壳聚糖基角膜细胞载体的制备及其细胞相容性

为探讨羟丙基壳聚糖基共混膜作为组织工程技术中角膜细胞培养载体的可行性, 分别制备了羟丙基壳聚糖/硫酸软骨素、羟丙基壳聚糖/明胶/硫酸软骨素以及羟丙基壳聚糖/氧化透明质酸/硫酸软骨素三种共混膜。测定其透光率、含水量和蛋白吸附性能; 在共混膜上培养兔角膜上皮细胞, 通过观察角膜上皮细胞在不同载体膜上的生长状态、贴附情况, 测定细胞活性以及上清液中乳酸脱氢酶的活性, 研究三种壳聚糖基载体膜片与角膜上皮细胞的相容性。膜片理化性质测定结果表明三种共混膜片具有良好的透明度, 适宜的含水量和较强的蛋白吸附性能; 细胞相容性实验结果表明羟丙基壳聚糖/明胶/硫酸软骨素共混膜对细胞的损伤最小, 有利于细胞在膜上的贴附和生长, 表现出良好的细胞相容性, 有望作为角膜细胞载体体外构建组织工程化角膜。     

Preparation and characterization on mechanical and antibacterial properties of chitsoan/cellulose blends

Polysaccharides-based membranes of chitosan and cellulose blends were prepared using trifluoroacetic acid as a co-solvent. Morphology and mechanical property of prepared membranes were studied by Instron and dynamic mechanical thermal analysis. The mechanical and dynamic mechanical thermal properties of the cellulose/chitosan blends appear to be dominated by cellulose, suggests that cellulose/chitosan blends were not well miscible. It is believed that the intermolecular hydrogen bonding of cellulose is supposed to be break down to form cellulose–chitosan hydrogen bonding; however, the intra-molecular and intra-strand hydrogen bonds hold the network flat. The reduced water vapor transpiration rate through the chitosan/cellulose membranes indicates that the membranes used as a wound dressing may prevent wound from excessive dehydration. The chitosan/cellulose blend membranes demonstrate effective antimicrobial capability against Escherichia coli and Staphylococcus aureus, as examined by the antimicrobial test. These results indicate that the chitosan/cellulose blend membranes may be suitable to be used as a wound dressing with antibacterial properties.     

Single crystals of chitosan

Lamellar single crystals of chitosan were prepared at 125 degrees C by adding ammonia to a low DP fraction of chitosan dissolved in water. The crystals gave sharp electron diffraction diagrams which could be indexed in an orthorhombic P2(1)2(1)2(1) unit cell with a = 8.07 A, b = 8.44 A, c = 10.34 A. The unit cell contained two anti-parallel chitosan chains and no water molecules. It was found that cellulose microfibrils from Valonia ventricosa could act as nuclei for inducing the crystallization of chitosan on cellulose. This produced a shish-kebab morphology.     

Cross-linking chitosan into UV-irradiated cellulose fibers for the preparation of antimicrobial-finished textiles

Chitosan cross-linked cellulose fibers were prepared using non-toxic procedures in order to confer antimicrobial properties to cellulose fibers. Citric acid was used as the cross-linker and NaH₂PO₄ as catalyst in previously UV-irradiated cellulose fibers. Further heat dried-cure process and washing with detergent, water and acetic acid (0.1 M) gave a maximum incorporation of chitosan of 27 mg per gram of functionalized textile. The thermogravimetric analysis of the material with the highest chitosan content showed an increased thermal stability compared to cellulose and chitosan. The UV-irradiation induced morphological changes, such as less entangled cellulose fibers, as observed by scanning electron microscopy, which was prompted to enhance the chitosan incorporation. The biomass and spore germination percentage of Penicillium chrysogenum and colony forming units per millilitre for Escherichia coli decreased significantly on the composed materials as compared to raw cellulose fiber and it was similar to that obtained with a commercial antimicrobial cellulose fiber.     

Biologically active fibers based on chitosan-coated lyocell fibers

The possibilities of obtaining biologically active cellulose–chitosan fibers were examined. An effective two-stage method was developed. The first stage involves the formation of dialdehyde cellulose by the potassium periodate oxidation of lyocell fibers, which is able to form Schiff’s base with chitosan. In the second stage, chitosan-coated lyocell fibers were prepared by subsequent treatment of oxidized lyocell fibers with a solution of chitosan in aqueous acetic acid. The impact of this two-stage protocol on the chemical and physical properties of lyocell fibers was evaluated by determining carbonyl group content, fineness and tensile strength of fibers, as well as chitosan content in the composite cellulose–chitosan fibers. Antibacterial activity of the chitosan-coated lyocell fibers against different pathogenens: Staphylococcus aureus and Escherichia coli, was confirmed in vitro experiments.     

Formation and properties of chitosan-cellulose nanocrystal polyelectrolyte-macroion complexes for drug delivery applications

This study examines a novel polyelectrolyte-macroion complex (PMC) between chitosan, a cationic polysaccharide, and cellulose nanocrystals (CNCs), anionic, cylindrical nanoparticles, for potential applications in drug delivery. CNCs were prepared by H(2)SO(4) hydrolysis of wood pulp. The formation of PMCs was monitored by turbidimetric titration. In titrations of a chitosan solution with a CNC suspension, the turbidity reached a plateau, but it had a maximum and then decreased when the direction of titration was reversed. PMC particles were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, dynamic light scattering, and laser Doppler electrophoresis. The particles were composed primarily of CNCs and ranged in size from a few hundred nanometers to several micrometers, depending on the cellulose/chitosan ratio. Particles formed at amino/sulfate group molar ratios >1 were nearly spherical in shape and positively charged, whereas particles formed at ratios <1 had well-defined nonspherical shapes and were negatively charged.     

Effect of sulfate groups from sulfuric acid hydrolysis on the thermal degradation behavior of bacterial cellulose

When used as fillers in polymer composites, the thermostability of cellulose crystals is important. Sulfate groups, introduced during hydrolysis with sulfuric acid, are suspected to diminish the thermostability. To elucidate the relationship between the hydrolysis conditions, the number of sulfate groups introduced, and the thermal degradation behavior of cellulose crystals, bacterial cellulose was hydrolyzed with sulfuric acid under different hydrolysis conditions. The number of sulfate groups in the crystals was determined by potentiometric titration. The thermal degradation behavior was investigated by thermogravimetric analysis. The sulfate group content increased with acid concentration, acid-to-cellulose ratio, and hydrolysis time. Even at low levels, the sulfate groups caused a significant decrease in degradation temperatures and an increase in char fraction confirming that the sulfate groups act as flame retardants. Profile analysis of the derivative thermogravimetric curves indicated thermal separation of the degradation reactions by the sulfate groups into low- and high-temperature processes. The Broido method was used to determine activation energies for the degradation processes. The activation energies were lower at larger amounts of sulfate groups suggesting a catalytic effect on the degradation reactions. For high thermostability in the crystals, low acid concentrations, small acid-to-cellulose ratios, and short hydrolysis times should be used.     

Polysaccharides with sulfate groups are human T cell mitogens and murine polyclonal B cell activators (PBAs) II. Cellulose sulfate and dextran sulfate with two different lower molecular weights

In our previous paper, we reported that various types of carrageenan, dextran sulfate and fucoidan, which are sulfated homopolysaccharides with high molecular weights, were human T cell mitogens and murine polyclonal B cell activators (PBAs) and that heparin, a sulfated heteropolysaccharide, was a very weak human mitogen and mouse PBA. Here we used cellulose sulfate (Mr 7-9 X 10(3], dextran sulfate with two different low molecular weights (Mr 5 X 10(3) and 8 X 10(3], two different condroitin sulfates (Mr 3.5 X 10(4], polyvinyl sulfate and polygalacturonic acid to investigate mitogenic activities of polysaccharides in detail. The following results were obtained. Low-molecular-weight sulfated homopolysaccharides, dextran sulfate and cellulose sulfate, were very weak or not human T cell mitogens. However, they were better murine PBAs. Sulfated heteropolysaccharides, chondroitin 4-sulfate and chondroitin 6-sulfate, hardly induced mitogenic changes in human T cells and mouse B cells, even though the molecular weight of these substances was more than 1 X 10(4). There were no other polymers examined so far which activated both human T cells and murine B cells. The relationship among molecular size, sulfate groups and lymphocyte activation is discussed in detail.     

Biomolecular Structure and Composition Changes in the Root System of Long-Lipped Serapias (Serapias vomeracea) After In Vitro Chitosan and Plant Growth Regulator Treatments

In the current study, effects of chitosan oligomers (CHI-OM) with different polymerization degrees (DP) between 2 and 15, and polymeric chitosan (CHI-P) with a DP of 70 were compared with kinetin (KIN), 6-benzylaminopurine (BAP), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and jasmonic acid (JAS) on the structure and composition of several biomolecules in the root system of Serapias vomeracea. Evaluation of molecular alterations in the root system of S. vomeracea through an infrared spectroscopic approach provided insight into the differentiation between action mechanisms of chitosan and commonly used plant growth regulators. The results revealed that CHI-P and JAS treatments at low concentrations might enhance the lignin content in the cell walls. Also, JAS, CHI-OM, and CHI-P treatments enhanced cell wall lignification. The water-associated cellulose content of the cell walls was affected by the DP of chitosan. Membrane lipid stabilization and protein content were enhanced after CHI-P, JAS, and KIN treatments, while BAP and CHI-OM treatments triggered lipid, protein, and cell wall polysaccharide synthesis. In particular, CHI-OM treatment also enhanced rhamnogalacturonan and β-galactan content as well as xyloglucans and glucomannans, while the auxin treatments had a similar impact only on glucomannan content. These findings suggest that chitosan may show either purine-based cytokinin-like or lipid-based jasmonic acid-like activity in the root system of S. vomeracea. Additionally, depending on its degree of polymerization, the effects of chitosan may vary on root system development in S. vomeracea.

     

Effect of dietary chitosans with different viscosity on plasma lipids and lipid peroxidation in rats fed on a diet enriched with cholesterol

To investigate the effect of dietary chitosan on lipid metabolism, male SD (Sprague-Dawley) rats were fed a cholesterol-enriched diet containing 5% cellulose (CE), 5% chitosan (CCS; high viscosity), or 5% chitosan (FCS; low viscosity) for 4 weeks. The two types of chitosan with a comparable degree of deacetylation had a different molecular weight and intrinsic viscosity. Significantly (p < 0.05) lower plasma total cholesterol, LDL-cholesterol and VLDL-cholesterol concentrations were observed in the rats fed on the chitosan diets. In addition, chitosan significantly increased the fecal cholesterol and triglyceride contents. Although no significant difference in body weight was found among the dietary groups, the rats fed on the chitosan diets had lower relative liver weight when compared with those fed on the cellulose diet. Both of the chitosan groups had significantly lower liver total lipid and total cholesterol contents compared to the cellulose group, although the FCS group was less effective. The plasma and liver thiobarbituric acid reactive substances (TBAR) values were similar in the CE and FCS groups, while the CCS group had increased liver TBAR values. Although a significant increase in liver glucose-6-phosphate dehydrogenase activity was observed in the CCS group, no significant change was found in the FCS group. The observed influence of chitosans with different viscosity on the plasma lipid level, liver lipids and lipid peroxidation suggests that, while the hypocholesterolemic action of chitosans with different viscosity was similar, changes in the liver lipids and liver peroxidation status depended on their molecular weight when the deacetylation degree was comparable.     

区域选择性改性的纤维素和壳聚糖衍生物及其血液相容性

通过对纤维素和壳聚糖的区域选择性改性,将内皮细胞表面硫酸乙酰肝素（ES—HS）分子结构中对其血液相容性有重要影响的官能团引入纤维素和壳聚糖的分子结构中,并将其通过离子键固定在部分阳离子化的纤维素膜上,以期模拟ES—HS的血液相容性。血小板吸附结果表明,6位改性的纤维素衍生物的吸附程度较高。在五种壳聚糖衍生物中,2位的NS03／NAc为6／4的衍生物表现出最低的血小板吸附。当保持壳聚糖2位的NS03／NAc值不变时,对6位进行完全磺酸酯化,也可有效减少血小板的吸附。     

Polymer coated liposomes for use in the oral cavity – a study of the in vitro toxicity,effect on cell permeability and interaction with mucin

In this study we investigated the in vitro toxicity, impact on cell permeability and mucoadhesive potential of polymer-coated liposomes intended for use in the oral cavity. A TR146 cell line was used as a model. The overall aim was to end up with a selection of safe polymer coated liposomes with promising mucoadhesive properties for drug delivery to the oral cavity. The following polymers were tested: chitosan, low-methoxylated pectin (LM-pectin), high-methoxylated pectin (HM-pectin), amidated pectin (AM-pectin), Eudragit, poly(N-isopropylacrylamide-co-methacrylic acid) (p(NIPAAM-co-MAA)), hydrophobically modified hydroxyethyl cellulose (HM-HEC), and hydrophobically modified ethyl hydroxyethyl cellulose (HM-EHEC). With chitosan as an exception, all the systems exhibited no significant effect on cell viability and permeability at the considered concentrations. Additionally, all the formulations showed to a varying degree an interaction with mucin (BSM type I-S); the positively charged formulations exhibited the strongest interaction, while the negatively and neutrally charged formulations displayed a moderate or low interaction. The ability to interact with mucin makes all the liposomal formulations promising for oromucosal administration. Although the chitosan-coated liposomes affected the cell viability, this formulation also influenced the cell permeability, which makes it an interesting candidate for systemic drug delivery from the oral cavity.     

Three D structures of chitosan

Crystal structures of two polymorphs of chitosan, tendon (hydrated) and annealed (anhydrous) polymorphs, have been reported. In both crystals, chitosan molecule takes up similar conformation (Type I form) to each other, an extended two-fold helix stabilized by intramolecular O3-O5 hydrogen bond, which is also similar to the conformation of chitin or cellulose. Three chitosan conformations other than Type I form have been found in the crystals of chitosan-acid salts. In the salts with acetic and some other acids, called Type II salts, chitosan molecule takes up a relaxed two-fold helix composed of asymmetric unit of tetrasaccharide. This conformation seems to be unstable because no strong intramolecular hydrogen bond like Type I form. Type II crystal changes to the annealed polymorph of chitosan by a spontaneous water-removing action of the acid. Chitosan molecule in its hydrogen iodide salt prepared at low temperature takes a 4/1 helix with asymmetric unit of disaccharide. The fourth chitosan conformation was found to be a 5/3 helix in chitosan salts with medical organic acids having phenyl group such as salicylic or gentisic acids. Similar conformation of chitosan molecule in the aspirin (acetylsalicylic acid) salt was suggested by a solid-sate NMR measurement.     

Modification of cellulose films by adsorption of CMC and chitosan for controlled attachment of biomolecules

The adsorption of human immunoglobulin G (hIgG) and bovine serum albumin (BSA) on cellulose supports were investigated. The dynamics and extent of related adsorption processes were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Amine groups were installed on the cellulose substrate by adsorption of chitosan from aqueous solution, which allowed for hIgG to physisorb from acid media and produced a functionalized substrate with high surface density (10 mg/m(2)). hIgG adsorption from neutral and alkaline conditions was found to yield lower adsorbed amounts. The installation of the carboxyl groups on cellulose substrate via carboxymethylated cellulose (CMC) adsorption from aqueous solution enhanced the physisorption of hIgG at acidic (adsorbed amount of 5.6 mg/m(2)) and neutral conditions. hIgG adsorption from alkaline conditions reduced the surface density. BSA was used to examine protein attachment on cellulose after modification with chitosan or carboxymethyl cellulose. At the isoelectric point of BSA (pI 5), both of the surface modifications enhanced the adsorption of this protein when compared to that on unmodified cellulose (a 2-fold increase from 1.7 to 3.5 mg/m(2)). At pH 4, the electrostatic interactions favored the adsorption of BSA on the CMC-modified cellulose, revealing the affinity of the system and the possibility of tailoring biomolecule binding by choice of the surface modifier and pH of the medium.     

Biodegradation of high molecular weight lignin under sulfate reducing conditions: lignin degradability and degradation by-products

This study is designed to investigate the biodegradation of high molecular weight (HMW) lignin under sulfate reducing conditions. With a continuously mesophilic operated reactor in the presence of co-substrates of cellulose, the changes in HMW lignin concentration and chemical structure were analyzed. The acid precipitable polymeric lignin (APPL) and lignin monomers, which are known as degradation by-products, were isolated and detected. The results showed that HMW lignin decreased and showed a maximum degradation capacity of 3.49 mg/l/day. APPL was confirmed as a polymeric degradation by-product and was accumulated in accordance with HMW lignin reduction. We also observed non-linear accumulation of aromatic lignin monomers such as hydrocinnamic acid. Through our experimental results, it was determined that HMW lignin, when provided with a co-substrate of cellulose, is biodegraded through production of APPL and aromatic monomers under anaerobic sulfate reducing conditions with a co-substrate of cellulose.     

N,N,N-三甲基壳聚糖甲基硫酸盐的合成及理化性质的测定

壳聚糖与甲醛、甲酸反应得到N,N-二甲基壳聚糖,然后以硫酸二甲酯为季铵化试剂反应得到N,N,N-三甲基壳聚糖甲基硫酸盐(TMCMS),用IR1、H NMR和元素分析对其结构进行了表征。元素分析结果表明其季铵化度为74.6%,差示扫描量热法和热重分析法结果表明其热稳定性比壳聚糖差,但其水溶性明显优于壳聚糖,25℃时在水中的溶解度可达20 mg/mL,浓度为2 mg/mL时在pH 3～12范围内无沉淀产生。     

Extraction and precipitation of chitosan from cell wall of zygomycetes fungi by dilute sulfuric acid

A new method was developed in this work for extraction of chitosan from the zygomycetes cell wall. It is based on the temperature-dependent solubility of chitosan in dilute sulfuric acid. Chitin is soluble in neither cold nor hot dilute sulfuric acid. Similarly chitosan is not soluble at room temperature but is dissolved in 1% H 2SO 4 at 121 degrees C within 20 min. The new method was developed to measure the chitosan content of the biomass and cell wall. The procedures were investigated by measuring phosphate, protein, ash, glucuronic acid, and degree of acetylation. The cell wall derivatives of fungus Rhizomucor pusillus were then examined by this new method. The results indicated 8% of the biomass as chitosan. After treatment with NaOH, the alkali-insoluble material (AIM) contained 45.3% chitosan. Treatment of AIM with acetic acid resulted in 16.5% acetic-acid-soluble material (AcSM) and 79.0% alkali- and acid-insoluble material (AAIM). AcSM is usually cited as pure chitosan, but the new method shows major impurities by, for example, phosphate. Furthermore, AAIM is usually considered to be the chitosan-free fraction, whereas the new method shows more than 76% of the chitosan present in AIM is found in AAIM. It might indicate the inability of acetic acid to separate chitosan from the cell wall.     

Anticoagulant activity of a sulfated chitosan

Chitin prepared from the shells of rice-field crabs (Somanniathelphusa dugasti) was converted into chitosan with a degree of acetylation of 0.21 and then sulfated with chlorosulfonic acid in N,N-dimethylformamide under semi-heterogeneous conditions to give 87% of water-soluble sulfated chitosan with degree of substitution (d.s) of 2.13. 1H NMR revealed the sulfate substitution at C-2, C-3 and C-6. Gel filtration on Sepharose CL-6B of the sulfated chitosan gave three fractions with average molecular weights of 7.1, 3.5, and 1.9 x 10(4). The three sulfated chitosan preparations showed strong anticoagulant activities, with the same mechanism of action observed for standard therapeutic heparin.     

Tyramine-O-sulfate, in addition to tyrosine-O-sulfate, is produced and secreted by HepG2 human hepatoma cells, but not by 3Y1 rat embryo fibroblasts

The spent media of HepG2 human hepatoma cells and 3Y1 rat embryo fibroblasts labeled with [35S]sulfate, upon ultrafiltration, were analyzed by a two-dimensional thin-layer separation procedure. Autoradiographs of the cellulose thin-layer plate revealed the presence of tyramine-O-[35S]sulfate in addition to tyrosine-O-[35S]sulfate in spent medium from human hepatoma cells. In contrast, only tyrosine-O-[35S]sulfate was observed in spent medium of 3Y1 rat fibroblasts. Using adenosine, 3'-phosphate, 5'-phospho[35S]sulfate as the sulfate donor, sulfotransferase(s) present in HepG2 cell homogenate catalyzed the sulfation of tyramine to tyramine-O-[35S]sulfate, but not the sulfation of tyrosine to tyrosine-O-[35S]sulfate. Endogenous aromatic amino acid decarboxylase present in HepG2 homogenate was shown to catalyze the decarboxylation of [3H]tyrosine to form [3H]tyramine while attempts to use it for the decarboxylation of tyrosine-O-sulfate to form tyramine-O-sulfate were unsuccessful. These results suggest that tyramine-O-sulfate may be derived from the de novo sulfation of tyramine, instead of the decarboxylation of tyrosine-O-sulfate.     

Formation of biocompatible nanoparticles by self-assembly of enzymatic hydrolysates of chitosan and carboxymethyl cellulose

A simple preparation method for biocompatible nanoparticles in high concentration (0.5 wt %) by self-assembly of chitosan and carboxymethyl cellulose hydrolysates was developed. Chitosan and carboxymethyl cellulose were hydrolyzed beforehand with chitosanase and cellulase respectively to make fragments having lower molecular weights. Nanoparticles were spontaneously formed only by mixing the two hydrolysate solutions. The particle size distribution was relatively narrow, about 200 nm in mean size. The mean particle size decreased from 226 nm to 165 nm with decreasing molecular weight of chitosan hydrolysate from 9.5 to 6.8 kDa. The mixing ratio of chitosan and carboxymethyl cellulose hydrolysates also affected particle size. Changes in particle size are discussed in relation to a possible mechanism of polyionic complexation. The chitosan-carboxymethyl cellulose nanoparticles were stably suspended over 1 week even under low pH (pH 3.0), high ionic strength (NaCl 1 M), or low temperature (4 degrees C) conditions.