Kinetics and mechanisms of depolymerization of alginate and chitosan in aqueous solution

The kinetics and mechanisms of depolymerization of aqueous chitosan and alginate solutions at elevated temperatures have been investigated. Chitosan salts of different degree of acetylation (F_A), type of counterions (-glutamate, -chloride) and degree of purity were studied. One commercially available highly purified sodium alginate sample with high content of guluronic acid (G) was also studied. Furthermore, the influence of oxygen, H⁺ and OH⁻ ions on the initial depolymerization rates was investigated. Depolymerization kinetics was followed by measuring the time courses of the apparent viscosity and the intrinsic viscosity. The initial rate constants for depolymerization were determined from the intrinsic viscosity data converted to a quantity proportional to the fraction of bonds broken. The activation energies of the chitosan chloride and chitosan glutamate solutions with pH close to 5 and the same degree of acetylation, F_A = 0.14, were determined from the initial rate constants to be 76 ± 13 kJ/mol and 80 ± 11 kJ/mol, respectively.The results reported herein suggest that the stability of aqueous chitosan and alginate solutions at pH values 5–8 will be influenced by oxidative–reductive depolymerization (ORD) as the primary mechanism as long as transition metal ions are presented in the samples. Acid – and alkaline depolymerization will be the primary mechanisms for highly purified samples.     

Preparation of low molecular weight chitosan using solution plasma system

The solution plasma system was introduced to treat chitosan solution in order to prepare low molecular weight chitosan. The plasma treatment time was varied from 0 min to 300 min. The plasma-treated chitosan was characterized including viscosity, molecular weight by GPC, and chemical characteristics by FT-IR. The results showed that after treated with plasma for 15-60 min, the viscosity of chitosan solution and apparent molecular weight of chitosans were remarkably decreased, compared to those of untreated sample. Longer treatment time had less effect on both viscosity and molecular weight of samples. Eventually, long treatment time (≥180 min) showed no influence on both viscosity and apparent molecular weight. This suggested that the degradation process of chitosan occurred during plasma treatment. FT-IR analysis revealed that chemical structure of chitosan was not affected by solution plasma treatment. TOF-MS results showed that chitooligosaccharides with the degree of polymerization of 2-8 were also generated by solution plasma treatment. The results suggested that solution plasma system could be a potential method for the preparation of low molecular weight chitosan and chitooligosaccharides.     

Low molecular weight chitosans--preparation by depolymerization with Aspergillus niger pectinase,and characterization

The viscosity of a chitosan solution was rapidly lowered in the presence of pectinase from Aspergillus niger at pH 3.0 and 37 degrees C. The low molecular weight chitosans (LMWC) had a molecular weight in the range 20,000-5000 Da. Circular dichroism spectra showed a decrease in the segment of acetylated glucosamine units, whereas X-ray diffraction and CP-MAS 13C NMR indicated higher crystallinity and polymorphism in LMWC. The latter on thermal drying resulted in structural alterations, and yielded an insoluble product. FT-IR and X-ray diffraction showed no evidence of either Schiff's base linkage or any annealed polymorph. CP-MAS 13C NMR showed marked changes in the chain conformations of LMWC, which are believed to be responsible for its loss of solubility and functionality.     

Low molecular weight chitosan is an efficient inhibitor of ribonucleases

RNase inhibitors are commonly used to block the RNase activity in manipulations with RNA-containing preparations. Recently RNase inhibitors, either synthetic or natural, have been intensively sought because they appeared to be promising for therapy of cancer and allergy. However, there is only a limited number of efficient RNase inhibitors. We have shown that a low molecular weight chitosan (M(r) approximately 6 kDa) inhibits activity of pancreatic RNase A and some bacterial RNases with inhibition constants in the range of 30-220 nM at pH 7.0 and ionic strength 0.14 M. The preferential contribution to the chitosan complex formation with RNases is due to establishment of 5-6 ion pairs. The results of this work show that polycations may efficiently inhibit ribonuclease activities.     

Improved blood biocompatibility of composite film of chitosan/carbon nanotubes complex

Single-walled carbon nanotubes are novel molecular-scale wires having excellent anti-adhesion properties with regard to platelets. On the other hand, chitosan is a partially de-acetylated derivative of chitin that has a critical role in cell attachment and growth. The aim of this study was to investigate how carbon nanotubes improve the blood biocompatibility of chitosan film. We prepared composite films with various concentrations of chitosan/carbon nanotubes (CS/CNTs) (1.3–6.3 wt%). The sample surfaces were characterized by Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and contact angle measurements. The surface characterization revealed that the surface of the CS/CNTs composite film became more hydrophobic with increasing amounts of CNTs. Cell attachment tests using bovine aortic endothelial cells (BAECs) indicated that CS/CNTs composite films retained their cell adhesion ability. The blood compatibility of the CS/CNTs composite films was evaluated using the blood platelet adhesion and activation tests in vitro. Platelet adhesion results confirmed that platelet adhesion and the formation of a platelet network were inhibited on composite films with higher concentrations of CNTs (5.1 wt%). Our experimental results show that the novel composite film containing CS/CNTs possesses two paradoxical characteristics, namely, good adherence of endothelial cells and minimum adherence and activation of platelets, making this film a promising antithrombogenic material for use in the biomedical field.     

A comparative study on hypolipidemic activities of high and low molecular weight chitosan in rats

The hypolipidemic activities of high (712.6kDa) and low (39.8kDa) molecular weight chitosan (HMWC and LMWC) were evaluated in rats fed high-fat diets. Thirty-two male Sprague-Dawley rats in four groups were fed on three high-fat diets with each of them containing HMWC, LMWC or cellulose (high-fat control), and a control normal-fat diet for eight weeks. Compared with HMWC group, LMWC group showed decreased body weight gain, serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), as well as decreased liver triglyceride (TG). Fecal fat and cholesterol of LMWC group was lower than those of HMWC group. However, the activities of liver and serum lipoprotein lipase (LPL) of LMWC group were increased compared with HMWC group. The obtained results suggested that hypolipidemic activity of LMWC was better than HMWC, which might be partially attributed to the increase of serum and liver LPL activities.     

The impact of dilute sulfuric acid on the selectivity of xylooligomer depolymerization to monomers

The disappearance of xylose and xylooligosaccharides with degrees of polymerization (DP) ranging from 2 to 5 was followed at 160 degrees C with sulfuric acid added to adjust the pH from near neutral to 1.45, and the impact on the yields of lower DP xylooligomers and xylose monomer was determined. In addition, the experimental data for the disappearance of these xylooligomers was kinetically modeled assuming first-order reaction kinetics for xylose degradation and xylooligomer hydrolysis to evaluate how the pH affected the selectivity of monomer formation from xylooligomers and direct oligomer degradation to unknown products. The yield of xylose from xylooligomers increased appreciably with increasing acid concentration but decreased with increasing xylooligomer DP at a given acid concentration, resulting in more acid being required to realize the same xylose yields for higher DP species. For example, the maximum xylose yields were 49.6%, 28.0%, 13.2% and 3.2% for DP values of 2, 3, 4, and 5, respectively, at pH 4.75. Kinetic modeling revealed that all the xylooligomers disappeared at a higher rate compared to xylose monomer and the disappearance rate constant increased with DP at all pH. The kinetics for lower DP oligomers of 2 and 3 showed that these species directly degrade to unknown compounds in the absence of acid. On the other hand, higher oligomers of DP 4 and 5 exhibited negligible losses to degradation products at all pH. Therefore, only xylooligomers of DP 2 and 3 were found to directly degrade to undesired products in the absence of acid, but more work is needed to determine how higher DP species behave. This study also revealed that the source of water and the material used for the construction of the reactor impacted xylose degradation kinetics.     

Mechanisms involved in enhancement of osteoclast formation and function by low molecular weight hyaluronic acid

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.     

Plausible molecular and crystal structures of chitosan/HI type II salt

Chitosan/HI type II salt prepared from crab tendon was investigated by X-ray fiber diffraction. Two polymer chains and 16 iodide ions (I(-)) crystallized in a tetragonal unit cell with lattice parameters of a = b = 10.68(3), c (fiber axis) = 40.77(13) A, and a space group P4(1). Chitosan forms a fourfold helix with a 40.77 A fiber period having a disaccharide as the helical asymmetric unit. One of the O-3... O-5 intramolecular hydrogen bonds at the glycosidic linkage is weakened by interacting with iodide ions, which seems to cause the polymer to take the 4/1-helical symmetry rather than the extended 2/1-helix. The plausible orientations of two O-6 atoms in the helical asymmetric unit were found to be gt and gg. Two chains are running through at the corner and the center of the unit cell along the c-axis. They are linked by hydrogen bonds between N-21 and O-61 atoms. Two out of four independent iodide ions are packed between the corner chains while the other two are packed between the corner and center chains when viewing through the ab-plane. The crystal structure of the salt is stabilized by hydrogen bonds between these iodide ions and N-21, N-22, O-32, O-61, O-62 of the polymer chains.     

Effect of deacetylation time on the preparation, properties and swelling behavior of chitosan films

The objectives of the study were to propose a two-stage microfluidization combined with an ultrafiltration (UF) treatment for chitosan mass production and the manipulation of molecular weight and its distribution. The proposed methods are based on the degradation rate and rate constant of various process variables studied. Results obtained were that the rate constants were faster during the earlier reaction period, were higher for those operating at a higher pressure, were better for using concurrent UF treatment to remove small degraded fragments, and the degradation rate constants were faster for 30 °C solutions than that for 50 or 0 °C. A two-stage microfluidization process is proposed. The first stage constitutes of the highest possible concentration solution with concurrent UF treatment at 50 °C, and recycled 5 times. The second stage consists of the highest possible concentration of solutions with concurrent UF treatment at 30 °C, and recycled 5 times.     

Anticoagulant and antithrombotic effects of low molecular weight heparin

Low molecular weight heparin (Mr 8 kDa) was prepared from conventional heparin (Mr 18 kDa) by the chromatography on DEAE-sephadex with the recovery of 56%. Low molecular weight heparin had less affinity to antithrombin III than unfractionated heparin and had less anticoagulant and anti-IIa activities. The anti-Xa activity of low molecular weight heparin exceed by 17% the activity of conventional heparin. In the experiments on rats it was determined that the biological half-life of low molecular weight heparin exceed two-fold that of the unfractionated heparin. In the modified model of the arteriovenous shunt thrombosis in normal and nephrotic syndrome rats it was shown that the low molecular weight heparin was the most efficient antithrombotic remedy in normal and decreased level of antithrombin III in the organism.     

Synthesis of methylated chitosan containing aromatic moieties: Chemoselectivity and effect on molecular weight

N-Arylated chitosans were synthesized via Schiff bases formed by the reaction between the primary amino group of chitosan with aromatic aldehydes followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Treatment of chitosan containing N,N-dimethylaminobenzyl and N-pyridylmethyl substituents with iodomethane under basic conditions led to quaternized N-(4-N,N-dimethylaminobenzyl) chitosan and quaternized N-(4-pyridylmethyl) chitosan. Methylation occurred at either N,N-dimethylaminobenzyl and N-pyridylmethyl groups before the residual primary amino groups of chitosan GlcN units were substituted. The total degree of quaternization of each chitosan varied depending on the extent of N-substitution (ES) and the sodium hydroxide concentration used in methylation. Increasing ES increased the total degree of quaternization but reduced attack at the GlcN units. N,N-dimethylation and N-methylation at the primary amino group of chitosan decreased at higher ES’s. Higher total degrees of quaternization and degrees of O-methylation resulted when higher concentrations of sodium hydroxide were used. The molecular weight of chitosan before and after methylation was determined by gel permeation chromatography under mild acidic condition. The methylation of the N,N-dimethylaminobenzyl derivative with iodomethane was accompanied by numerous backbone cleavages and a concomitant reduction in the molecular weight of the methylated product was observed. The antibacterial activity of water-soluble methylated chitosan derivatives was determined using Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria; minimum inhibitory concentrations (MIC) of these derivatives ranged from 32 to 128 μg/mL. The presence of the N,N-dimethylaminobenzyl and N-pyridylmethyl substituents on chitosan backbone after methylation did not enhance the antibacterial activity against S. aureus. However, N-(4-N,N-dimethylaminobenzyl) chitosan with degree of quaternization at the aromatic substituent and the primary amino group of chitosan of 17% and 16–30%, respectively, exhibited a slightly increased antibacterial activity against E. coli.     

Kinetics and efficiency of chitosan reacetylation

Chitosan reacetylation kinetics and efficiency were studied in water-methanol (MeOH) mixtures. The polymer was dissolved using acetic acid and acetic anhydride was used for reacetylation. Combining second-order kinetics and acid-base dissociation equations of chitosan, and using acetic anhydride hydrolysis rates determined by conductivity measurements, reacetylation reaction rate constants of 187, 108, 46 min⁻¹ M⁻¹ were found in 0, 50 and 80% MeOH (v/v), respectively. Contrary to previous literature, it was found that improvement in reacetylation efficiency in the presence of MeOH is mainly due to an increase of acetic acid pK_a by MeOH that limits the ionization of the polymer in the course of the reaction rather than to a decreased acetic anhydride hydrolysis rate, as previously thought. Based on these insights, the model developed in this study was able to predict the significantly reduced efficiency of the reaction for a large extent of reacetylation, without requiring any steric hindrance from the acetyl group. Conditions to maximize the reaction efficiency for a large extent of reacetylation were identified.     

N-Deacetylation and depolymerization reactions of chitin/chitosan: Influence of the source of chitin

The deacetylation and depolymerization reactions of chitin/chitosan from three crustacean species (Paralomis granulosa, Lithodes antarcticus and Palinurus vulgaris) were evaluated under the same conditions. The average molecular weight and the mole fraction of N-acetylated units were the parameters studied in the resulting chitosans. During the N-deacetylation process P. granulosa, L. antarcticus and P. vulgaris follow a pseudo-first order kinetics and their apparent rate constants are very similar. However, the degradation rate of chitosan in the first 45 min of this process is higher for P. vulgaris. The depolymerization process follows a pseudo-first order kinetics for the three species, but in the first 9 min P. vulgaris shows a slightly lower depolymerization rate. Hence, depending on the ash contents, crystallinity and the physicochemical characteristics of chitin from these sources, the obtained chitosans show different qualities.     

The effect of low molecular weight chitosan on bone resorption in vitro and in vivo.

We studied the effect of low molecular weight chitosan (LMWC) on the formation of osteoclast-like multinucleated cells (OCLs) in the co-culture of mouse osteoblastic cells and bone marrow cells in the presence of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. LMWC at 440 microg/ml inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive OCLs induced by 1alpha,25(OH)2D3. We prepared OCLs in the co-culture of osteoblastic cells and bone marrow cells. The effect of LMWC on pit formation by OCLs was examined using dentin slices, and LMWC inhibited pit formation at 440 microg/ml. Oral administration of the LMWC to ovariectomized rats prevented a decrease in bone mineral density (BMD) of the lumbar vertebra without affecting the body and uterus weights. These results suggested that LMWC prevented a decrease in BMD in vivo by inhibiting osteoclastic bone resorption.     

High molecular weight chitosan and sodium alginate effect on secretory acid proteinase of Candida albicans

The effect of high molecular weight chitosan (HMWCh) and sodium alginate (NaAL) on acid proteinase secretion of Candida albicans (one of culture collection and five isolates) was evaluated. The secretion of acid proteinase was induced in the presence and the absence of these polymers in different concentrations and their enzymatic activity was determined. HMWCh and NaAL significantly diminished the enzymatic activity (>76% for the collection strains and > 89% for the isolates, p < 0.05). HMWCh did not modify protein concentrations, but NaAL did. It can be concluded that both polymers can inhibit the proteinase activity of Candida albicans.     

Hydrodynamic and molecular characteristics of polyelectrolyte complexes between sodium dextransulfate and chitosan hydrochloride

Hydrodynamic and molecular characteristics of particles of polyelectrolyte complexes (PEC) between sodium dextransulfate and chitosan hydrochloride were studied by various physicochemical methods (high-rate sedimentation, viscosimetry, turbidimetry, and diffusion). As was shown, the complex formation is accompanied by increase in the average sizes with simultaneous changes in the shape of the particles of the investigated PEC. According to the proposed polycomplexes model, side by side aggregation of the taken macromolecules could cause disordering of adjacent helical parts of polyanion-matrix that facilitates the formation of sphere-like polycomplexes particles.