Surface-mediated self-assembly of click-reactive cello-oligosaccharides for fabricating functional nonwoven fabrics.

Polymer fabrics are versatile materials used in various fields. Surface modification methods for hydrophobic polymer fibers have been developed to endow the materials with water wettability and functionality. Nevertheless, it remains a challenge to freely introduce functional groups to polymer fiber surfaces in a simple manner. Herein, we report the decoration of nonwoven fabric surfaces with azidated cello-oligosaccharide assemblies via molecular self-assembly. Cello-oligosaccharides with a terminal azido group were enzymatically synthesized and allowed to self-assemble in polyolefin, polyester, and vinylon nonwoven fabrics. It was found that the functional oligosaccharides formed bark-like assemblies on the nonwoven fiber surfaces, probably through heterogeneous nucleation. The hydrophilic oligosaccharide assemblies made the hydrophobic nonwoven surfaces water-wettable. Moreover, the azido group at oligosaccharide terminal was available for the post-functionalization of the modified nonwovens. In fact, an antigen was successfully conjugated to the modified nonwovens via the click chemistry. The antigen-conjugated nonwovens were useful for the specific and quantitative detection of a corresponding antibody. Our findings demonstrate the great potential of cello-oligosaccharide assembly for the functionalization of fabrics and other polymeric materials.

This study developed a novel and simple method for modifying surfaces of polymer nonwoven fabrics based on the self-assembly of azidated cello-oligosaccharides to fabricate water-wettable and click-reactive functional materials.

Synthetic Nanocelluloses Fluorescently Responsible to Enzymatic Degradation.

Crystalline assemblies of cellulose and cellulose derivatives that can be synthetically produced by in vitro enzymatic reactions in a bottom-up manner have attracted increasing attention as chemically designable functional nanomaterials (e.g., synthetic nanocelluloses). In this study, we demonstrate the preparation and characterization of alkyl ß-celluloside assemblies loaded with fluorescent molecules, which are fluorescently responsible to the enzymatic degradation of the cellulose moieties. The fluorescent properties are afforded to the assemblies by their bilayer-structured nanosheet morphologies realized through the uptake of environmentally responsive fluorescent molecules (namely, Nile Red (NR)). Incubation of the NR-loaded n-octyl ß-celluloside (CEL-C8) assembly with cellulase resulted in decreases in the fluorescence intensities. This suggests that NR molecules were released into the aqueous phase through enzymatic degradation of the cellulose moieties of CEL-C8 molecules in the assembly. The fluorescence decrease rates were clearly dependent on the concentration and source of cellulase. Fluorescence decreases through enzymatic degradation were again observed in the presence of contaminant proteins. These observations revealed the high potential of alkyl ß-celluloside assemblies loaded with fluorescent molecules as fluorescently responsible cellulase substrates for cellulase detection assays by simply measuring changes in the fluorescence intensities. Moreover, the assemblies were revealed as carriers for the controlled release of loaded molecules triggered by enzymatic degradation.

Aqueous Suspensions of Cellulose Oligomer Nanoribbons for Growth and Natural Filtration-Based Separation of Cancer Spheroids.

In vitro growth of cancer spheroids (CSs) and the subsequent separation of CSs from a 2D or 3D cell culture system are important for fundamental cancer studies and cancer drug screening. Although biopolymer-based or synthetic hydrogels are suitable candidates to be used as 3D cell culture scaffolds, alternatives with better processing capabilities are still required to set up cell culture microenvironment. In this study, we show that aqueous suspensions of crystalline nanoribbons composed of cellulose oligomers have a potential for CS growth and separation. The nanoribbon suspensions in serum-containing cell culture media fixed single cancer cells and CSs with large sizes in a 3D space, leading to suspension cultures for CS growth corresponding to culture time. Well-grown CSs were easily separated from the suspensions by natural filtration using a mesh filter with a suitable pore size. Cell viability tests revealed negligible cytotoxicity of the nanoribbons. In addition, physical damages to CSs by the separation procedures were negligible. Stable suspensions of biocompatible nanomaterials will thus provide novel microenvironments for growth and separation of diverse cell aggregates.

Affinity-based thermoresponsive fluorescence switching of proteins conjugated with a polymer-binding peptide.

The affinity-based thermoresponsive fluorescence switching of proteins conjugated with a polymer-binding peptide is demonstrated. The specific affinity of the peptide and thermoresponsive structural transitions of the polymer are essential for reliable fluorescence switching behavior.

Temperature-Directed Assembly of Crystalline Cellulose Oligomers into Kinetically Trapped Structures during Biocatalytic Synthesis.

Crystalline polysaccharides, such as cellulose and chitin, can form superior assemblies in terms of physicochemical stability and mechanical properties. However, their use as molecular building blocks for self-assembled materials is rare, possibly because each crystalline polysaccharide has its own unique monomer unit, preventing molecular design for controlling the self-assembly. Herein, we demonstrate the temperature-directed assembly of crystalline cellulose oligomers into kinetically trapped structures, namely, precipitated nanosheets, nanoribbon network hydrogels, and dispersed nanosheets (in descending order of temperature). It was found that enzymatically synthesized cellulose oligomers self-assembled in situ into those structures depending on the synthetic temperatures. Mechanistic studies suggested that the formation of the nanoribbon networks and the dispersed nanosheets at lower temperatures were driven by synergy between the decreased hydrophobic effect and the simultaneously induced self-crowding effect. Furthermore, nanoribbon network formation was exploited for the construction of cellulose oligomer-based hybrid gels with colloidal particles. Our findings promote the development of robust self-assembled materials composed of crystalline polysaccharides with highly ordered nano-to-macroscale structures.

Enzyme-Catalyzed Bottom-Up Synthesis of Mechanically and Physicochemically Stable Cellulose Hydrogels for Spatial Immobilization of Functional Colloidal Particles.

The dispersion stabilization of colloidal particles and subsequent construction of functional materials are of great interest in areas ranging from colloid chemistry to materials science. A promising strategy is the spatial immobilization of colloidal particles within gel scaffolds. However, conventional gels readily deform and even collapse when changes in environmental conditions occur. Herein, we describe the enzyme-catalyzed bottom-up synthesis of mechanically and physicochemically stable nanoribbon network hydrogels composed of crystalline cellulose oligomers in which cellulose nanocrystals (CNCs) as model colloidal particles are immobilized spatially. The stiffness of the hydrogels increased with the amount of CNCs incorporated. Filling the void space of the hydrogels with hydrophobic polymers resulted in polymer nanocomposites with excellent mechanical properties. The nanoribbon networks will be useful for demonstrating the potential functions of colloidal particles.

Self-Assembly of Cellulose Oligomers into Nanoribbon Network Structures Based on Kinetic Control of Enzymatic Oligomerization.

The ability to chemically synthesize desired molecules followed by their in situ self-assembly in reaction solution has attracted much attention as a simple and environmentally friendly method to produce self-assembled nanostructures. In this study, α-d-glucose 1-phosphate monomers and cellobiose primers were subjected to cellodextrin phosphorylase-catalyzed reverse phosphorolysis reactions in aqueous solution in order to synthesize cellulose oligomers, which were then in situ self-assembled into crystalline nanoribbon network structures. The average degree-of-polymerization (DP) values of the cellulose oligomers were estimated to be approximately 7-8 with a certain degree of DP distribution. The cellulose oligomers crystallized with the cellulose II allomorph appeared to align perpendicularly to the base plane of the nanoribbons in an antiparallel manner. Detailed analyses of reaction time dependence suggested that the production of nanoribbon network structures was kinetically controlled by the amount of water-insoluble cellulose oligomers produced.

A Bottom-Up Synthesis of Vinyl-Cellulose Nanosheets and Their Nanocomposite Hydrogels with Enhanced Strength.

Extracted nanocellulose from natural resources commonly requires modification before it is used as an effective nanofiller. In the present study, through an enzymatic polymerization of α-d-glucose 1-phosphate from the primer 2-(glucosyloxy)ethyl methacrylate (GEMA), a novel type of two-dimensional methacrylate-containing cellulose nanosheets (CNS) with a thickness of about 6 nm, named as GEMA-CNS, was directly synthesized under a mild condition by a "bottom-up" method. The structure and morphology of GEMA-CNS were characterized by 1H-nuclear magnetic resonance (NMR), matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF MS), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Afterward, the obtained GEMA-CNS was covalently incorporated into poly(ethylene glycol) matrix through thiol-ene Michael addition, fabricating a series of GEMA-CNS-based nanocomposite hydrogels. The addition of GEMA-CNS effectively improved the mechanical strength and altered the internal network structures of hydrogels; additionally, the swelling/biodegradation behaviors of gels in phosphate buffer saline (pH 7.4) at 37 °C were affected to some degree. This species of property-tunable hydrogels with GEMA-CNS dosage demonstrates potential applications in tissue engineering. The current presentation opens a new road for direct enzymatic preparation of reactive nanocellulose and its novel applications in nanocomposite materials.

Multidimensional Self-Assembled Structures of Alkylated Cellulose Oligomers Synthesized via in Vitro Enzymatic Reactions.

The self-assembly of biomolecules into highly ordered nano-to-macroscale structures is essential in the construction of biological tissues and organs. A variety of biomolecular assemblies composed of nucleic acids, peptides, and lipids have been used as molecular building units for self-assembled materials. However, crystalline polysaccharides have rarely been utilized in self-assembled materials. In this study, we describe multidimensional self-assembled structures of alkylated cellulose oligomers synthesized via in vitro enzymatic reactions. We found that the alkyl chain length drastically affected the assembled morphologies and allomorphs of cellulose moieties. The modulation of the intermolecular interactions of cellulose oligomers by alkyl substituents was highly effective at controlling their assembly into multidimensional structures. This study proposes a new potential of crystalline oligosaccharides for structural components of molecular assemblies with controlled morphologies and crystal structures.

Enzymatic Synthesis of Oligo(ethylene glycol)-Bearing Cellulose Oligomers for in Situ Formation of Hydrogels with Crystalline Nanoribbon Network Structures.

Enzymatic synthesis of cellulose and its derivatives has gained considerable attention for use in the production of artificial crystalline nanocelluloses with unique structural and functional properties. However, the poor colloidal stability of the nanocelluloses during enzymatic synthesis in aqueous solutions limits their crystallization-based self-assembly to greater architectures. In this study, oligo(ethylene glycol) (OEG)-bearing cellulose oligomers with different OEG chain lengths were systematically synthesized via cellodextrin phosphorylase-catalyzed oligomerization of α-d-glucose l-phosphate monomers against OEG-bearing ß-d-glucose primers. The products were self-assembled into extremely well-grown crystalline nanoribbon network structures with the cellulose II allomorph, potentially due to OEG-derived colloidal stability of the nanoribbon's precursors, followed by the in situ formation of physically cross-linked hydrogels. The monomer conversions, average degree of polymerization, and morphologies of the nanoribbons changed significantly, depending on the OEG chain length. Taken together, our findings open a new avenue for the enzymatic reaction-based facile production of novel cellulosic soft materials with regular nanostructures.

Dispersion of boron nitride nanotubes in aqueous solution by simple aromatic molecules.

We report that the simple aromatic molecules successfully disentangle and disperse multi-walled boron nitride nanotubes (BNNTs) in aqueous solutions through pi-pi stacking interactions. Aromatic molecules such as derivatives of naphthalene, anthracene, and pyrene were used as dispersants and components for construction of nanohybrids. Spectroscopic analyses of water-dispersed BNNTs revealed that not only numbers of aromatic rings but also substituted functional groups were essential for dispersion capabilities. It was suggested that greater numbers of aromatic rings and a carboxylic acid as the substituted group showed higher dispersion capabilities among the molecules used in this study. Detailed microscopic analyses using atomic force microscopy and transmission electron microscopy showed certain isolation capabilities of the aromatic molecules for BNNTs in aqueous solution. Moreover, fluorescence spectra indicated that BNNTs and the aromatic molecules have different electronic states after hybrid formation. Our results using the simple aromatic molecules will be utilized as a basic data for dispersion of BNNTs and construction of disentangled BNNT nanohybrids effectively.

Difference in protein adsorption onto polymer films with or without thermal annealing.

Protein adsorptions onto non-annealed (NA) and thermally annealed (TA) polyetherimide films were examined by surface plasmon resonance measurements. Proteins adsorbed onto the NA films with smaller adsorption constants in comparison with the TA films. Neutron reflectivity measurements of the two films suggested that the outermost region of the NA films swelled with larger amounts of water molecules than the TA films. It is plausible that the aforementioned difference in the protein adsorption properties is derived from the difference in the interfacial aggregation structures of the two films.

Freeze-Dryable, Stable, and Click-Reactive Nanoparticles Composed of Cello-oligosaccharides for Biomolecular Sensing.

Nanoparticles have been widely used as platforms for biomolecular sensing because of their high specific surface area and attractive properties depending on their constituents and structures. Nevertheless, it remains challenging to develop nanoparticulate sensing platforms that are easily storable without aggregation and conjugatable with various ligands in a simple manner. Herein, we demonstrate that nanoparticulate assemblies of cello-oligosaccharides with terminal azido groups are promising candidates. Azidated cello-oligosaccharides can be readily synthesized via the enzyme-catalyzed oligomerization reaction. This study characterized the assembled structures of azidated cello-oligosaccharides produced during the enzymatic synthesis and revealed that the terminal azidated cello-oligosaccharides formed rectangular nanosheet-shaped lamellar crystals. The azido groups located on the nanosheet surfaces were successfully exploited for antigen conjugation via the click chemistry. The resultant antigen-conjugated nanosheets allowed for the quantitative and specific detection of a corresponding antibody, even in 10% serum, owing to the antifouling properties of cello-oligosaccharide assemblies against proteins. It was found that the functionalized nanosheets were redispersible in water after freeze-drying. This remarkable characteristic is attributed to the well-hydrated saccharide residues on the nanosheet surfaces. Moreover, the antibody detection capability did not decline after the thermal treatment of the functionalized nanosheets in a freeze-dried state. Our findings contribute to developing convenient nanoparticulate biomolecular sensing platforms.

Tunable thermal diffusivity of silk protein assemblies based on their structural control and photo-induced chemical cross-linking.

Silk, which has excellent mechanical properties and is lightweight, serves as a structural material in natural systems. However, the structural and functional applications of silk in artificial systems have been limited due to the difficulty in controlling its properties. In this study, we demonstrate the tunable thermal diffusivity of silk-based assemblies (films) based on secondary structural control and subsequent cross-linking. We found that the thermal diffusivity of the silk film is increased by the formation of ß-sheet structures and dityrosine between Tyr residues adjacent to the ß-sheet structures. Our results demonstrate the applicability of silk proteins as material components for thermally conductive biopolymer-based materials.

Distinguishing anti-PEG antibodies by specificity for the PEG terminus using nanoarchitectonics-based antibiofouling cello-oligosaccharide platforms.

The conjugation of poly(ethylene glycol) (PEG) to therapeutic proteins or nanoparticles is a widely used pharmaceutical strategy to improve their therapeutic efficacy. However, conjugation can make PEG immunogenic and induce the production of anti-PEG antibodies, which decreases both the therapeutic efficacy after repeated dosing and clinical safety. To address these concerns, it is essential to analyze the binding characteristics of anti-PEG antibodies to PEG. However, distinguishing anti-PEG antibodies is still a difficult task. Herein, we demonstrate the use of antibiofouling cello-oligosaccharide assemblies tethering one-terminal methoxy oligo(ethylene glycol) (OEG) ligands for distinguishing anti-PEG antibodies in a simple manner. The OEG ligand-tethering two-dimensional crystalline cello-oligosaccharide assemblies were stably dispersed in a buffer solution and had antibiofouling properties against nonspecific protein adsorption. These characteristics allowed enzyme-linked immunosorbent assays (ELISAs) to be simply performed by cycles of centrifugation/redispersion of aqueous dispersions of the assemblies. The simple assays revealed that the specific OEG ligand-tethering assemblies could distinguish anti-PEG antibodies to detect a specific antibody that preferentially binds to the methoxy terminus of the PEG chain with 3 repeating ethylene glycol units. Furthermore, quantitative detection of the antibodies was successfully performed with high sensitivity even in the presence of serum. The detectable and quantifiable range of antibody concentrations covered those required clinically. Our findings open a new avenue for analyzing the binding characteristics of anti-PEG antibodies in biological samples.

Interfacial jamming of surface-alkylated synthetic nanocelluloses for structuring liquids.

Nanocelluloses derived from natural cellulose sources are promising sustainable nanomaterials. Previous studies have reported that nanocelluloses are strongly adsorbed onto liquid-liquid interfaces with the concurrent use of ligands and allow for the structuring of liquids, that is, the kinetic trapping of nonequilibrium shapes of liquids. However, the structuring of liquids using nanocelluloses alone has yet to be demonstrated, despite its great potential in the development of sustainable liquid-based materials that are biocompatible and environmentally friendly. Herein, we demonstrated the structuring of liquids using rectangular sheet-shaped synthetic nanocelluloses with surface alkyl groups. Synthetic nanocelluloses with ethyl, butyl, and hexyl groups on their surfaces were readily prepared following our previous reports via the self-assembly of enzymatically synthesized cello-oligosaccharides having the corresponding alkyl groups. Among the alkylated synthetic nanocelluloses, the hexylated nanocellulose was adsorbed and jammed at water-n-undecane interfaces to form interfacial assemblies, which acted substantially as an integrated film for structuring liquids. These phenomena were attributed to the unique structural characteristics of the surface-hexylated synthetic nanocelluloses; their sheet shape offered a large area for adsorption onto interfaces, and their controlled surface hydrophilicity/hydrophobicity enhanced the affinity for both liquid phases. Our findings promote the development of all-liquid devices using nanocelluloses.

Suspension Culture System for Isolating Cancer Spheroids using Enzymatically Synthesized Cellulose Oligomers.

Isolating cancer cells from tissues and providing an appropriate culture environment are important for a better understanding of cancer behavior. Although various three-dimensional (3D) cell culture systems have been developed, techniques for collecting high-purity spheroids without strong stimulation are required. Herein, we report a 3D cell culture system for the isolation of cancer spheroids using enzymatically synthesized cellulose oligomers (COs) and demonstrate that this system isolates only cancer spheroids under coculture conditions with normal cells. CO suspensions in a serum-containing cell culture medium were prepared to suspend cells without settling. High-purity cancer spheroids could be separated by filtration without strong stimulation because the COs exhibited antibiofouling properties and a viscosity comparable to that of the culture medium. When human hepatocellular carcinoma (HepG2) cells, a model for cancer cells, were cultured in the CO suspensions, they proliferated clonally and efficiently with time. In addition, only developed cancer spheroids from HepG2 cells were collected in the presence of normal cells by using a mesh filter with an appropriate pore size. These results indicate that this approach has potential applications in basic cancer research and cancer drug screening.

Antibacterial Synthetic Nanocelluloses Synergizing with a Metal-Chelating Agent.

Antibacterial materials composed of biodegradable and biocompatible constituents that are produced via eco-friendly synthetic strategies will become an attractive alternative to antibiotics to combat antibiotic-resistant bacteria. In this study, we demonstrated the antibacterial properties of nanosheet-shaped crystalline assemblies of enzymatically synthesized aminated cellulose oligomers (namely, surface-aminated synthetic nanocelluloses) and their synergy with a metal-chelating antibacterial agent, ethylenediaminetetraacetic acid (EDTA). Growth curves and colony counting assays revealed that the surface-aminated cellulose assemblies had an antibacterial effect against Gram-negative Escherichia coli (E. coli). The cationic assemblies appeared to destabilize the cell wall of E. coli through electrostatic interactions with anionic lipopolysaccharide (LPS) molecules on the outer membrane. The antibacterial properties were significantly enhanced by the concurrent use of EDTA, which potentially removed metal ions from LPS molecules, resulting in synergistic bactericidal effects. No antibacterial activity of the surface-aminated cellulose assemblies was observed against Gram-positive Staphylococcus aureus even in the presence of EDTA, further supporting the contribution of electrostatic interactions between the cationic assemblies and anionic LPS to the activity against Gram-negative bacteria. Analysis using quartz crystal microbalance with dissipation monitoring revealed the attractive interaction of the surface-aminated cellulose assembly with LPS Ra monolayers artificially produced on the device substrate.

A Visible Light Cross-Linked Underwater Hydrogel Adhesive with Biodegradation and Hemostatic Ability.

Hydrogel adhesives with integrated functionalities are still required to match their ever-expanding practical applications in the field of tissue repair and regeneration. A simple and effective safety strategy is reported, involving an in situ injectable polymer precursor and visible light-induced cross-linking. This strategy enables the preparation of a hydrogel adhesive in a physiological environment, offering wet adhesion to tissue surfaces, molecular flexibility, biodegradability, biocompatibility, efficient hemostatic performance, and the ability to facilitate liver injury repair. The proposed one-step preparation process of this polymer precursor involves the mixing of gelatin methacryloyl (GelMA), poly(thioctic acid) [P(TA)], poly(acrylic acid)/amorphous calcium phosphate (PAAc/ACP, PA) and FDA-approved photoinitiator solution, and a subsequent visible light irradiation after in situ injection into target tissues that resulted in a chemically-physically cross-linked hybrid hydrogel adhesive. Such a combined strategy shows promise for medical scenarios, such as uncontrollable post-traumatic bleeding.

Peptides as new smart bionanomaterials: molecular-recognition and self-assembly capabilities.

Biomolecules express exquisite properties that are required for molecular recognition and self-assembly on the nanoscale. These smart capabilities have developed through evolution and such biomolecules operate based on smart functions in natural systems. Recently, these remarkable smart capabilities have been utilized in not only biologically related fields, but also in materials science and engineering. A peptide-screening technology that uses phage-display systems has been developed based on this natural smart evolution for the generation of new functional peptide bionanomaterials. We focused on peptides that specifically bound to synthetic polymers. These polymer-binding peptides were screened by using a phage-display peptide library to recognize nanostructures that were derived from polymeric structural features and were utilized for possible applications as new bionanomaterials. We also focused on self-assembling peptides with ß-sheet structures that formed nanoscale, fibrous structures for applications in new bottom-up nanomaterials. Moreover, nanofiber-binding peptides were also screened to introduce the desired functionalities into nanofibers without the need for additional molecular design. Our approach to construct new bionanomaterials that employ peptides will open up excellent opportunities for the next generation of materials science and technology.