Influence of chitosan-sodium alginate pretreated with ultrasound on the enzyme activity, viscosity and structure of papain.

BACKGROUND: Ultrasound treatment has been shown to be an effective technique for improving the activity of immobilized enzymes. However, its mechanism is unclear. RESULTS: The effect of ultrasonic pretreated chitosan-sodium alginate (CHI-ALG) on the enzymatic activity of papain was investigated via a single factor (temperature, time, frequency, power) experiment. The maximum relative enzyme activity of papain was observed when it was mixed with ultrasound pretreated CHI-ALG at 135 kHz, 0.25 W cm-2 and 50 °C for 20 min, during which the relative activity increased by 72.14% compared to untreated CHI-ALG. Viscosity analysis of papain mixed with CHI-ALG pretreated and untreated with ultrasound revealed that stronger association interactions between the polymers were formed compared to the untreated sample. Fluorescence and circular dichroism spectra indicated that the ultrasonic pretreatment of CHI-ALG increased the number of tryptophan on the papain surface and also increased the content of α-helix by 6.97% and decreased the content of ß-sheet by 3.45% compared to the untreated solution. CONCLUSION: The results of the present study indicate that papain combined with CHI-ALG pretreated with the appropriate ultrasound could be effective technique for improving the activity of immobilized enzymes as a result of changes in its structure and intermolecular interactions. It is important to extend the application of CHI-ALG gel in the immobilized enzyme industry. © 2016 Society of Chemical Industry.

Light-responsive iron(III)-polysaccharide coordination hydrogels for controlled delivery.

Visible-light responsive gels were prepared from two plant-origin polyuronic acids (PUAs), alginate and pectate, coordinated to Fe(III) ions. Comparative quantitative studies of the photochemistry of these systems revealed unexpected differences in the photoreactivity of the materials, depending on the polysaccharide and its composition. The roles that different functional groups play on the photochemistry of these biomolecules were also examined. Mannuronic-rich alginates were more photoreactive than guluronic acid-rich alginate and than pectate. The microstructure of alginates with different mannuronate-to-guluronate ratios changed with polysaccharide composition. This influenced the gel morphology and the photoreactivity. Coordination hydrogel beads were prepared from both Fe-alginate and Fe-pectate. The beads were stable carriers of molecules as diverse as the dye Congo Red, the vitamin folic acid, and the antibiotic chloramphenicol. The photoreactivity of the hydrogel beads mirrored the photoreactivity of the polysaccharides in solution, where beads prepared with alginate released their cargo faster than beads prepared with pectate. These results indicate important structure-function relationships in these systems and create guidelines for the design of biocompatible polysaccharide-based materials where photoreactivity and controlled release can be tuned on the basis of the type of polysaccharide used and the metal coordination environment.

Influence of amino acids, buffers, and ph on the γ-irradiation-induced degradation of alginates.

Alginate-based biomaterials and medical devices are commonly subjected to γ-irradiation as a means of sterilization, either in the dry state or the gel (hydrated) state. In this process the alginate chains degrade randomly in a dose-dependent manner, altering alginates' material properties. The addition of free radical scavenging amino acids such as histidine and phenylalanine protects the alginate significantly against degradation, as shown by monitoring changes in the molecular weight distributions using SEC-MALLS and determining the pseudo first order rate constants of degradation. Tris buffer (0.5 M), but not acetate, citrate, or phosphate buffers had a similar effect on the degradation rate. Changes in pH itself had only marginal effects on the rate of alginate degradation and on the protective effect of amino acids. Contrary to previous reports, the chemical composition (M/G profile) of the alginates, including homopolymeric mannuronan, was unaltered following irradiation up to 10 kGy.

Effects of gamma irradiation and moist heat for sterilization on sodium alginate.

Polysaccharides, such as alginates, are already being used as carriers for drug delivery. The physicochemical and biological properties of alginates may be affected via irradiation and thermal treatments. To explore and compare effects of two kinds of sterilization methods, gamma irradiation and moist heat, on sodium alginate (SA), physicochemical and biological properties of SA powder and solutions were investigated after sterilization. Human umbilical vein endothelial cells (HUVEC) was used to assess the cytotoxicity of the SA after sterilization. The research showed that 25 kGy gamma ray can effectively sterilize microorganism. Both gamma irradiation and moist heat hardly affect the native pH of SA. Compared to irradiation sterilization, moist heat sterilization showed smaller changes in intrinsic viscosity for all SA samples and lead to less glycosidic bond breaking of SA powders. The moist heat sterilization can cause the main chain scission and double bonds formation of the SA solutions. Cytotoxicity studies demonstrated that sterilized SA powers and SA solutions treated by gamma ray sterilization can increase the viability of HUVEC. However, SA solutions treated by moist heat sterilization were found to present severe cytotoxicity. The research results may provide interesting future advancements toward the development of SA-based products for biomedical applications.

Radiolytically depolymerized sodium alginate improves physiological activities, yield attributes and composition of essential oil of Eucalyptus citriodora Hook.

Eucalyptus citriodora Hook. is highly valued for its citronellal-rich essential oil (EO) extracted from its leaves. Hence, escalated EO production of eucalyptus is the need of hour. Marine polysaccharides (sodium alginate) are processed through gamma radiation of particular intensity, to obtain the irradiated sodium alginate (ISA). A pot experiment was conducted to study the effect of foliar application of ISA on growth, biochemical, physiological, EO yield and composition of E. citriodora. The treatments were applied as: foliar spray of deionized water only (control), seed soaked with ISA (90 mg L(-1)) and foliar spray of ISA with 30, 60, 120 and 240 mg L(-1). The treatment 6 (spray of ISA at 120 mg L(-1)) showed the highest value for most of the parameters studied. It also enhanced the EO content (33.3%), EO yield (86.7%), citronellal content (63.4%) and citronellal yield (205.5%) as compared to the control.

Photo-activated ionic gelation of alginate hydrogel: real-time rheological monitoring of the two-step crosslinking mechanism.

We examine the gelation of alginate undergoing ionic crosslinking upon ultraviolet (UV) irradiation using in situ dynamic rheology. Hydrogels are formed by combining alginate with calcium carbonate (CaCO3) particles and a photoacid generator (PAG). The PAG is photolyzed upon UV irradiation, resulting in the release of free calcium ions for ionic crosslinking. The viscous and elastic moduli during gelation are monitored as a function of the UV irradiation intensity, exposure time, alginate concentration, and the ratio between alginate and calcium carbonate. Gel time decreases as irradiation intensity increases because a larger concentration of PAG is photolyzed. Interestingly, dark curing, the continuing growth of microstructure in the absence of UV light, is observed. In some instances, the sample transitions from a solution to a gel during the dark curing phase. Additionally, when exposed to constant UV irradiation after the dark curing phase, samples reach the same plateau modulus as samples exposed to constant UV without dark curing, implying that dark curing does not affect the gelation mechanism. We believe the presence of dark curing is the result of the acidic environment persisting within the sample, allowing CaCO3 to dissociate, thereby releasing free Ca(2+) ions capable of binding with the available appropriate ionic blocks of the polymer chains. The growth of microstructure is then detected if the activation barrier has been crossed to release sufficient calcium ions. In this regard, we calculate a value of 30 J that represents the activation energy required to initiate gelation.

Terminal sterilization of alginate hydrogels: efficacy and impact on mechanical properties.

Terminal, or postprocessing, sterilization of composite biomaterials is crucial for their use in wound healing and tissue-engineered devices. Recent research has focused on optimizing traditional biomaterial formulations to create better products for commercial and academic use which incorporate hydrophobic compounds or secondary gel networks. To use a hydrogel in a clinical setting, terminal sterilization is necessary to ensure patient safety. Lyophilization, gamma-irradiation, and ethylene oxide treatment all have negative consequences when applied to alginate scaffolds for clinical use. Here, we aim to find alternative terminal sterilization methods for alginate and alginate-based composite hydrogels which maintain the structure of composite alginate networks for use in biomedical applications. A thorough investigation of the effect of common sterilization methods on swollen alginate-based hydrogels has not been reported and therefore, this work examines autoclaving, ethanol washing, and ultraviolet light as sterilization techniques for alginate and alginate/Pluronic® F68 composite hydrogels. Preservation of structural integrity is evaluated using shear rheology and analysis of water retention, and efficacy of sterilization is determined via bacterial persistence within the hydrogel. Results indicate that ethanol sterilization is the best method of those investigated because ethanol washing results in minimal effects on mechanical properties and water retention and eliminates bacterial persistence. Furthermore, this study suggests that ethanol treatment is an efficacious method for terminally sterilizing interpenetrating networks or other composite hydrogel systems.

Preparation of oligoalginate plant growth promoter by γ irradiation of alginate solution containing hydrogen peroxide.

Degraded alginate compounds with molecular weights of 7-26, 40-77, or 11-26 kDa were obtained by γ irradiation, hydrogen peroxide (5% H(2)O(2)) treatment, or a combination treatment involving ionizing radiation and H(2)O(2), respectively. The 14 kDa oligoalginate, prepared by the combined method, promoted the growth of mustard greens and lettuce at an optimal concentration of 75 mg/L. The growth promotion effects of the oligoalginate prepared by γ irradiation in the presence of H(2)O(2) were statistically equivalent to those of the oligoalginate prepared by γ irradiation only. The combination of γ irradiation and H(2)O(2) reduced the required irradiation dosage by a factor of 9 relative to the oligoalginate produced by γ irradiation only. The combination treatment (γ irradiation/H(2)O(2)) may be carried out on a large scale at low cost to produce oligoalginate for use as a plant growth promoter in agricultural industries.

Stiffness of photocrosslinked RGD-alginate gels regulates adipose progenitor cell behavior.

Adipose progenitor cells (APCs) are widely investigated for soft tissue reconstruction following tumor resection; however, the long-term success of current approaches is still limited. In order to develop clinically relevant therapies, a better understanding of the role of cell-microenvironment interactions in adipose tissue regeneration is essential. In particular, the effect of extracellular matrix (ECM) mechanics on the regenerative capability of APCs remains to be clarified. We have used artificial ECMs based on photocrosslinkable RGD-alginate to investigate the adipogenic and pro-angiogenic potential of 3T3-L1 preadipocytes as a function of matrix stiffness. These hydrogels allowed us to decouple matrix stiffness from changes in adhesion peptide density or extracellular Ca(2+) concentration and provided a physiologically relevant 3D culture context. Our findings suggest that increased matrix rigidity promotes APC self-renewal and angiogenic capacity, whereas, it inhibits adipose differentiation. Collectively, this study advances our understanding of the role of ECM mechanics in adipose tissue formation and vascularization and will aid in the design of efficacious biomaterial scaffolds for adipose tissue engineering applications.

Hemostasis for severe hemorrhage with photocrosslinkable chitosan hydrogel and calcium alginate.

In patients with severe hemorrhage, complications such as shock or death may occur if the patient is not treated appropriately and expeditiously. To create a hemostat kit for severe hemorrhage, ultraviolet light irradiation was applied to photocrosslinkable chitosan hydrogel and calcium alginate. As a hemorrhage model, the femoral arteries and veins of anesthetized rats were cut. Hemodynamics and hematological parameters including red blood cell (RBC) count, hemoglobin concentration, hematocrit, white blood cell (WBC) count, and platelet count, and serum parameters including aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured as a marker of hemostasis. In rats for which no procedure was used, death occurred within 30 min. By using the hydrogel hemostat, the survival rate rose to 75% or more. RBC count, hemoglobin, hematocrit, and platelet levels were not significantly changed for 3 days. WBC count increased 1 day after hemostasis. AST and ALT increased 1 day after hemostasis, but it decreased 3 days later. The photocrosslinkable chitosan hydrogel and calcium alginate were biodegraded at 3 and 28 days, respectively, by neutrophils and keratinocyte chemoattractant.

Patterning alginate hydrogels using light-directed release of caged calcium in a microfluidic device.

This paper describes a simple reversible hydrogel patterning method for 3D cell culture. Alginate gel is formed in select regions of a microfluidic device through light-triggered release of caged calcium. In the pre-gelled alginate solution, calcium is chelated by DM-nitrophen (DM-n) to prevent cross-linking of alginate. After sufficient UV exposure the caged calcium is released from DM-n causing alginate to cross-link. The effect of using different concentrations of calcium and chelating agents as well as the duration of UV exposure is described. Since the cross-linking is based on calcium concentration, the cross-linked alginate can easily be dissolved by EDTA. We also demonstrate application of this capability to patterned microscale 3D co-culture using endothelial cells and osteoblastic cells in a microchannel.

Photochemical synthesis of biopolymer coated Aucore-Agshell type bimetallic nanoparticles.

Spherical Au nanoparticles have been prepared in the presence of a biopolymer, sodium alginate using UV-photoactivation technique. The particles are sodium alginate coated and are extremely stable. These Au nanoparticles have been used as seed for the synthesis of Aucore-Agshell type bimetallic nanoparticles. Sodium alginate is a carbohydrate-based biopolymer. In this synthesis it acts both as a reducing agent and a stabilizer for the evolved particles. Therefore, no extra capping agent is required from outside to make the generated particle stable. By varying the seed to silver ion ratios and using photoactivation technique Aucore-Agshell type bimetallic nanoparticles with various sizes and compositions have been synthesized. The method is very simple and reproducible and does not need any manipulative skill. Characterizations of these bimetallic nanoparticles have been done from their UV-visible spectroscopy, TEM/EDX, and AFM results. UV-visible extinction spectra reveal that the seed particles have an absorption maxima approximately 527 nm, attributed to the surface plasmon of the pure gold nanoparticles. From the TEM images the particle size of the gold seed particles was calculated to be 8.6 nm. The growth of bimetallic nanoparticles with time has been monitored. The finally evolved bimetallic Aucore-Agshell nanoparticles have a size in the range between approximately 10-14 nm. The particles are very stable and may have the potential for biological and catalytic applications.

Gamma-irradiation of lyophilised wound healing wafers.

Lyophilised wafers are being developed as drug delivery systems that can be applied directly to the surface of suppurating wounds. They are produced by the freeze-drying of polymer solutions and gels. This study investigates the possibility of sterilising these glassy, solid dosage forms with gamma-irradiation and determining the rheological properties of rehydrated wafers post-irradiation. One series of wafers was formulated using sodium alginate (SA) modified with increasing amounts of methylcellulose (MC), the other being composed of xanthan gum (XG) and MC. Batches were divided into three lots, two of which were exposed to 25 and 40 kGrays (kGy) of Cobalt-60 gamma-irradiation, respectively, the third being retained as a non-irradiated control. Apparent viscosities of solutions/gels resulting from the volumetric addition of distilled water to individual wafers were determined using continuous shear, flow-rheometry. Flow behaviour on proprietary suppurating surfaces was also determined. Large reductions in viscosity were apparent for irradiated SA samples while those of XG appeared to be largely unaffected. In addition, an increase in the yield stress of xanthan formulations was observed. Xanthan wafers appeared to withstand large doses of irradiation with no detrimental effect on the rheology of reconstituted gels. This offers the possibility of manufacturing sterilisable delivery systems for wounds.

Preparation of nanosized gold particles in a biopolymer using UV photoactivation.

Gold nanoparticles have been prepared by UV photoactivation in the presence of a biopolymer, sodium alginate. The particles are characterized by UV-vis spectra and TEM studies. Both particle size and the UV-visible absorption peak are dependent on the sodium alginate concentration. The effects of various other parameters such as change of light source, cell material of the reaction chamber, heating effect, irradiation time, and HAuCl4 concentration are studied. The particles are spherical and in situ stabilized by the biopolymer. The method is very simple and reproducible.

Aging and microwave effects on alginate/chitosan matrices.

The influence of microwave irradiation on the drug release properties of freshly prepared and aged alginate, alginate-chitosan and chitosan beads was investigated. The beads were prepared by extrusion method with sulphathiazole as a model drug. The dried beads were subjected to microwave irradiation at 80 W for 10 min, 20 min or three consecutive cycles of 10 and 20 min, respectively. The profiles of drug dissolution, drug content, drug stability, drug polymorphism, drug-polymer interaction, polymer crosslinkage and complexation were determined by dissolution testing, drug content assay, differential scanning calorimetry and Fourier transform infra-red spectroscopy. The chemical stability of drug embedded in beads was unaffected by microwave conditions and length of storage time. The release property of drug was mainly governed by the extent of polymer interaction in beads. The aged alginate beads required intermittent cycles of microwave irradiation to induce drug release retarding effect in contrast to their freshly prepared samples. Unlike the alginate beads, the level of polymer interaction was higher in aged alginate-chitosan beads than the corresponding fresh beads. The drug release retarding property of aged alginate-chitosan beads could be significantly enhanced through subjecting the beads to microwave irradiation for 10 min. No further change in drug release from these beads was observed beyond 30 min of microwave irradiation. Unlike beads containing alginate, the rate and extent of drug released from the aged chitosan beads were higher upon treatment by microwave in spite of the higher degree of polymer interaction shown by the latter on prolonged storage. The observation suggested that the response of polymer matrix to microwave irradiation in induction of drug release retarding property was largely affected by the molecular arrangement of the polymer chains.

Rheological characterization and turbidity of riboflavin-photosensitized changes in alginate/GDL systems.

Riboflavin (RF) in combination with light, in the wavelength range of 310-800 nm, is used to induce degradation of alginic acid gels. Light irradiation of alginate solutions in the presence of RF under aerobic conditions causes scission of the polymer chains. In the development process of a new drug delivery system, RF photosensitized degradation of alginic acid gels is studied by monitoring changes in the turbidity and rheological parameters of alginate/glucono-delta-lactone (GDL) systems with different concentrations of GDL. Addition of GDL induces gel formation of the samples by gradually lowering the pH-value of the system. The turbidity is measured and the cloud point determined. The turbidity starts to increase after shorter times with enhanced concentration of GDL. Enhanced viscoelasticity is detected with increasing GDL concentration in the post-gel regime, but small differences are detected at the gel point. The incipient gel is 'soft' and has an open structure independent on the GDL concentration. In the post-gel regime solid-like behavior is observed, this is more distinct for the systems with high GDL concentrations. The effect of photosensitized RF on alginate/GDL systems decreases with increasing amount of GDL in the system. The same trend is detected whether the systems are irradiated in the pre-gel or in the post-gel regime.

Biological effect of radiation-degraded alginate on flower plants in tissue culture.

Alginate with a weight-average molecular mass (Mw) of approx. 9.04 x 10(5) Da was irradiated at 10-200 kGy in 4% (w/v) aqueous solution. The degraded alginate product was used to study its effectiveness as a growth promoter for plants in tissue culture. Alginate irradiated at 75 kGy with an Mw of approx. 1.43 x 10(4) Da had the highest positive effect in the growth of flower plants, namely limonium, lisianthus and chrysanthemum. Treatment of plants with irradiated alginate at concentrations of 30-200 mg/l increased the shoot multiplication rate from 17.5 to 40.5% compared with control. In plantlet culture, 100 mg/l irradiated alginate supplementation enhanced shoot height (9.7-23.2%), root length (9.7-39.4%) and fresh biomass (8.1-19.4%) of chrysanthemum, lisianthus and limonium compared with that of the untreated control. The survival ratios of the transferred flower plantlets treated with irradiated alginate were almost the same as the control value under greenhouse conditions. However, better growth was attained for the treated plantlets.

Effect of gamma-irradiation on degradation of alginate.

The aqueous solution of alginate was irradiated by 60Co gamma-rays in the dose range of 10-500 kGy. To assess the effect of irradiation on the degradation of alginate, the irradiation-induced changes in the viscosity, molecular weight, color, monomer composition, and sequence were measured. The molecular weight of raw alginate was reduced from 300000 to 25000 when irradiated at 100 kGy. The degradation rate decreased and the chain breaks per molecule increased with increasing irradiation dose. The viscosity of irradiated alginate solution reached a near minimum as low as at 10 kGy. No appreciable color changes were observed in the samples irradiated at up to 100 kGy, but intense browning occurred beyond 200 kGy. The 13C NMR spectra showed that homopolymeric blocks, MM and GG, increased and the M/G ratio decreased with irradiation. Considering both the level of degradation and the color change of alginate, the optimum irradiation dose was found to be 100 kGy.