rodlike molecules, undergo a jamming transition at a critical concentration. This concentration shows a strong dependence on the length of the molecules, which supports the high thickening effect of xanthan. When both, agarose and xanthan are mixed, the gel structure becomes very different. The gelling process is now determined by one step only. It is proposed that the jamming xanthan molecules prevent the formation of the aggregates of the agarose gel. The gels themselves appear then less elastic, and should yield a better mouth feeling.     

Rheological properties of mixtures of κ-carrageenan from Hypnea musciformis and galactomannan from Cassia javanica

Mixed gels of κ-carrageenan (κ-car) from Hypnea musciformis and galactomannans (Gal) from Cassia javanica (CJ) and locust bean gum (LBG) were compared using dynamic viscoelastic measurements and compression tests. Mixed gels at 5 g/l of total polymer concentration in 0.1 M KCl showed a synergistic maximum in viscoelastic measurements for κ-car/CJ and κ-car/LBG at 2:1 and 4:1 ratios, respectively. The synergistic maximum obtained from compression tests carried out for mixed gels at 10 g/l of total polymer concentration in 0.25 M KCl was the same for both κ-car/CJ and κ-car/LBG gels. An enhancement in the storage modulus (G′) and the loss modulus (G″) was observed in the mechanical spectra for the mixtures in relation to κ-car. The proportionally higher increase in G″ compared with G′, as indicated by the values of the loss tangent (tan δ), suggests that the Gal adhere non-specifically to the κ-car network.     

Inhomogeneous alginate gel spheres: an assessment of the polymer gradients by synchrotron radiation-induced X-ray emission, magnetic resonance microimaging, and mathematical modeling

It has been previously demonstrated that calcium alginate gels prepared by dialysis often exhibit a concentration inhomogeneity being the polymer concentration considerably lower in the center of the gel than at the edges. Inhomogeneity may be a preferred structure in microcapsules due to low porosity and higher stability so that it is interesting to evaluate the polymer gradient in spherically symmetrical small alginate beads (1.0-0.7 mm diameter) obtained in different conditions. In this paper, two complementary techniques have been used to investigate this aspect. The concentration gradient of alginate has been analyzed by measuring both the spatial distribution of calcium ions in sections of alginate gel spheres, by means of x-ray fluorescence spectroscopy, and the T2 relaxation behavior on intact gel beads using magnetic resonance microimaging. The experimentally determined gradients from three-dimensional gels provide data to reevaluate the parameter estimates in the recently reported mathematical model for alginate gel formation (A. Mikkaelsen and A. Elgsaeter, Biopolymers, 1995, Vol. 36, pp. 17-41). The model may account for the gels being less inhomogeneous when nongelling sodium or magnesium ions are added during gelation.     

Thermoreversible gelation of aqueous mixtures of pectin and chitosan. Rheology

The synergistic interaction between pectin and chitosan in aqueous acid solution and in the gel phase has been studied by oscillatory shear measurements. Mixtures of pectin and chitosan form thermoreversible gels over a broad composition range by lowering the temperature. The value of the gelation temperature depends on the composition of the mixture, with low values for mixtures with low pectin contents. For incipient gels, a power law can describe the frequency dependence of the complex viscosity, with power law exponents close to -1. The gel evolution of pectin-chitosan mixtures upon a temperature quench below the gel point has been studied. Evidence is provided for a relation between gelation and phase separation in the process of temperature-induced gelation of pectin-chitosan mixtures. A simple model is proposed to rationalize the gelation process in these systems.     

Adhesive thermosensitive gels for local delivery of viral vectors

Local delivery of viral vectors can enhance the efficacy of therapies by selectively affecting necessary tissues and reducing the required vector dose. Pluronic F127 is a thermosensitive polymer that undergoes a solution–gelation (sol–gel) transition as temperature increases and can deliver vectors without damaging them. While pluronics can be spread over large areas, such as the surface of an organ, before gelation, they lack sufficient adhesivity to remain attached to some tissues, such as the surface of the heart or mucosal surfaces. Here, we utilized blends of pluronic F127 and polycarbophil (PCB), a mucoadhesive agent, to provide the necessary adhesivity for local delivery of viral vectors to the cardiac muscle. The effects of PCB concentration on adhesive properties, sol–gel temperature transition and cytocompatibility were evaluated. Rheological studies showed that PCB decreased the sol–gel transition temperature at concentrations >1% and increased the adhesive properties of the gel. Furthermore, these gels were able to deliver viral vectors and transduce cells in vitro and in vivo in a neonatal mouse apical resection model. These gels could be a useful platform for delivering viral vectors over the surface of organs where increased adhesivity is required.     

Competitive inhibition evidence for specific intermolecular interactions in hyaluronate solutions

Intermolecular associations in hyaluronate systems have been investigated by a competitive inhibition approach, monitored by low deformation oscillatory measurements of dynamic viscosity and rigidity. Solutions of sodium hyaluronate isolated by a mild procedure from rooster combs showed, under physiological conditions of pH and ionic strength, coupling behaviour typical of a transient polymer network. On addition of an equal concentration of hyaluronate segments (~60 disaccharide units), all evidence of coupling is lost and the solution rheology approximates closely to that typical of isolated chains. Such behaviour is clearly incompatible with entanglement coupling, such as occurs between synthetic polymers in solution, but parallels the behaviour of gelling polysaccharides, where co-operative interchain association is known to occur. Similar inhibition is observed in hyaluronate viscoelastic “putties” and “gels”, where further intermolecular association is promoted by suppression of interchain electrostatic repulsion and reduction of water activity. The effects are particularly pronounced for hyaluronate of lower molecular weight, where crosslinking in putties and gels may approach the minimum requirements for a cohesive network. No inhibition is observed with very short chain segments (~4 disaccharide residues) nor with longer segments (~400 disaccharides). On the basis of this evidence we suggest that hyaluronate chains interact by formation of specific co-operative junctions analogous to those characterised for plant structural polysaccharides, but having substantially shorter lifetimes. The magnitude of rheological changes on suppression of these fleeting associations by competitive inhibition suggests that they are likely to dominate the physical properties of hyaluronate in vivo.     

Agarose-acrylamide gradient gel electrophoresis of proteins

A new agarose-acrylamide gradient slab gel electrophoresis system is described. The preparation of this new gel has been facilitated by the use of agarose with a relatively low gelation temperature. Fractionation of marker proteins and crosslinked proteins from a subcellular cytoskeletal preparation on agarose-acrylamide gradient gels is compared to that found using other acrylamide gel electrophoresis systems.     

Preparation of GDP-D-mannose specifically labeled with tritium in the D-mannosyl moiety

A method, called “bidirectional transfer”, has been described for the transfer of DNA and RNA from agarose or polyacrylamide gels onto diazobenzyloxymethyl (DBM)-paper or nitrocellulose filters. The gels were sandwiched between either two nitrocellulose filters or two diazobenzyloxymethyl-papers. Next, the nucleic acids were allowed to diffuse out of the gels onto the filters. In this way, duplicate blots were obtained from a single gel. The bidirectional transfer of DNA or RNA from 0.5 to 1% agarose gels was complete and nearly quantitative after 1 h of transfer. DNA fragments from 5% polyacrylamide gels were efficiently blotted after 36 h onto nitrocellulose filters using bidirectional transfer. The fragments were transferred with good resolution and were shown to be efficient substrates for homologous [³²P]DNA probes.