Structural Analyses of Polysaccharides Extracted from Cyanobacterial Extracellular Gels and Oriented Liquid Crystalline Microfiber Processing by Poly(vinyl alcohol)-Assisted Electrospinning.

Sacran is a supergiant cyanobacterial polysaccharide that forms mesogenic supercoil rods that exhibit liquid crystalline (LC) gels at deficient concentrations of around 0.5 wt%, and has several bioactive stimuli-responsive functions. Here, we attempted to form oriented microfibers of sacran by electrospinning, following structural analyses of the sacran rods. A heterogeneous acid-hydrolysis method using a protonated cation-exchange resin was adopted to examine the short-time exposition of concentrated acid to sacran rods. From the supernatant, the oligomeric fraction that was soluble in water and methanol was isolated. The oligomeric fraction had a main sugar ratio of α-Glc:ß-Glc:α-Xyl:ß-Xyl:α-Rha of 2:5:1.5:1.5:4 (Glc:Xyl:Rha = 7 (=4 + 3):3:4), and it was speculated that the sacran structure includes rhamnoglucan and xyloglucan (4:3), which are generally rigid enough to exhibit LC. To make oriented microfibers of LC sacran, solubility testing was performed on sacran to find good new solvents of polyhydroxy alcohols such as ethylene glycol, 1,2-propanediol, and glycerol. The oriented film was prepared from a sacran aqueous solution where calcium compound particles deposited on the film are different from polyhydroxy alcohol solutions. Although sacran could not form microfibers by itself, polymer composite microfibers of sacran with poly(vinyl alcohol) were prepared by electrospinning. Cross-polarizing microscopy revealed the molecular orientation of the microfibers.

Metal-Assisted Injection Spinning of Ultra Strong Fibers from Megamolecular LC Polysaccharides.

The molecular orientation of liquid crystalline (LC) hydrogels has the potential to induce a range of functionalities that can deliver great mechanical strength. Sacran is a supergiant LC polysaccharide isolated from the cyanobacterium Aphanothece sacrum with a high amount of anionic functional groups such as sulfates and carboxylates. In this article, ultra-strong sacran hydrogels and their dried fibers were produced by cross-linking under injection flow with trivalent metal ions such as Al3+, Cr3+, Fe3+, In3+, and rare-earth metal ions such Er3+ and Sr3+. Crossed-polarizing microscopy and X-ray diffraction imaging revealed a uniaxial molecular orientation in the LC gel fiber, resulting in outstanding mechanical characteristics.

Dataset of Sac/CNF-Ag nanocomposites for antibacterial properties.

The data in this article includes a variety of analysis, including swelling, viscosity and FT-IR to better understand the thermal-mechanical, viscoelastic and swelling properties of sacran, CNF, and Ag nanoparticles individually and collectively. The fabrication method of Sacran, CNF, and Sac/CNF-Ag composite films is also demonstrated in this data item and are related to the research article "Facile design of antibacterial sheets of sacran and nanocellulose". This data article summarizes all the information so that it is evident how silver nanoparticle-polysaccharide hydrogels might be employed as an on-demand dressings due to their proven ability to reduce bacterial viability.

Cyanobacterial supra-polysaccharide: Self-similar hierarchy, diverse morphology, and application prospects of sacran fibers.

The biological functions of polysaccharides are influenced by their chemistry and chain conformation, which have resulted in various functional applications and new uses for polysaccharides in recent years. Sacran is an intriguing ampholytic polysaccharide with several key properties such as metal adsorption, anti-inflammatory nature, and transdermal drug-carrying capacity. It has an extremely high molecular weight over 107 g/mol, which is much higher than those of the previously reported microbial polysaccharides. In particular, it has a remarkable self-orienting characteristic over a large length scale, which could produce a bundle with twisted morphologies from the nanoscale to the microscale with diameters of ~1 µm and lengths of >800 µm. In this review, morphological variations, as well as novel self-organization and hierarchical self-assembly are comprehensively discussed. Sacran fibers deform into various forms, such as two- and three-dimensional flexible fibers and micro-nano fragments, during their evaporation. The self-assembly and disassembly of the sacran are explained in terms of the preparation process and factors that influence the morphology. This review will pave the way for the development of novel modules such as humidity-sensitive actuators, micro-patterned cell scaffolds, and uniaxially oriented membranes.

Super-Moisturizing Materials from Morphological Deformation of Suprapolysaccharides.

The evaporative interface on polysaccharides has evolved to form hierarchical structures with moisture sensitivity to enable organisms to live in drying environments. Here, the discovery of the morphological instability of polysaccharides, especially the reversible self-assembly/disassembly between micron-fibers and microparticles in response to changes in aquatic environments, is reported. This is similar but different to the dynamic instability observed in cytoskeletal proteins, in terms of an accompanying the polymeric deformation. The formation of the polymeric fibers containing crystalline structures can be flexibly controlled by controlling the polymer concentration and salt concentration in aqueous mixtures. Moreover, the microparticles having crosslinking points in the interior acquire the ability to retain a larger number of water molecules in drying environments and behave as super-moisturizing materials.

Physiological and genomic analysis of newly-isolated polysaccharide synthesizing cyanobacterium Chroococcus sp. FPU101 and chemical analysis of the exopolysaccharide.

A unicellular cyanobacterium that produces a large amount of exopolysaccharide (EPS) was isolated. The isolate, named Chroococcus sp. FPU101, grew between 20 and 30°C and at light intensities between 10 and 80 µmol m-2 s-1. Purified EPS from Chroococcus sp. FPU101 had a molecular size of 5.9 × 103 kDa and contained galactose, rhamnose, fucose, xylose, mannose, glucose, galacturonic acid, and glucuronic acid at a molar ratio of 17.2:15.9:14.1:11.0:9.6:9.5:13.0:9.7. The EPS content significantly increased when the NaCl concentration in the medium was increased from 1.7 to 100 mM. However, high NaCl concentrations did not significantly affect the molecular size or chemical composition of the EPS. The genes wza, wzb, wzc, wzx, wzy, and wzz that are involved in EPS synthesis were conserved in the genome of Chroococcus sp. FPU101, which was sequenced in this study. These results suggest that the Wzy-dependent pathway is potentially involved in EPS production in this organism.

Interfacial self-assembly of polysaccharide rods and platelets bridging over capillary lengths.

HYPOTHESIS: Generation of long-range ordering of colloidal particles through anisotropic interactions is of growing interest in material designing. At submicron-scale, routine works use synthetic spheres or rods but the knowledge pertaining to assembly of binary combination of particles is severely restricted. Improved understanding of the fundamental aspects that drive self-assembly, can lead to robust strategies for fabrication of topographically oriented films. EXPERIMENT: The fluidical geometry of a liquid crystalline (LC) solution of polysaccharide consisting of micron-sized rod and platelet units was explored. The solutions, characterized for their rheological behavior, were evaporated from a rectangular cavity. The assembly and orientation of the units was monitored by polarizing microscopy and the interparticle capillary forces approximated mathematically. FINDINGS: The units deposited into an uninterrupted membrane upon interfacial evaporation, forming a bridge along the 8 mm gap, linking the substrates. The membrane, composed of a lamellar structure, was uniaxially oriented along the direction of the gap. The rheological estimations corroborated an extremely high value of viscosity with the presence of crosslinking junctions in this solution when compared to a solution with only rod units, capable of bridging a maximum of 1 mm. It has been demonstrated for the very first time that the presence of platelet-units contributes lateral capillary interactions and assist rod-units towards a wider, self-assembled structure.

Vapor-Sensitive Materials from Polysaccharide Fibers with Self-Assembling Twisted Microstructures.

Polysaccharides play a variety of roles in nature, including molecular recognition and water retention. The microscale structures of polysaccharides are seldom utilized in vitro because of the difficulties in regulating self-assembled structures. Herein, it is demonstrated that a cyanobacterial polysaccharide, sacran, can hierarchically self-assemble as twisted fibers from nanoscale to microscale with diameters of ≈1 µm and lengths >800 µm that are remarkably larger than polysaccharides previously reported. Unlike other rigid fibrillar polysaccharides, the sacran fiber is capable of flexibly transforming into two-dimensional (2D) snaking and three-dimensional (3D) twisted structures at an evaporative air-water interface. Furthermore, a vapor-sensitive film with a millisecond-scale response time is developed from the crosslinked polymer due to the spring-like behavior of twisted structures. This study increases understanding of the functions of fibers in nature and establishes a novel approach to the design of environmentally adaptive materials for soft sensors and actuators.

Epidermal growth factor in sacran hydrogel film accelerates fibroblast migration.

Epidermal growth factor (EGF) accelerates epidermal regeneration, and it is widely studied as a wound-healing agent. However, the special carrier for the topical administration of EGF is urgently needed to deliver EGF on the wound site. In a preceding study, sacran hydrogel film (Sac-HF) showed a possible use as a dressing material for wound healing, as well as a good capability as a drug carrier. In the current study, we prepared Sac-HF containing EGF (Sac/EGF-HF) and then characterized their physicochemical properties, including thickness, swelling ratio, degradability, tensile strength, and morphology. In addition, we have also conducted thermal and crystallography studies using differential scanning calorimetry (DSC) and X-ray diffraction, respectively. Furthermore, we investigated the in vitro influence of Sac/EGF-HF on cell migration using a fibroblast cell line. Morphology study confirmed that the casting method used for the film preparation resulted in a homogeneous film of Sac/EGF-HF. Furthermore, EGF significantly increased the thickness, tensile strength, and degradability of Sac/EGF-HF compared to Sac-HF. Sac/EGF-HF had a lower swelling ability compared to Sac-HF; this result corroborated the tensile strength result. Interestingly, X-ray diffraction and DSC results showed that Sac/EGF-HF had an amorphous shape. The in vitro studies revealed that Sac/EGF-HF induced the fibroblast migration activity. These results conclude that Sac/EGF-HF has the potential properties of HF for biomedical applications.

Cationic Polymer Brush/Giant Polysaccharide Sacran Assembly: Structure and Lubricity.

A highly effective aqueous lubrication strategy employing electrostatic assembly of a negatively charged ultrahigh molecular weight natural polysaccharide named "sacran" and a positively charged poly[2-(methacryloyloxy)ethyltrimethylammonium chloride] (PMTAC) brush was investigated. The PMTAC brush was compressed through the adsorption of sacran to produce the layered structure of a PMTAC brush/sacran hybrid bottom layer and a poorly hydrated sacran top layer. The dynamic friction coefficients of the PMTAC brush were drastically reduced in salt-free sacran aqueous solutions, and the lubrication mode transition from the brush-lubrication regime to hydrodynamic lubrication was promoted. The electrostatic assembly was inhibited by the addition of NaCl into the lubricant solutions, leading to the loss of the lubrication effect. The hydrodynamic lubrication would be encouraged by the local viscosity enhancement at the friction boundary due to the poorly hydrated and highly viscous PMTAC brush/sacran hybrid film produced by the spontaneous electrostatic assembly.

Structure and Properties of Hybrid Film Fabricated by Spin-Assisted Layer-by-Layer Assembly of Sacran and Imogolite Nanotubes.

A free-standing (biomacomolecule/synthetic inorganic nanotubes) hybrid film was fabricated through an alternative layer-by-layer (LBL) assembly of sacran and imogolite nanotubes. Sacran is a natural polysaccharide extracted from the cyanobacterium Aphanothece sacrum, while imogolite is a natural tubular aluminosilicate clay found in volcano ash. The hybrid film thickness increased linearly with the number of the bilayers, because of the interaction between the negatively charged surface of sacran and the positively charged surface of imogolite. UV-vis spectroscopy indicated that the LBL film exhibited good transparency. The surface morphology of the LBL film was smooth in the micrometer scale; many imogolite nanotubes were adsorbed onto the sacran layer, while no imogolite clusters were observed. Furthermore, the structure, stability, gas permeability, and mechanical properties of the LBL films were investigated.

Rheopectic Behavior for Aqueous Solutions of Megamolecular Polysaccharide Sacran.

The rheopectic behavior of sacran aqueous solutions, a natural giant molecular polysaccharide with a molecular weight of 1.6 × 107 g/mol, was investigated. When a low shear was applied to 1.0 wt.% sacran solution, the shear viscosity increased from 7.2 to 34 Pas. The increment in the viscosity was enhanced as the shear rate decreased. The shear viscosity was independent of the time at a shear rate of 0.8 s-1; simultaneously, thixotropic behavior was observed at shear rates higher than 1.0 s-1. A crossover was observed at 0.15 wt.% for the concentration dependence of both the viscosity increase and zeta potential, which was the vicinity of the helix transition concentration or gelation concentration. It was clear that the molecular mechanism for the rheopexy was different at lower and higher regions of the crossover concentration.

Sacran Hydrogel Film Containing Keratinocyte Growth Factor Accelerates Wound Healing by Stimulating Fibroblast Migration and Re-epithelization.

Regenerative therapy with keratinocyte growth factor (KGF) is a novel therapeutic approach for treatment of chronic wounds. However, KGF cannot be used directly to the wound site due to its physicochemical instability. In previous study, sacran, a natural megamolecular polysaccharide, showed potential properties as a biomaterial for hydrogel film in wound healing. In this study, we fabricated sacran hydrogel film containing KGF (Sac/KGF-HF) and evaluated the effects of Sac/KGF-HF on fibroblasts migration and re-epithelialization process. We successfully prepared a homogenous and -amorphous Sac/KGF-HF by a casting method. In addition, Sac/KGF-HF had a high swelling ratio and flexibility. Sac/KGF-HF promoted a migration process of NIH3T3 cells and improved wound healing ability in mice with a percentage of wound closure reaching 90.4% at 9 d. Interestingly, the addition of KGF in Sac-HF considerably increased the number of epithelial cells compared to control, which is important in the re-epithelialization process. It could be concluded that KGF in Sac-HF has the potential for promoting Sac-HF abilities in wound healing process.

[Optical Second-harmonic Observation of Stimulated Sacran Aggregates].

Second-order nonlinear optical images of aggregates of the ampholytic megamolecular polysaccharide sacran under various stimuli were observed by optical second-harmonic generation (SHG) microscopy. SHG intensity microspots of several tens of micrometers in size are seen for sacran cotton-like lumps and fibers and they have very clear incident polarization dependence. In these microspots, the sacran molecules are oriented in concentric multilayers. We also observed SHG images of sacran prepared in a needle-ring electrode with applied voltage, where SHG was enhanced near the negatively biased needle electrode. We also observed SHG signals near the cast film edges of sacran. The appearance of the SHG image suggested a phase-separation structure in sacran aggregates. These results show that sacran molecules aggregate in several different ways.

[Industrial and Medical Material Applications of a Mega-polysaccharide Derived from Cyanobacteria].

Polysaccharides (PS) are one form of biomass occurring in great abundance on earth and are promising organic material alternatives to petroleum-based ones. We have focused on PS produced by cyanobacteria. Aphanothece sacrum, which is a freshwater unicellular cyanobacterium, produces large amounts PS as a main component of the extracellular matrix. We successfully extracted the PS sacran from A. sacrum biomaterials and demonstrated that sacran contains carboxyls and sulfate groups. Furthermore, the constituent sugars of the sacran extracted were determined to be fucose, rhamnose, xylose, arabinose, mannose, glucose, galactose, glucuronic acid, galacturonic acid, and galactosamine. The sequence of sacran is still under investigation. In addition, we confirmed that the sacran was a supergiant with a high absolute molecular weight of 2.35×107 g/mol. Sacran shows a self-orienting behavior in dilute solution at a concentration range over 0.25 wt% to form a liquid crystalline structure. Using this property, it is possible to prepare a sacran cast film with an orientation structure, especially swelling in the thickness direction, to form anisotropic hydrogels. The anisotropy and degree of swelling of the hydrogels could be controlled by varying the heating temperature of the sacran cast file. Furthermore, sacran could be combined successfully with rayon as new fiber materials, and the sacran-rayon complex materials showed increased water absorption compared with the original rayon. This article introduces various applications of sacran in the industrial and medical fields.

Physiological properties and genetic analysis related to exopolysaccharide (EPS) production in the fresh-water unicellular cyanobacterium Aphanothece sacrum (Suizenji Nori).

The clonal strains, phycoerythrin(PE)-rich- and PE-poor strains, of the unicellular, fresh water cyanobacterium Aphanothece sacrum (Suringar) Okada (Suizenji Nori, in Japanese) were isolated from traditional open-air aquafarms in Japan. A. sacrum appeared to be oligotrophic on the basis of its growth characteristics. The optimum temperature for growth was around 20°C. Maximum growth and biomass increase at 20°C was obtained under light intensities between 40 to 80 µmol m-2 s-1 (fluorescent lamps, 12 h light/12 h dark cycles) and between 40 to 120 µmol m-2 s-1 for PE-rich and PE-poor strains, respectively, of A. sacrum . Purified exopolysaccharide (EPS) of A. sacrum has a molecular weight of ca. 104 kDa with five major monosaccharides (glucose, xylose, rhamnose, galactose and mannose; ≥85 mol%). We also deciphered the whole genome sequence of the two strains of A. sacrum. The putative genes involved in the polymerization, chain length control, and export of EPS would contribute to understand the biosynthetic process of their extremely high molecular weight EPS. The putative genes encoding Wzx-Wzy-Wzz- and Wza-Wzb-Wzc were conserved in the A. sacrum strains FPU1 and FPU3. This result suggests that the Wzy-dependent pathway participates in the EPS production of A. sacrum.

Micropatterned Cell Orientation of Cyanobacterial Liquid-Crystalline Hydrogels.

Control of cell extension direction is crucial for the regeneration of tissues, which are generally composed of oriented molecules. The scaffolds of highly oriented liquid crystalline polymer chains were fabricated by casting cyanobacterial mega-saccharides, sacran, on parallel-aligned micrometer bars of polystyrene (PS). Polarized microscopy revealed that the orientation was in transverse direction to the longitudinal axes of the PS bars. Swelling behavior of the micropatterned hydrogels was dependent on the distance between the PS bars. The mechanical properties of these scaffolds were dependent on the structural orientation; additionally, the Young's moduli in the transverse direction were higher than those in the parallel direction to the major axes of the PS bars. Further, fibroblast L929 cells were cultivated on the oriented scaffolds to be aligned along the orientation axis. L929 cells cultured on these scaffolds exhibited uniaxial elongation.

Micelle-Mediated Self-Assembly of Microfibers Bridging Millimeter-Scale Gap To Form Three-Dimensional-Ordered Polysaccharide Membranes.

Micelle-mediated three-dimensional-ordered polysaccharide membranes are constructed by introducing cationic/anionic surfactant into a liquid crystalline polysaccharide solution. Upon drying mixtures of the polysaccharide solution with the surfactant such as cetyltrimethylammonium bromide or sodium dodecyl sulfate (SDS), the polymeric microfibers deposit as a nucleus to form a membrane, bridging millimeter-scale gap with high probability. In particular, in a solution with SDS micellar structures, the microscale fibers with diameter ∼1 µm disassemble into nanoscale fibers with diameter ∼50 nm. This transformation allows the polymeric network to become finer in nanoscale, and the vertical membrane is formed much more easily than that from a pure polysaccharide solution. Furthermore, it is clarified that the vertical membrane has been successfully formed with three-dimensionally ordered microstructures with a linearly oriented and layered structure. This method will shed light on the preparation of hybrid materials with biocompatibility and responsivity to stimuli such as magnetics, electrics, and optics via hybridization with nanomaterials dispersed by surfactants.

Surface-Selective Control of Cell Orientation on Cyanobacterial Liquid Crystalline Gels.

Liquid crystalline hydrogels (LCGs) with layer structures and oriented pores were created using sacran which is a cyanobacterial heteropolysaccharide possessing functional sulfate, carboxylate, and amide groups in common with glycosaminoglycan. The LCG biocompatibility with L929 mouse fibroblasts was confirmed under the appropriate conditions. Enhanced growth and proliferation of L929 cells without exhibiting any toxicity were confirmed. The water contact angle and protein adsorption ability on the LCG were well-controlled by the cross-linking degree. Additionally, fibroblasts were finely oriented on the LCG side face where layer edges made a striped morphology on its surface, whereas the flat top faces of the LCG did not induce any specific cell orientation.

[Super Liquid Crystalline Polysaccharides Produced by Ultimately-ecological Microreactors].

　Cyanobacteria fix carbon dioxide and nitrogen from the atmosphere using solar energy to produce various biomolecules, and thus are regarded as ultimately ecological microreactors. Sacran is a cyanobacterial polysaccharide with a very high molecular weight of 29 Mg/mol, which is extracted from Aphanothece sacrum cyanobacterium mass-cultivated in freshwater environments such as river or spring. Sacran is a water-soluble heteropolysaccharide comprising more than 6 kinds of sugar residues and contains 12% sulfate anionic groups and 27% carboxyls. Sacran has a super-absorbent function of water, which can retain 6000 mL for 1 g specimen, due to very long hydrating chains. The value is much higher than hyaluronic acid or conventional super-absorbent polymers. Sacran exhibits self-orienting behavior in dilute solution at a concentration range over 0.25 wt%, which is quite low when compared with conventional liquid crystalline polysaccharides. Mesogenic helical chains of sacrans have extremely high aspect ratios of 1600 for highly persistent lengths of 32 micrometer. Through the liquid crystallinity, sacran solution shows a shear-thinning behavior and the solution spread over a substrate such as biological skin very efficiently to create a thin layer. Applied on atopic dermatitis skin sacran solution exerts excellent moisturizing effect and anti-itching action.