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.

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.

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.

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.

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.

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.

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.

Identification of Water-Soluble Polymers through Machine Learning of Fluorescence Signals from Multiple Peptide Sensors.

Recently, there has been growing concern about the discharge of water-soluble polymers (especially synthetic polymers) into the environment. Therefore, the identification of water-soluble polymers in water samples is becoming increasingly crucial. In this study, a chemical tongue system to simply and precisely identify water-soluble polymers using multiple fluorescently responsive peptide sensors was demonstrated. Fluorescence spectra obtained from the mixture of each peptide sensor and water-soluble polymer were changed depending on the combination of the polymer species and peptide sensors. Water-soluble polymers were successfully identified through the supervised or unsupervised machine learning of multidimensional fluorescence signals from the peptide sensors.

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.

Photo-reactive polymers for the immobilisation of epidermal growth factors.

Photo-reactive polymers are important for biomaterials, including devices with a 3D-structure. Here, different types of photo-reactive polymers were prepared and utilised for immobilisation of growth factors. They were synthesised by conjugation of gelatin with the azidophenyl group or by copolymerisation of the azidophenyl group-coupled methacrylate with poly(ethylene glycol) methacrylate. The azidophenyl content and the zeta potential of the prepared polymers were measured. After spin coating of polymers, the thickness and the water contact angle of coated layers were measured. The amount of the immobilised epidermal growth factor (EGF) was determined using fluorescence labelling. Cell adhesion responded to the nature of photo-reactive polymers but did not depend on the immobilised EGF. However, cell growth was dependent on the amount of immobilised EGF and was significantly affected by the nature of photo-reactive polymers. The study shows that the properties of the photo-immobilisation matrix significantly influence the biological activity.

Nanospiked paper: Microfibrous cellulose materials nanostructured via partial hydrolysis and self-assembly.

Nanocelluloses, such as cellulose nanofibers and nanocrystals, are sustainable nanomaterials that are generally extracted from natural raw materials in a top-down manner. These nanomaterials and their assemblies are facilitating new applications of biopolymers. However, creating nanostructures from conventional cellulosic materials including paper and cloth remains challenging. Herein, we report an approach for bottom-up nanostructuring of conventional microfibrous cellulose materials via a molecular self-assembly strategy. As a precursor cellulose material, paper was allowed to swell with aqueous phosphoric acid for the partial dissolution and hydrolysis of cellulose while maintaining its microfibrous structure. The generated cello-oligosaccharides in a dissolved state started to self-assemble upon adding water as a coagulant, resulting in nanospike-like assemblies on the microfiber surfaces. The resultant nanospiked papers were found to serve as a precursor for synthesizing silver nanoparticle-cellulose composites with bactericidal activities. Our findings promote the development of cellulose-based functional materials with nanostructures designed via molecular self-assembly.

Alkyl chain length-dependent protein nonadsorption and adsorption properties of crystalline alkyl ß-celluloside assemblies.

Cellulose-based crystalline assemblies artificially constructed in a bottom-up manner are attracting increasing attention as chemically stable and functionally designable nano- to macroscale materials. However, basic knowledge of how such crystalline assemblies interact with biomolecules and how to control them via molecular design is still limited. In this study, we investigated the protein adsorption properties of crystalline lamella assemblies composed of alkyl ß-cellulosides (namely, ethyl, n-butyl, and n-hexyl ß-cellulosides) or plain cellulose, which all have an antiparallel molecular arrangement. It was found that the adsorption of proteins was observed only for the n-hexyl ß-celluloside assembly, while it was hardly observed for other assemblies. The n-hexyl groups appeared to be ordinarily embedded in the assembly surface in an aqueous phase, while, when in contact with proteins, n-hexyl groups appeared to be tethered to promote protein adsorption. All-atom molecular dynamics simulations supported the contradictory protein adsorption properties. The basic knowledge obtained herein is highly valuable for controlling the interactions of cellulose-based synthetic assemblies with proteins for designing new biological applications.

Identification of Water-Soluble Polymers through Discrimination of Multiple Optical Signals from a Single Peptide Sensor.

The pollution of water environments is a worldwide concern. Not only marine pollution by plastic litter, including microplastics, but also the spillage of water-soluble synthetic polymers in wastewater have recently gained increasing attention due to their potential risks to soil and water environments. However, conventional methods to identify polymers dissolved in water are laborious and time-consuming. Here, we propose a simple approach to identify synthetic polymers dissolved in water using a peptide-based molecular sensor with a fluorophore unit. Supervised machine learning of multiple fluorescence signals from the sensor, which specifically or nonspecifically interacted with the polymers, was applied for polymer classification as a proof of principle demonstration. Aqueous solutions containing different polymers or multiple polymer species with different mixture ratios were identified successfully. We found that fluorophore-introduced biomolecular sensors have great potential to provide discriminative information regarding water-soluble polymers. Our approach based on the discrimination of multiple optical signals of water-soluble polymers from peptide-based molecular sensors through machine learning will be applicable to next-generation sensing systems for polymers in wastewater or natural environments.

Control of parallel versus antiparallel molecular arrangements in crystalline assemblies of alkyl ß-cellulosides.

HYPOTHESIS: The precise control of parallel versus antiparallel molecular arrangements in synthetic assemblies of biorelated molecules is an attractive research focus from both scientific and technological viewpoints. However, little is known about cellulose-based synthetic assemblies. We hypothesized the existence of potential parameters, such as temperature, salt concentration, salt species, and solvent species, for controlling the molecular arrangement in assemblies of alkyl ß-cellulosides with different alkyl chain lengths. EXPERIMENTAL: The self-assembly of alkyl ß-cellulosides was triggered by neutralization-induced water insolubilization. The crystal structures of the cellulose moieties in the assemblies were characterized by attenuated total reflection-Fourier transform infrared absorption spectroscopy and wide-angle X-ray diffraction measurements. The morphologies of the assemblies were also characterized by scanning electron, atomic force, and transmission electron microscopy. FINDINGS: The temperature for the self-assembly, the concentration and species of inorganic salt in the self-assembly solution, and the solvent species (namely, the addition of water-miscible organic solvents into the self-assembly solution) strongly affected the molecular arrangement of the assemblies. The observations suggested that hydrophobic effects between the alkyl groups of the alkyl ß-cellulosides and/or interactions of the alkyl ß-cellulosides with solvent species were potential factors for controlling the molecular arrangement.

Self-assembly of cellulose for creating green materials with tailor-made nanostructures.

Inspired by living systems, biomolecules have been employed in vitro as building blocks for creating advanced nanostructured materials. In regard to nucleic acids, peptides, and lipids, their self-assembly pathways and resulting assembled structures are mostly encoded in their molecular structures. On the other hand, outside of its chain length, cellulose, a polysaccharide, lacks structural diversity; therefore, it is challenging to direct this homopolymer to controllably assemble into ordered nanostructures. Nevertheless, the properties of cellulose assemblies are outstanding in terms of their robustness and inertness, and these assemblies are attractive for constructing versatile materials. In this review article, we summarize recent research progress on the self-assembly of cellulose and the applications of assembled cellulose materials, especially for biomedical use. Given that cellulose is the most abundant biopolymer on Earth, gaining control over cellulose assembly represents a promising route for producing green materials with tailor-made nanostructures.

Structured liquids with interfacial robust assemblies of a nonionic crystalline surfactant.

HYPOTHESIS: The structuring of liquids, that is, the kinetic trapping of nonequilibrium shapes of liquid-liquid interfaces, shows great promise for various applications, especially all-liquid devices. The strategies reported thus far to stabilize such unstable states include interfacial jamming of large colloidal particles and interfacial coassembly of elaborate molecules and/or nanoparticles. However, the structuring of liquids using a simple molecular surfactant has not been sufficiently demonstrated. We hypothesized that a surfactant with strong intermolecular interactions would form interfacial assemblies that behave substantially as solid particles for the structuring of liquids. EXPERIMENTS: n-Octyl cello-oligosaccharide, a novel nonionic surfactant developed recently was explored as a candidate because of the ability of cello-oligosaccharides to form robust crystalline assemblies. Interfacial assembly of the nonionic crystalline surfactant was investigated for various water-organic solvent interfaces via pendant drop tensiometry and emulsification. FINDINGS: The crystalline surfactant was found to self-assemble and form a crystalline monolayer at water-organic solvent interfaces, allowing arrested shape changes of the liquid-liquid interfaces. Irregular-shaped liquid droplets were successfully created under various solution conditions, such as various organic solvents for the oil phase and the water phase with high ionic strengths and harsh pH values.

Discovery of Surfactant-Like Peptides from a Phage-Displayed Peptide Library.

Peptides with specific affinities for various materials have been identified in the past three decades and utilized in materials science and engineering. A peptide's capability to specifically interact with materials is not naturally derived but screened from a biologically constructed peptide library displayed on phages or cells. To date, due to limitations in the screening procedure, the function of screened peptides has been primarily limited to the affinity for target materials. Herein, we demonstrated the screening of surfactant-like peptides from a phage-displayed peptide library. A screened phage clone displaying a peptide showed high activity for accumulating at emulsion surfaces with certain assembled structures, resulting in stable emulsions. The surface tension for the solution of the chemically synthesized peptide decreased with increasing peptide concentration, demonstrating certain surface activity, which corresponded to the ability to decrease the surface tension of liquids (e.g., water), owing to the accumulation of molecules at the air-liquid or liquid-liquid interface. Peptides with a randomized sequence did not lower the surface tension, indicating the essential role of amino acid sequences in surface activity. Our strategy for identifying novel functional peptides from a phage-displayed peptide library can be used to expand the applicability of peptidyl materials and biosurfactants.

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.