Characterization of Different Salt Forms of Chitooligosaccharides and Their Effects on Nitric Oxide Secretion by Macrophages.

In this paper, chitooligosaccharides in different salt forms, such as chitooligosaccharide lactate, citrate, adipate, etc., were prepared by the microwave method. They were characterized by SEM, FTIR, NMR, etc., and the nitric oxide (NO) expression was determined in RAW 264.7 cells. The results showed that pure chitooligosaccharide was an irregular spherical shape with rough surface, and its different salt type products are amorphous solid with different honeycomb sizes. In addition to the characteristic absorption peaks of chitooligosaccharides, in FTIR, the characteristic absorption of carboxyl group, methylene group, and aromatic group in corresponding acid appeared. The characteristic absorption peaks of carbon in carboxyl group, hydrogen and carbon in methyl, methylene group, and aromatic group in corresponding acid also appeared in NMR. Therefore, the sugar ring structure and linking mode of chitooligosaccharides did not change after salt formation of chitooligosaccharides. Different salt chitooligosaccharides are completely different in promoting NO secretion by macrophages, and pure chitooligosaccharides are the best.

Extraction, characterization and comparison of chitins from large bodied four Coleoptera and Orthoptera species.

Chitins were extracted from large insect species of order Coleoptera (Lucanus cervus (Linnaeus, 1758) (Lucanidae) and Polyphylla fullo (Linnaeus, 1758) (Scarabaeidae) and order Orthoptera (Bradyporus (Callimenus) sureyai Ünal, 2011) (Tettigonidae) and Gryllotalpa gryllotalpa (Linnaeus, 1758) (Gryllotalpidae)) for the first time. Fourier Transform Infrared Spectrometry (FT-IR) confirms that isolation of chitin is successful. Yields of chitins on dry basis from P. fullo, L. cervus, G. gryllotalpa and B. (C.) sureyai are 11.3%, 10.9%, 10.1% and 9.8% respectively. Thermogravimetric Analysis (TGA) showed a variety of thermal stability of chitin samples from 614 °C to 748 °C with a small percent of ash. X-ray diffraction (XRD) data showed a crystallinity index percent from 80.6% to 85.2%. Scanning Electron Microscope (SEM) was examined for surface characterization determining as fibrous and porous for all species and changes from nm scales to µm scales. Elemental analysis has been applied to determine the elemental composition of chitin and nitrogen percent was relatively low for all specimens than expected. It is detected that examined insects have α-chitin form from XRD and FT-IR data. If these species can be grown in the laboratory, adults of them could be accepted as promising alternative chitin sources without negative effects on biodiversity.

Fabrication of magnetic and fluorescent chitin and dibutyrylchitin sub-micron particles by oil-in-water emulsification.

Chitin is a carbohydrate polymer with unique pharmacological and immunological properties, however, because of its unwieldy chemistry, the synthesis of discreet sized sub-micron particles has not been well reported. This work describes a facile and flexible method to fabricate biocompatible chitin and dibutyrylchitin sub-micron particles. This technique is based on an oil-in-water emulsification/evaporation method and involves the hydrophobization of chitin by the addition of labile butyryl groups onto chitin, disrupting intermolecular hydrogen bonds and enabling solubility in the organic solvent used as the oil phase during fabrication. The subsequent removal of butyryl groups post-fabrication through alkaline saponification regenerates native chitin while keeping particles morphology intact. Examples of encapsulation of hydrophobic dyes and nanocrystals are demonstrated, specifically using iron oxide nanocrystals and coumarin 6. The prepared particles had diameters between 300-400nm for dibutyrylchitin and 500-600nm for chitin and were highly cytocompatible. Moreover, they were able to encapsulate high amounts of iron oxide nanocrystals and were able to label mammalian cells. STATEMENT OF SIGNIFICANCE: We describe a technique to prepare sub-micron particles of highly acetylated chitin (>90%) and dibutyrylchitin and demonstrate their utility as carriers for imaging. Chitin is a polysaccharide capable of stimulating the immune system, a property that depends on the acetamide groups, but its insolubility limits its use. No method for sub-micron particle preparation with highly acetylated chitins have been published. The only approach for the preparation of sub-micron particles uses low acetylation chitins. Dibutyrylchitin, a soluble chitin derivative, was used to prepare particles by oil in water emulsification. Butyryl groups were then removed, forming chitin particles. These particles could be suitable for encapsulation of hydrophobic payloads for drug delivery and cell imaging, as well as, adjuvants for vaccines.

The effect of chitin size, shape, source and purification method on immune recognition.

The animal immune response to chitin is not well understood and needs to be investigated further. However, this is a challenging topic to study because of the technical difficulties in purifying chitin, and because this material usually comes associated with contaminating components that can activate the immune system. In this study, improvements to previously described purification protocols were investigated for chitin obtained from different sources, including commercial shellfish, Candida albicans yeast and hyphal cell walls, as well as cell walls of the filamentous fungi Aspergillus fumigatus and Mucor circinelloides. The immune response to these different chitin preparations was tested using human peripheral blood mononuclear cells. In agreement with previous literature, small chitin particles of an average size of 0.2 µm were not immunogenic. On the other hand, bigger chitin particles induced in some cases a pro-inflammatory response. The results of this work suggest that not only the purity and size of the chitin particles, but also their shape can influence immune recognition.

Elucidating the biosynthesis of chitin filaments and their configuration with specific proteins and electron microscopy.

To deepen the knowledge of chitin synthesis, a yeast mutant has been used as a model. Purified chitin synthase I-containing vesicles (chitosomes) with a diameter of 85 to 120 nm are identified by electron microscopy to eject tiny fibers upon addition of UDP-N-acetylglucosamine. The filigree of extruded filaments fused gradually into a large three-dimensional network, which is degradable by a chitinase. The network is targeted and restructured by the Streptomyces chitin-binding protein CHB1, which has a very high affinity only for alpha-chitin. Within the chitosomes, filaments are found to be highly condensed within consecutive oval fibroids, which are specifically targeted by the alpha-chitin-binding protein. The presented data give new insights to the generation of chitin filaments with an antiparallel (alpha) configuration. [image: see text]

Controlled release of Bordetella bronchiseptica dermonecrotoxin (BBD) vaccine from BBD-loaded chitosan microspheres in vitro.

Chitosan microspheres were prepared by ionic gelation process with sodium sulfate for nasal vaccine delivery. Bordetella Bronchiseptica Dermonecrotoxin (BBD) as a major virulence factor of a causative agent of atrophic rhinitis (AR) was loaded to the chitosan microspheres for vaccination. Morphology of BBD-loaded chitosan microspheres was observed as spherical shapes. The average particle sizes of the BBD-loaded chitosan microspheres were about 2.69 microm. More BBD was released with an increase of molecular weight of chitosan and with an increase of medium pH in vitro due to weaker intermolecular interaction between chitosan and BBD. Tumor necrosis factor-alpha (TNFalpha) and nitric oxide (NO) from RAW264.7 cells stimulated with BBD-loaded chitosan microspheres were gradually secreted, suggesting that released BBD from chitosan microspheres had immune stimulating activity of AR vaccine.

Studies of male sexual tubes in hermit crabs (crustacea, decapoda, anomura, paguroidea). I. Morphology of the sexual tube in Micropagurus acantholepis (Stimpson, 1858), with comments on function and evolution.

The external morphology and internal structure of the male sexual tube of the hermit crab Micropagurus acantholepis, a member of the family Paguridae from Australian waters, is described in detail using histological thick sectioning and scanning and transmission electron microscopy techniques. This is the first in-depth study of a sexual tube in the Paguroidea, a group where a remarkable number of genera (55.9% in the family Paguridae) with species having these intriguing sexual structures are known. In M. acantholepis a sexual tube is present on the left side, whereas only a gonopore is present on the right side. The tube is used for the delivery of spermatophores to the female and consists of a sheath of cuticular origin surrounding an internal, functional extension of the posterior vas deferens. Pedunculate spermatophores were observed within the lumen and partially extruding from the terminal opening of the tube in preserved specimens. The tube protrudes from the left coxa of the fifth pereopod as an elongate 3-mm-long, hollow, coiled structure with a terminal opening. Exteriorly the tube consists of a conspicuous thick chitinous cuticular ridge throughout its length, and a thin chitinous cuticle with sparse, regularly arranged simple setae. Interior to the cuticle, the tube contains loose connective tissue, secretory cells, oblique muscle, circular muscle, and epithelial cells. The latter cells line a central lumen that runs the length of the sexual tube. The morphology, cellular composition, and function of the tube are discussed.

Accelerating enzymatic hydrolysis of chitin by microwave pretreatment.

Response surface analysis was used to determine optimum conditions [2% (w/v) chitin, 57.5 degrees C, 38 min] for microwave irradiation of chitin to improve its enzymatic hydrolysis. V(max)/K(m) of cabbage chitinase toward untreated and microwave-irradiated chitin was found to be 21.1 and 31.7 nmol h(-1) mg(-2) mL, respectively. Similar improvement was observed in the case of pectinase in its unusual catalytic activity of chitin degradation. It was found that a greater extent of chitin hydrolysis by chitinase was possible after the substrate chitin was irradiated with microwaves.

Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering.

Porous scaffolds for skin tissue engineering were fabricated by freeze-drying the mixture of collagen and chitosan solutions. Glutaraldehyde (GA) was used to treat the scaffolds to improve their biostability. Confocal laser scanning microscopy observation confirmed the even distribution of these two constituent materials in the scaffold. The GA concentrations have a slight effect on the cross-section morphology and the swelling ratios of the cross-linked scaffolds. The collagenase digestion test proved that the presence of chitosan can obviously improve the biostability of the collagen/chitosan scaffold under the GA treatment, where chitosan might function as a cross-linking bridge. A detail investigation found that a steady increase of the biostability of the collagen/chitosan scaffold was achieved when GA concentration was lower than 0.1%, then was less influenced at a still higher GA concentration up to 0.25%. In vitro culture of human dermal fibroblasts proved that the GA-treated scaffold could retain the original good cytocompatibility of collagen to effectively accelerate cell infiltration and proliferation. In vivo animal tests further revealed that the scaffold could sufficiently support and accelerate the fibroblasts infiltration from the surrounding tissue. Immunohistochemistry analysis of the scaffold embedded for 28 days indicated that the biodegradation of the 0.25% GA-treated scaffold is a long-term process. All these results suggest that collagen/chitosan scaffold cross-linked by GA is a potential candidate for dermal equivalent with enhanced biostability and good biocompatibility.

Growth factor releasing porous poly (epsilon-caprolactone)-chitosan matrices for enhanced bone regenerative therapy.

Drug releasing porous poly(epsilon-caprolactone) (PCL)-chitosan matrices were fabricated for bone regenerative therapy. Porous matrices made of biodegradable polymers have been playing a crucial role as bone substitutes and as tissue-engineered scaffolds in bone regenerative therapy. The matrices provided mechanical support for the developing tissue and enhanced tissue formation by releasing active agent in controlled manner. Chitosan was employed to enhance hydrophilicity and biocompatibility of the PCL matrices. PDGF-BB was incorporated into PCL-chitosan matrices to induce enhanced bone regeneration efficacy. PCL-chitosan matrices retained a porous structure with a 100-200 microm pore diameter that was suitable for cellular migration and osteoid ingrowth. NaHCO3 as a porogen was incorporated 5% ratio to polymer weight to form highly porous scaffolds. PDGF-BB was released from PCL-chitosan matrices maintaining therapeutic concentration for 4 week. High osteoblasts attachment level and proliferation was observed from PCL-chitosan matrices. Scanning electron microscopic examination indicated that cultured osteoblasts showed round form and spread pseudopods after 1 day and showed broad cytoplasmic extension after 14 days. PCL-chitosan matrices promoted bone regeneration and PDGF-BB loaded matrices obtained enhanced bone formation in rat calvarial defect. These results suggested that the PDGF-BB releasing PCL-chitosan porous matrices may be potentially used as tissue engineering scaffolds or bone substitutes with high bone regenerative efficacy.

Design and evaluation of a mucoadhesive therapeutic agent delivery system for postoperative chemotherapy in superficial bladder cancer.

The most common treatment method is known as the transurethral resection (TUR) for the therapy of bladder cancer. Unfortunately, because of the recurrency of the tumoral tissues after TUR the chemotherapy or immunotherapy should be performed. In these kind of therapies the pharmacotherapeutics are infused intravesically into the bladder after TUR periodically (i.e. upto 6--36 weeks, each week). But these therapies are having very big problems (i.e. disturbancy to patients, adjustment of the suitable dosage, loss of active agents without using etc.). An alternative approach can be proposed as to design a pharmacotherapeutic agent delivery system, which will supply the suitable dosage of the agent for a certain time period to solve those problems. In this study; the pharmacotherapeutic agent (i.e. Mytomycin-C) delivery system was prepared by using a mucoadhesive polymer (i.e. chitosan) in the form of cylindirical geometry to facilitate the insertion of the carrier for in vivo studies. The chitosan carriers were prepared by cross-linking during the solvent evaporation technique. In the preparation of the chitosan carriers the chitosan polymers with different molecular weights, different amounts of cross-linker (i.e. glutaraldehyde) and different amounts of pharmacotherapeutic agent were used to obtain desired attachment onto the bladder wall and optimum release rate of the agent. On the other hand because of the gelous structure of the chitosan, the swelling behaviour of the polymer was evaluated by gravimetric determinations in aqueous media periodically.

Mg2+, Mn2+, and Co2+ stimulate Entamoeba histolytica to produce chitin-like material.

The mechanism of Entamoeba histolytica cyst cell wall synthesis is not well understood. Previous research has shown that cyst-like structures formed in the presence of chitin synthase cofactors (Mg2+, Mn2+, and Co2+) resist 1% sodium dodecyl sulfate lysis (RCLS), whereas those formed in the absence of cofactors (CLS) do not, and trophozoites are immediately destroyed. This suggests that E. histolytica is able to synthesize chitin, initiating a differentiation process under axenic conditions. To test this hypothesis, polysaccharide hydrolysates from E. histolytica trophozoites, CLS, or RCLS were analyzed with high-performance liquid chromatography. The major components found in all 3 preparations were N-acetylglucosamine (NAG) and glucose (GLC), with RCLS possessing 129 and 180 times more NAG and 2.4 and 2.0 more GLC than trophozoites and CLS, respectively. After 36 hr of incubation with chitinase (16 U/ml) in a hypotonic medium (50 mOsm/kg), 68% of RCLS was lysed, and 100% lost affinity for calcofluor white M2R. The RCLS polysaccharides bound wheat germ agglutinin and appeared as long and thin or short and thick fibers. Accordingly, Mg2+, Mn2+, and Co2+ stimulated E. histolytica to synthesize a chitin-like material.

Effect of a composite membrane of chitosan and poloxamer gel on postoperative adhesive interactions.

Excessive postoperative adhesion formation is a major result of surgery. The adhesion reduction effects of a chitosan membrane and poloxamer gel barrier were measured in a rat peritoneal model. Forty-four male Sprague-Dawley rats were divided into four groups (control, poloxamer, chitosan, and poloxamer+chitosan sandwich). Two cm2 of cecal serosa and the adjacent abdominal wall were abraded. The denuded cecum was covered with either a chitosan membrane, a poloxamer gel, chitosan in a sandwich configuration with poloxamer on both sides, or neither (control group) and apposed to the abdominal wall. Fourteen days after surgery adhesions were graded using a whole-number scoring system of zero to five. Adhesion strength was determined using a whole-number system of one to four. Adhesion area was measured on a continuous scale of adhesion severity. Adhesion grades were highest in the control group (5.00 +/- 0.00) and were significantly (P < 0.05) lower in the poloxamer group (3.50 +/- 1.35), the chitosan group (1.64 +/- 1.63), and the poloxamer+chitosan group (1.18 +/- 1.25). The two chitosan-containing groups also had significantly (P < 0.05) reduced adhesion grades in comparison with the poloxamer group. Adhesion area in both chitosan-containing groups was reduced in comparison with control and adhesion strength was reduced significantly (P < 0.05) in all groups compared with control. The poloxamer+chitosan group had significantly (P < 0.05) reduced adhesion strength versus poloxamer only. There was a significant (P < 0.05) linear correlation (r = 0.931, P < 0.001) between adhesion grade and adhesion strength. We conclude that chitosan and the combination of poloxamer+chitosan were shown to effectively reduce adhesion area, grade, and strength.

In vitro expression of cartilage-specific markers by chondrocytes on a biocompatible hydrogel: implications for engineering cartilage tissue.

Natural cartilage tissue has a limited self-regenerative capacity; thus, strategies to replenish the lost cartilage are desired in reconstructive and plastic surgery. Tissue-engineered cartilage using biodegradable polymeric scaffolds is one such approach gaining wide attention. We have earlier demonstrated the biocompatible nature and ability of chitosan-gelatin hydrogel to maintain differentiated populations of respiratory epithelial cells. The aim of the present study was to evaluate its suitability as a substratum for inducing chondrocyte growth and differentiation. Electron microscopic (SEM) analysis of freeze-dried hydrogels showed a highly porous morphology with interconnections as seen in cross section. Chondrocytes were observed to attach and exhibited a differentiated phenotype with proper cell-cell contact on three-dimensional freeze-dried hydrogels. When cultured on two-dimensional hydrogel films they showed higher growth rates (4-6%) compared with a polystyrene (TCPS) control until 6 days (p > 0.05), which slowed down after 10 days. Immunofluorescent microscopic studies revealed that chondrocytes on hydrogel films exhibited comparable expression of beta1 integrin (CD29) to TCPS controls, indicating the ability of the hydrogel substrate to maintain normal expression of beta1 integrin. RT-PCR analysis of chondrocytes grown on hydrogel films showed that chondrocytes express the mRNA for extracellular matrix proteins such as collagen type IIalpha1 (COL IIalpha1), COL III, COL IXalpha3. Expression of COL I was less prominent than COL II as indication of differentiation. Expression of COL X could not be detected, suggesting an absence of chondrocyte hypertrophy. Chondrocytes also showed weak mRNA expression of aggrecan, a cartilage-specific proteoglycan. All of these results point out the ability of the chitosan-gelatin hydrogel to induce the expression of mRNAs for cartilage-specific extracellular matrix proteins by nasal septal chondrocytes. This hydrogel needs to be further evaluated for its ability to support chondrocyte-specific marker expression to explore the possibility of forming a tissue resembling natural cartilage in vitro.

Novel beta-emitting poly(ethylene terephthalate) surface modification.

Restenosis after percutaneous interventions in coronary and peripheral arteries leads to repeat procedures and surgery in a significant number of patients. We have previously demonstrated that irradiation of an arterial site using an endovascular source (brachytherapy) is highly effective in preventing the restenotic process. To this end, a novel beta radiation delivery system was developed, based on the adsorption of (32)P (o-phosphoric acid) by pH-sensitive chitosan hydrogel on a poly(ethylene terephthalate) (PET) balloon surface. The PET balloon surface was treated with oxygen plasma and coated with chitosan hydrogel. Covalent bonds, ionic bonds, and hydrogen bonds all contribute to the adhesion between chitosan hydrogel and PET. In the aqueous phosphoric acid (PA) solution, the -NH(2) groups of chitosan were protonated by PA and the adsorption of PA occurred at the same time. The effect of PA concentration and temperature on adsorption efficiency and kinetics were studied. More than 70% PA was adsorbed on the sample surface in 0.2 mM PA solution. The surface of samples was also investigated by attenuated total reflection-Fourier transform infrared spectroscopy and scanning electron microscopy. PET surface may be modified to carry high activity beta emitters; such materials may be useful in a therapeutic setting

5-fluorouracil loaded chitosan microspheres for chemoembolization.

In this study, chitosan microspheres were prepared by a suspension cross-linking technique. A petroleum ether/mineral oil mixture was used as the suspension medium which includes an emulsifier, e.g. Tween-80. Glutaraldehyde was used as the cross-linker. 5-Fluorouracil was incorporated in the matrix for the possible use of the microspheres in chemoembolization. The size and size distribution of the chitosan microspheres varied in the size range of 100-200 microns, by changing the emulsifier concentration, stirring rate, chitosan/solvent ratio and drug/chitosan solution ratio. In summary, the size and size distribution of the microspheres decreased when the emulsifier concentration and stirring rate were increased. Smaller microspheres with narrower size distributions were obtained when the chitosan/solvent ratio and drug/chitosan ratio were lower. It was possible to load the chitosan microspheres with 5-FU to a concentration of 10.4 mg 5-FU/g chitosan. Around 60% of the loaded drug was released within the first 24 h, then the release rate became much slower.

Formulation and biological activity of antineoplastic proteoglycans derived from Mycobacterium vaccae in chitosan nanoparticles.

Although heat-killed suspensions of Mycobacterium vaccae have been tested clinically against tuberculosis and cancer, from a pharmaceutical perspective it would be advantageous to utilize isolated active components rather than the heat-degraded bacterial materials. In our laboratory we have isolated from M. vaccae a number of high-molecular-weight proteoglycans with considerable immunological and antineoplastic activity. The structure of one of these, PS4A, obtained by extraction with boiling water, seems to consist of a basic unit with a 20-kDa protein core to which are attached glucans and O-methylated 4-kDa polysaccharides. The molecular weight is (approx.) 50 kDa, but because of self-association, that of the recovered high-molecular-weight fraction is greater than 150 kDa. A similar, but even larger, molecule (PS4alpha, MW approximately 20 MDa) is obtained by cold extraction with 8 M urea. Both are active in-vivo against an S-180 murine sarcoma model but have no activity in-vitro, suggesting an antitumour effect involving activated macrophages. For this reason gelatin nanoparticles are unsuitable as a vehicle but chitosan seemed to be a promising alternative. In this report we describe the production of stable 600-700-nm diameter nanoparticles of chitosan without organic solvents. Adsorption and release of bovine serum albumin seemed to be affected by the charge of the two reactants and at high doses not all adsorbate was released. PS4A, because of structural and compositional differences, had to be loaded on to the chitosan by freeze drying a suspension of the nanoparticles in a solution of the drug. After a rapid (burst) release phase, the rate of release into water was steady for the next 4 h, but not all the drug was released. In-vivo it was evident that PS4A and PS4alpha were equally active in solution or when formulated in the chitosan nanoparticles. These results show that chitosan nanoparticles, readily prepared without the use of organic solvents, are a suitable vehicle for the delivery of these immunostimulants from M. vaccae; the formulations might find application as antitumour agents.

Perspectives of molecular and cellular electron tomography.

After a general introduction to three-dimensional electron microscopy and particularly to electron tomography (ET), the perspectives of applying ET to native (frozen-hydrated) cellular structures are discussed. In ET, a set of 2-D images of an object is recorded at different viewing directions and is then used for calculating a 3-D image. ET at a resolution of 2-5 nm would allow the 3-D organization of structural cellular components to be studied and would provide important information about spatial relationships and interactions. The question of whether it is a realistic long-term goal to visualize or--by sophisticated pattern recognition methods--identify macromolecules in cells frozen in toto or in frozen sections of cells is addressed. Because of the radiation sensitivity of biological specimens, a prerequisite of application of ET is the automation of the imaging process. Technical aspects of automated ET as realized in Martinsried and experiences are presented, and limitations of the technique are identified, both theoretically and experimentally. Possible improvements of instrumentation to overcome at least part of the limitations are discussed in some detail. Those means include increasing the accelerating voltage into the intermediate voltage range (300 to 500 kV), energy filtering, the use of a field emission gun, and a liquid-helium-cooled specimen stage. Two additional sections deal with ET of isolated macromolecules and of macromolecular structures in situ, and one section is devoted to possible methods for the detection of structures in volume data.