Crystalline behavior of chitosan organic acid salts.

The crystal structures of chitosan acid salts were studied by X-ray diffraction measurements on a fiber diagram and a new procedure to obtain an anhydrous polymorph of chitosan was found. The salts prepared by immersing a chitosan into a mixture of acid solution and isopropanol were classified into two types (Types I and II) depending on their conformation. Molecular conformation of the Type I salt retains the extended 2-fold helical structure of the original chitosan, but that of Type II salt is a twisted 2-fold helix. All the Type II salts changed to the anhydrous "Annealed" polymorph of chitosan when soaking in 75% aqueous isopropanol, but when the Type I salts were immersed in the solution, they returned to the hydrated "Tendon" polymorph which is that of the original chitosan. The strange transformation observed in Type II salt may be related to the stability of the molecular conformation of chitosan in the salt.     

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.     

Conformational Behavior of Chitosan in the Acetate Salt: An X-Ray Study

A well-defined X-ray fiber pattern of chitosan acetate was obtained by immersing a tendon chitosan, prepared from a crab tendon chitin by a solid-state N-deacetylation, in an aqueous acetic acid-isopropanol solution at 110°C. This pattern was very similar to that of chitosan salts with some inorganic acids, such as HF, HCl, and H₂SO₄, in which chitosan chains form an 8/5 helix, indicating that chitosan acetate also take up this conformation. This information may give an influential clue to the chitosan conformation in the aqueous acetic acid solution, the most popular solvent for chitosan. However, after one month of storage of the chitosan acetate, the fiber pattern, the density and its IR spectrum changed to those of the anhydrous polymorph of chitosan, suggesting that the acetic acid was removed accompanied with water molecules from the crystal during storage and that the polymorph can be obtained not only by annealing chitosan, but also through the chitosan acetate.     

Dependence on the Preparation Procedure of the Polymorphism and Crystallinity of Chitosan Membranes

Differences in the polymorphism and crystallinity of chitosan were found in membranes prepared by different procedures when examined by X-ray diffraction measurements for four samples of chitosan differing in the degree of polymerization. When an acetic acid solution of chitosan was dried in air and then soaked in an alkaline solution (method A), both hydrated and anhydrous polymorphs of chitosan were present in the resulting membranes; the latter polymorph made chitosan insoluble in common solvents of chitosan, and its crystallinity increased with decreasing chitosan molecular weight. When a highly concentrated chitosan solution in aqueous acetic acid was neutralized with an alkaline solution (method B), no anhydrous polymorphs were detected in the membrane because of incomplete drying. When aqueous formic acid was used as the solvent, behavior basically similar to that in aqueous acetic acid was observed. In contrast, even with method A, aqueous hydrochloric acid gave a chitosan membrane having very little anhydrous crystallinity. The crystalline polymorph called “1–2”, which has been proposed to be one of four chitosan polymorphs, is considered to be a mixture of hydrated and anhydrous crystals.     

Fourth 3D structure of the chitosan molecule: conformation of chitosan in its salts with medical organic acids having a phenyl group

Chitosan salts with two medical organic acids having phenyl groups (salicylic and gentisic acids) exhibited fiber diffraction patterns of a new type of crystal which does not compare with known types I and II. The crystals, called type III salts, showed a fiber repeat of 2.550 nm and a meridional reflection at the 5th layer line. These results coupled with a conformational analysis indicate the chain conformation of chitosan with the salts to be a 5/3 helix, this helix differing from those of type I (an extended two-fold helix) and type II (a relaxed two-fold helix or a 4/1 helix). The fiber patterns of all the type III salts were similar. This observation has also been found with type II salts and is an indication that the acid ions are not arranged in regular positions in the crystals. A comparison of solid-state 13C-NMR spectra of the gentisic acid salt and the aspirin salt, which could not be crystallized, suggests that, in the latter salt, the chitosan molecules also formed a 5/3 helix.     

Two different molecular conformations found in chitosan type II salts

The type II structure of chitosan acidic salts prepared from crab tendon in solid state was studied using an X-ray fiber diffraction technique together with the linked-atom least-squares (LALS) technique. The cylindrical Patterson method was applied to confirm the molecular conformation of the chitosan. It was shown that there are two different helical conformations for type II salts. One is the relaxed twofold helix having a tetrasaccharide as an asymmetric unit as found in chitosan.HCl salt, which was previously reported as a conformation of chitosan.HCOOH salt. The other is the fourfold helix having a disaccharide as an asymmetric unit newly found in chitosan.HI salt.     

X‐ray crystal structure of anhydrous chitosan at atomic resolution

We determined the crystal structure of anhydrous chitosan at atomic resolution, using X‐ray fiber diffraction data extending to 1.17 Å resolution. The unit cell [a = 8.129(7) Å, b = 8.347(6) Å, c = 10.311(7) Å, space group P2₁2₁2₁] of anhydrous chitosan contains two chains having one glucosamine residue in the asymmetric unit with the primary hydroxyl group in the gt conformation, that could be directly located in the Fourier omit map. The molecular arrangement of chitosan is very similar to the corner chains of cellulose II implying similar intermolecular hydrogen bonding between O6 and the amine nitrogen atom, and an intramolecular bifurcated hydrogen bond from O3 to O5 and O6. In addition to the classical hydrogen bonds, all the aliphatic hydrogens were involved in one or two weak hydrogen bonds, mostly helping to stabilize cohesion between antiparallel chains. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 361–368, 2016.     

The solution behavior of poly-L-proline: I. Light scattering studies in water and concentrated aqueous neutral salt solutions

The solution bebavior of poly-L -proline Form II has been studied in water and aqueous salt solutions by both elastic and quasi-elastic light -scattering techniques. The results of this study suggest that polyproline Form II can exist in water at 24 °C as an associated polymer complex and that certain salts which do not appear to affect the helix integrity, e.g., guanidinium-HCl, resutl in dissociation of the aggregate. Other neutral salts, of the variety effective in mediating unfolding of the Form II helix (e.g., 4M NaClO₄) also induce aggregate dissociation, but 4M CaCl₂ results in enhanced aggregation of polyproline. Kinetic experiments indicate that a time of 20 hours is necessary for the completion of the “large” to “small” transformation (at 22°C) which is induced by the addition of 4M NaClO₄. Thus it appears that neutral salts additives in aqueous solutions of polyproline influence both the state of aggregation and the conformation of this polymer.     

Poly(N-isopropylacrylamide) surface-grafted chitosan membranes as a new substrate for cell sheet engineering and manipulation

The immobilization of poly(N-isopropylacrylamide) (PNIPAAm) on chitosan membranes was performed in order to render membranes with thermo-responsive surface properties. The aim was to create membranes suitable for cell culture and in which confluent cell sheets can be recovered by simply lowering the temperature. The chitosan membranes were immersed in a solution of the monomer that was polymerized via radical initiation. The composition of the polymerization reaction solvent, which was a mixture of a chitosan non-solvent (isopropanol) and a solvent (water), provided a tight control over the chitosan membranes swelling capability. The different swelling ratio, obtained at different solvent composition of the reaction mixture, drives simultaneously the monomer solubility and diffusion into the polymeric matrix, the polymerization reaction rate, as well as the eventual chain transfer to the side substituents of the pyranosyl groups of chitosan. A combined analysis of the modified membranes chemistry by proton nuclear magnetic resonance ((1)H-NMR), Fourier transform spectroscopy with attenuated total reflection (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) showed that it was possible to control the chitosan modification yield and depth in the solvent composition range between 75% and 100% of isopropanol. Plasma treatment was also applied to the original chitosan membranes in order to improve cell adhesion and proliferation. Chitosan membranes, which had been previously subjected to oxygen plasma treatment, were then modified by means of the previously described methodology. A human fetal lung fibroblast cell line was cultured until confluence on the plasma-treated thermo-responsive chitosan membranes and cell sheets were harvested lowering the temperature.     

Oral gene delivery: design of polymeric carrier systems shielding toward intestinal enzymatic attack

The gastrointestinal tract poses a variety of morphological and physiological barriers to the expression of target genes. The aim of this study was to evaluate the stability of cationic polymer/pDNA nanoparticles toward salts and enzymes of the intestinal fluid. Within this study, a chitosan-enzyme inhibitor conjugate has been generated and characterized. Based on this conjugate, nanoparticles with pDNA were generated to enhance transfection rate in oral gene delivery. The enzyme inhibitor aurintricarboxylic acid (ATA) was covalently bound to chitosan to improve the enzymatic stability of nanoparticles formed with this polymer and pDNA. Chitosan-ATA/pDNA nanoparticles showed a size of 98.5 +/- 26 nm and a zeta potential of -13.26 +/- 0.24 mV (n = 3-4). Stability studies with salt solution, lysozyme, DNase, and freshly collected porcine intestinal fluid showed that chitosan-ATA/pDNA nanoparticles are significantly (p < 0.05) more stable than unmodified chitosan/pDNA nanoparticles. Apart from improved stability, chitosan-ATA/pDNA nanoparticles showed a 2.6-fold higher transfection rate than chitosan/pDNA nanoparticles in the Caco-2 cell line, thus creating a promising carrier for orally administered therapeutic genes.     

Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation

The effect of inorganic salts such as sodium chloride on the hydrolysis of chitosan in a microwave field was investigated. While it is known that microwave heating is a convenient way to obtain a wide range of products of different molecular weights only by changing the reaction time and/or the radiation power, the addition of some inorganic salts was shown to effectively accelerate the degradation of chitosan under microwave irradiation. The molecular weight of the degraded chitosan obtained by microwave irradiation was considerably lower than that obtained by traditional heating. Moreover, the molecular weight of degraded chitosan obtained by microwave irradiation assisted under the conditions of added salt was considerably lower than that obtained by microwave irradiation without added salt. Furthermore, the effect of ionic strength of the added salts was not linked with the change of molecular weight. FTIR spectral analyses demonstrated that a significantly shorter time was required to obtain a satisfactory molecular weight by the microwave irradiation-assisted inorganic salt method than by microwave irradiation without inorganic salts and conventional technology.     

Spinning of hydroalcoholic chitosan solutions

We investigated the spinning of hydroalcoholic chitosan solutions. The dope composition was optimized in order to obtain a continuous alcogel fiber by water evaporation on heating the extruded hydroalcoholic solution. This alcogel fiber was then neutralized in aqueous alkali baths and washed in water to eliminate the residual alcohol and salts before final drying. Depending on the alcohol content in the filament at the neutralization step, on specific alcohol–chitosan interactions and on the nature and concentration of the coagulation base, the process yielded semicrystalline chitosan fibers with different proportions of anhydrous and hydrated allomorphs. Contrarily to the classical annealing method, the formation of mainly anhydrous crystals was obtained without significant molecular weight decrease by neutralizing the polymer in hydrophobic conditions. The control of allomorph content was shown to be related to the hydrophobicity of the solvent (alcohol fraction) at the neutralization step.     

Crystallization behavior of glucomannan

Five different glucomannan samples were recrystallized from dilute solution. Depending on the experimental conditions, the crystals obtained could be identified as corresponding to the mannan I (anhydrous precipitate of more or less regular lozenge-shaped crystals) or mannan II (hydrated gel-forming pseudo-fibrillar precipitate). High-molecular-weight material, low temperature of crystallization, or a polar crystallization medium favored the mannan II polymorph, whereas a low-molecular weight, a high temperature of crystallization, and a crystallization medium of low polarity yielded the mannan I polymorph. Since the base-plane unit-cell dimensions are fairly constant with respect to variation of glucose, it is likely that isomorphous replacement of mannose by glucose occurs in glucomannan crystallization; the data also indicate that perfection of the glucomannan crystals was reduced in specimens having a high glucose:mannose ratio. The oriented crystallization of glucomannan on cellulose microfibrils was also studied under conditions where the mannan I polymorph was obtained. This gave shish-kebab structures that were characterized.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence: II. Localization of type I and type II collagen during long bone development

The transition of type I and type II collagens during cartilage and bone development in the chick embryo was studied by immunofluorescence using antibodies against type I or type II collagens. Type II collagen was found in all cartilaginous structures which showed metachromatic staining. Type I collagen appeared in the perichondrium of the tibia at stage 28 and was also found in osteoid, periosteal and enchondral bone after decalcification, periosteum, and tendons, ligaments, and capsules.Using the immunohistological method it was possible to identify specific collagen types in areas undergoing rapid proliferation and collagen transition, such as diaphyseal and epiphyseal perichondrium, or in enchondral osteogenesis. During enchondral ossification type I collagen is deposited onto the eroded surface of cartilage. It partially diffuses into the cartilage matrix forming a “hybrid” collagen matrix with type II collagen, which is a site for subsequent ossification. During appositional growth of diaphyseal cartilage and differentiation of epiphyseal perichondrium into articular cartilage, perichondral cells switch from type I to type II collagen synthesis when differentiating into chondroblasts. In the transition zones, chondroblasts are imbedded in a “hybrid” matrix consisting of a mixture of type I and type II collagens.     

Effects of preparation method on reversible conformational changes induced by neutral salts in spin-labeled erythrocyte membranes

Addition of “chaotropic” neutral salts to spin-labeled erythrocyte membranes produced changes in membrane conformation proportional to the known activities of the salts: KSCN > CaCl₂ ≥ KNO₃ ? NaCl. The effects had a threshold of about 0.10 m and increased through 0.60 m for all salts. Above 0.6 m other changes due to dielectric effects and/or protein loss began.The chaotropic effects were reversible on removal of the chaotropic ion, for human (or bovine) membranes prepared with a buffer ionic strength of 0.02. For membranes prepared with a buffer ionic strength of 0.005 the changes were irreversible. Exposure to salt concentration greater than 1 m also produced irreversible changes, probably due to solubilization. The results appear to show that the water-soluble membrane proteins are essential to maintain the “native” membrane conformation.     

Spirometra species from Asia: Genetic diversity and taxonomic challenges

Despite considerable controversy concerning the taxonomy of species within the genus Spirometra, human sparganosis and spirometrosis mainly in Asia and Europe has long been confidently ascribed to Spirometra erinaceieuropaei. Recently, the mitochondrial genomes of purported “S. erinaceieuropaei”, “Spirometra decipiens” and “Spirometra ranarum” from Asia have been determined. However, it has been pointed out that the morphological criteria used for identifying these species are unsuitable and thus these identifications are questionable. In the present study, therefore, Spirometra samples from Asia were re-examined based on mitochondrial cytochrome c oxidase subunit 1 gene sequences and the identification of these species was discussed. Haplotype network and phylogenetic analyses revealed that: i) two distinct Spirometra species, Type I and Type II, are present in Asia and neither of which is close to likely European “S. erinaceieuropaei”; ii) Type I is genetically diverse and widely distributed, however Type II is known so far from Japan and Korea; iii) “S. decipiens” and “S. ranarum” reported from Asia are conspecific with Type I; iv) Type I is probably conspecific with Spirometra mansoni, and Type II may represent an undescribed species.     

Alkaline chitosan solutions

Rigid and transparent hydrogels were obtained upon pouring chitosan salt solutions into saturated ammonium hydrogen carbonate. Incubation at 20 degrees C for 5 days yielded chitosan carbamate ammonium salt, Chit-NHCO(2)(-)NH(4)(+) a chemical species that either by hydrolysis or by thermal treatment decomposed to restore chitosan in free amine form. Chitosans of different degrees of acetylation, molecular sizes and origins (squid and crustaceans) were used as hydrochloride, acetate, glycolate, citrate and lactate salts. Their hydrogels obtained in ammonium hydrogen carbonate yielded chitosan solutions at pH values as high as 9.6, from which microspheres of regenerated chitosans were obtained upon spray-drying. These materials had a modest degree of crystallinity depending on the partial acylation that took place at the sprayer temperature (168 degrees C). Citrate could cross-link chitosan and impart insolubility to the microspheres. Chloride on the contrary permitted to prepare microspheres of chitosan in free amine form. By the NH(4)HCO(3) treatment, the cationicity of chitosan could be reversibly masked in view of mixing chitosan with alginate in equimolar ratio without coacervation. The clear and poorly viscous solutions of mixed chitosan carbamate and alginate were spray-dried at 115 degrees C to manufacture chitosan-alginate microspheres having prevailing diameter approx 2 micron.     

Photoresponsive polypeptides: Photochromism and conformation of poly (L-glutamic acid) containing spiropyran units

Spirobenzopyran units were bound to the side chains of poly (L -glutamic acid) and partially methylated poly(L -glutamate)s. The modified polymers were found to exhibit “reverse photochromism” in hexafluoro-2-propanol (HFP), so the samples kept in the dark were characterized by an intense absorption band in the visible range of the spectrum, which was completely erased upon exposure to sunlight or irradiation at 500–550 nm. The CD spectra showed that the macromolecules adopted a random coil conformation in the dark, whereas the bleached solutions after exposure to light displayed the typical CD pattern of the α-helix. The back reaction in the dark was accompanied by the progressive decrease of the helix content and recovery of the original disordered conformation. The photoinduced conformational changes resulted in large and reversible viscosity variations. When spiropyran side chains were converted to “spiropyran salts” of trifluoroacetic acid, the system was still photochromic, but the macromolecules were disordered both in the dark and light conditions. However, when appropriate amounts of methanol were added as a cosolvent to the HFP solutions, the system responded to light, giving reversible variations of the α-helix content. Irradiation at appropriate solvent compositions allowed modulation of the extent of the photoresponse. © 1993 John Wiley & Sons, Inc.     

Effect of chitosan on tobacco mosaic virus（TMV） accumulation,hydrolase activity,and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus（TMV） in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV（2 μg/mL） and chitosan（1 mg/mL） were lower in the early period of infection（3 days after inoculation）, by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases（proteases, RNases） in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid（PTA）-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles（18 nm in diameter, 300 nm long）, these suspensions contained abnormal（swollen, “thin” and “short”） virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that “thin” virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.     

Androgen effects on vocal muscle structure in a teleost fish with inter- and intra-sexual dimorphism

The plainfin midshipman fish Porichthys notatus has both interand intra-sexual dimorphism in the sound-producing (vocal or sonic) muscles attached to the swimbladder wall. The “Type I” and “Type II” male morphs differ in that dramatic structural changes related to sexual maturity occur in the mass, the area of mitochondria-filled sarcoplasm, and the myofiber number of the sonic muscles of Type I males, but not in those of Type II males (nor of females). Androgen implantation for 9 weeks markedly increased the relative sonic muscle size in juvenile males, juvenile females, and Type II males, whereas estradiol or cholesterol treatment did not. The principal androgen effect on myofiber structure was an increase in the area of mitochondria-filled sarcoplasm. The ratio of sarcoplasm area to myofibril area (Sr/Mf) increased by 1.4- to 2-fold in myofibers of all androgen-treated groups, with the greatest structural change occurring in juvenile males. When androgen implants were removed from juvenile males, the muscle mass and Sr/Mf ratio reverted toward the unimplanted juvenile phenotype. Total fiber number in sonic muscle increased significantly in juvenile males following androgen implantation but did not detectably change in juvenile females or Type II males. These results suggest: (1) sonic muscle in Porichthys notatus is an androgen target tissue, (2) fiber structure and fiber number are androgen-sensitive features, and (3) there exist sex- and morph-specific patterns of sonic muscle responsiveness to androgen implants. © 1993 Wiley-Liss, Inc.