Nanocellulose aerogels as 3D amyloid templates.

Proteins in solution tend to coat solid surfaces upon exposure. Depending on the nature of the surface, the environmental conditions, and the nature of the protein these adsorbed proteins may self-assemble into ordered, fibre-like structures called amyloids. Nanoparticulate surfaces, with their high surface to volume ratio, are particularly favourable to amyloid formation. Most prior research has focussed on either inorganic or organic nanoparticles in solution. In this research, we instead focus on aerogels created from TEMPO-oxidized cellulose nanofibers (TO-CNF) to serve as bio-based, three-dimensional amyloid templates with a tuneable surface chemistry. Previous research on the use of cellulose as a protein adsorption template has shown no evidence of a change in the secondary protein structure. Herein, however, with the aid of the reducing agent TCEP, we were able to induce the formation of amyloid-like 'worms' on the surface of TO-CNF aerogels. Furthermore, we demonstrate that the addition of the TO-CNF aerogel can also induce bulk aggregation under conditions where it previously did not exist. Finally, we show that the addition of the aerogel increases the rate of 'worm' formation in conditions where previous research has found a long lag-phase. Therefore, TO-CNF aerogels are shown to be excellent templates for inducing ordered protein aggregation.

2D foam film coating of antimicrobial lysozyme amyloid fibrils onto cellulose nanopapers.

Amyloid fibrils made from inexpensive hen egg white lysozyme (HEWL) are bio-based, bio-degradable and bio-compatible colloids with broad-spectrum antimicrobial activity, making them an attractive alternative to existing small-molecule antibiotics. Their surface activity leads to the formation of 2D foam films within a loop, similar to soap films when blowing bubbles. The stability of the foam was optimized by screening concentration and pH, which also revealed that the HEWL amyloid foams were actually stabilized by unconverted peptides unable to undergo amyloid self-assembly rather than the fibrils themselves. The 2D foam film was successfully deposited on different substrates to produce a homogenous coating layer with a thickness of roughly 30 nm. This was thick enough to shield the negative charge of dry cellulose nanopaper substrates, leading to a positively charged HEWL amyloid coating. The coating exhibited a broad-spectrum antimicrobial effect based on the interactions with the negatively charged cell walls and membranes of clinically relevant pathogens (Staphylococcus aureus, Escherichia coli and Candida albicans). The coating method presented here offers an alternative to existing techniques, such as dip and spray coating, in particular when optimized for continuous production. Based on the facile preparation and broad spectrum antimicrobial performance, we anticipate that these biohybrid materials could potentially be used in the biomedical sector as wound dressings.

Nanocomposites of cellulose nanofibers incorporated with carvacrol via stabilizing octenyl succinic anhydride-modified ɛ-polylysine.

Food packaging plays an extremely important role in the global food chain, allowing for products to be shipped across long distances without spoiling. However, there is an increased need to both reduce plastic waste caused by traditional single-use plastic packaging and improve the overall functionality of packaging materials to extend shelf-life even further. Herein, we investigate composite mixtures based on cellulose nanofibers and carvacrol via stabilizing octenyl-succinic anhydride-modified epsilon polylysine (MɛPL-CNF) for active food packaging applications. The effects of epsilon polylysine (εPL) concentration and modification with octenyl-succinic anhydride (OSA) and carvacrol are evaluated with respect to composites morphology, mechanical, optical, antioxidant, and antimicrobial properties. We find that both increased εPL concentration and modification with OSA and carvacrol lead to films with increased antioxidant and antimicrobial properties, albeit at the expense of reduced mechanical performance. Importantly, when sprayed onto the surface of sliced apples, MεPL-CNF-mixtures are able to successfully delay/hinder enzymatic browning, suggesting the potential of such materials for a range of active food packaging applications.

Amyloid fibril-nanocellulose interactions and self-assembly.

Amyloid fibrils from inexpensive food proteins and nanocellulose are renewable and biodegradable materials with broad ranging applications, such as water purification, bioplastics and biomaterials. To improve the mechanical properties of hybrid amyloid-nanocellulose materials, their colloidal interactions need to be understood and tuned. A combination of turbidity and zeta potential measurements, rheology and atomic force microscopy point to the importance of electrostatic interactions. These interactions lead to entropy-driven polyelectrolyte complexation for positively charged hen egg white lysozyme (HEWL) amyloids with negatively charged nanocellulose. The complexation increased the elasticity of the amyloid network by cross-linking individual fibrils. Scaling laws suggest different contributions to elasticity depending on nanocellulose morphology: cellulose nanocrystals induce amyloid bundling and network formation, while cellulose nanofibrils contribute to a second network. The contribution of the amyloids to the elasticity of the entire network structure is independent of nanocellulose morphology and agrees with theoretical scaling laws. Finally, strong and almost transparent hybrid amyloid-nanocellulose gels were prepared in a slow self-assembly started from repulsive co-dispersions above the isoelectric point of the amyloids, followed by dialysis to decrease the pH and induce amyloid-nanocellulose attraction and cross-linking. In summary, the gained knowledge on colloidal interactions provides an important basis for the design of functional biohybrid materials based on these two biopolymers.

The Colloidal Properties of Nanocellulose.

Nanocelluloses are anisotropic nanoparticles of semicrystalline assemblies of glucan polymers. They have great potential as renewable building blocks in the materials platform of a more sustainable society. As a result, the research on nanocellulose has grown exponentially over the last decades. To fully utilize the properties of nanocelluloses, a fundamental understanding of their colloidal behavior is necessary. As elongated particles with dimensions in a critical nanosize range, their colloidal properties are complex, with several behaviors not covered by classical theories. In this comprehensive Review, we describe the most prominent colloidal behaviors of nanocellulose by combining experimental data and theoretical descriptions. We discuss the preparation and characterization of nanocellulose dispersions, how they form networks at low concentrations, how classical theories cannot describe their behavior, and how they interact with other colloids. We then show examples of how scientists can use this fundamental knowledge to control the assembly of nanocellulose into new materials with exceptional properties. We hope aspiring and established researchers will use this Review as a guide.

Phase Behavior, Self-Assembly, and Adhesive Potential of Cellulose Nanocrystal-Bovine Serum Albumin Amyloid Composites.

Structural organization is ubiquitous throughout nature and contributes to the outstanding mechanical/adhesive performance of organisms including geckoes, barnacles, and crustaceans. Typically, these types of structures are composed of polysaccharide and protein-based building blocks, and therefore, there is significant research interest in using similar building blocks in the fabrication of high-performance synthetic materials. Via evaporation-induced self-assembly, the organization of cellulose nanocrystals (CNCs) into a chiral nematic regime results in the formation of structured CNC films with prominent mechanical, optical, and photonic properties. However, there remains an important knowledge gap in relating equilibrium suspension behavior to dry film structuring and other functional properties of CNC-based composite materials. Herein, we systematically investigate the phase behavior of composite suspensions of rigid CNCs and flexible bovine serum albumin (BSA) amyloids in relation to their self-assembly into ordered films and structural adhesives. Increasing the concentration of BSA amyloids in the CNC suspensions results in a clear decrease in the anisotropic fraction volume percent via the preferential accumulation of BSA amyloids in the isotropic regime (as a result of depletion interactions). This translates to a blue shift or compression of the chiral nematic pitch in dried films. Finally, we also demonstrate the synergistic adhesive potential of CNC-BSA amyloid composites, with ultimate lap shear strengths in excess of 500 N/mg. We anticipate that understanding the systematic relationships between material interactions and self-assembly in suspension such as those investigated here will pave the way for a new generation of structured composite materials with a variety of enhanced functionalities.

Controlling lipid crystallization across multiple length scales by directed shear flow.

The crystallization behavior of lipids is relevant in many fields such as adipose tissue formation and regeneration, forensic investigations and food production. Using a lipid model system composed of triacylglycerols, we study the formation of crystalline structures under laminar shear flows across various length scales by polarized light-, scanning electron-, and atomic force microscopy, as well as laser diffraction spectroscopy. The shear rate during crystallization γ̇cryst influences the acyl-chain length structure and promotes domain growth into the flow direction thereby transforming the crystallites from oblate into prolate particles. Concentration dependent aggregation of crystallites into clusters is the rate limiting step for floc and floc network formation. At high γ̇cryst, fast crystallite cluster formation at smaller equilibrium diameters is promoted. The high crystallite cluster concentration induces their aggregation into flocs which form weak networks. At low γ̇cryst, floc generation is limited by the low amount of crystallite clusters leading to slow growth of larger flocs and forming of strong networks. The findings in this work have potential implications ranging from the design of injectable soft tissue fillers for adipose tissue regeneration, to the crystalline network formation in microorganism derived lipids, up to a more energy-efficient production of chocolate confectionery.

The rheology and foamability of crystal-melt suspensions composed of triacylglycerols.

The rheology of triacylglycerol (TAG) crystal-melt suspensions (CMSs) consisting of anhydrous milk fat (AMF), cocoa butter (CB), and palm kernel oil (PKO) as function of crystallization shear rate cryst and crystal volume fraction ΦSFC is investigated by in-line ultrasound velocity profiling - pressure difference (UVP-PD) rheometry. Measurements up to ΦSFC = 8.8% are presented. Below the percolation threshold Φc, no yield stress τ0 is observed and the viscosity η scales linearly with ΦSFC. Above Φc, a non-linear dependency of both τ0 and η as function of ΦSFC is apparent. For AMF and CB, the increase in cryst leads to a decrease in η and τ0 as function of ΦSFC, whereas for PKO based CMSs the opposite is the case. Scanning electron microscopy (SEM) and polarized light microscopy (PLM) relate these rheological findings to the microstructure of the investigated CMSs by taking the effective aspect ratio aeff and the concept of the effective crystal volume fraction ΦeffSFC into account. Foam formation by dynamically enhanced membrane foaming (DEMF) is performed directly after crystallization and reveals that depending on the CMS rheology and crystallite-, crystallite cluster- and crystal floc microstructure, a wide range of gas volume fractions between 0.05-0.6 are achievable.

Biohybrid Nanocellulose-Lysozyme Amyloid Aerogels via Electrostatic Complexation.

Modern science is increasingly turning to nature for inspiration to design sustainable biomaterials in a smart and effective way. Herein, we describe biohybrid aerogels based on electrostatic complexation between cellulose and proteins-two of the most abundant natural polymers on Earth. The effects of both particle surface charge and particle size are investigated with respect to aerogel properties including the morphology, surface area, stability, and mechanical strength. Specifically, negatively charged nanocellulose (cellulose nanocrystals and cellulose nanofibers) and positively charged lysozyme amyloid fibers (full-length and shortened via sonication) are investigated in the preparation of fibrillar aerogels, whereby the nanocellulose component was found to have the largest effect on the resulting aerogel properties. Although electrostatic interactions between these two classes of charged nanoparticles allow us to avoid the use of any cross-linking agents, the resulting aerogels demonstrate a simple additive performance as compared to their respective single-component aerogels. This lack of synergy indicates that although electrostatic complexation certainly leads to the formation of local aggregates, these interactions alone may not be strong enough to synergistically improve bulk aerogel properties. Nevertheless, the results reported herein represent a critical step toward a broader understanding of biohybrid materials based on cellulose and proteins.

Ultrafine Cellulose Nanofiber-Assisted Physical and Chemical Cross-Linking of MXene Sheets for Electromagnetic Interference Shielding.

Transition metal carbides and nitrides (MXenes) have shown great potential for constructing thin, high-performance electromagnetic interference (EMI) shields. The challenges with these materials involve the weak interfacial interactions of MXenes, which results in inferior mechanical properties and structure of the MXene films and a conductivity/EMI shielding performance decay related to the poor MXene oxidation stability. Numerous efforts have been devoted to improving the mechanical properties or oxidation stability of the films, which always comes at the expense of EMI shielding performance. Here, ultrafine (≈1.4 nm) cellulose nanofibers are employed to achieve physical and chemical dual cross-linking of MXene (PC-MXene) nanosheets. The procedure involves drying of flexible and highly conductive PC-MXene films at ambient pressure and is energy-efficient and scalable. Compared to the MXene films, the PC-MXene films show significantly improved mechanical strength, hydrophobicity, oxidation stability, and are waterproof, without compromising the excellent EMI shielding effectiveness (SE). Moreover, the freestanding PC-MXene films reach a thickness of merely 0.9 µm and exhibit a high SE of 33.3 dB, which cannot be achieved by pure MXene films. This leads to ultrahigh thickness-specific SE and surface-specific SE values of 37 000 dB mm-1 and 148 000 dB cm2  g-1 respectively, significantly surpassing those of previously reported MXene-based films.

Self-Assembly Pathways and Antimicrobial Properties of Lysozyme in Different Aggregation States.

Antimicrobial resistance in microorganisms will cause millions of deaths and pose a vast burden on health systems; therefore, alternatives to existing small-molecule antibiotics have to be developed. Lysozyme is an antimicrobial enzyme and has broad-spectrum antimicrobial activity in different aggregated forms. Here, we propose a reductive pathway to obtain colloidally stable amyloid-like worm-shaped lysozyme nanoparticles (worms) from hen egg white lysozyme (HEWL) and compare them to amyloid fibrils made in an acid hydrolysis pathway. The aggregation of HEWL into worms follows strongly pH-dependent kinetics and induces a structural transition from α-helices to ß-sheets. Both HEWL worms and amyloid fibrils show broad-spectrum antimicrobial activity against the bacteria Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-negative), and the fungus Candida albicans. The colloidal stability of the worms allows the determination of minimum inhibitory concentrations, which are lower than that for native HEWL in the case of S. aureus. Overall, amyloid fibrils have the strongest antimicrobial effect, likely due to the increased positive charge compared to native HEWL. The structural and functional characterizations of HEWL worms and amyloids investigated herein are critical for understanding the detailed mechanisms of antimicrobial activity and opens up new avenues for the design of broad-spectrum antimicrobial materials for use in various applications.

Chitin-amyloid synergism and their use as sustainable structural adhesives.

Structural adhesives are relevant to many engineering applications, especially those requiring load-bearing joints with high lap shear strength. Typical adhesives are synthesized from acrylics, epoxies, or urethanes, which may pose a burden to sustainability and the environment. In nature, the interfacial interactions between chitin and proteins are used for structural purposes and as a bio-cement, resulting in materials with properties unmatched by their man-made counterparts. Herein, we show that related supramolecular interactions can be harnessed to develop high strength green adhesives based on chitin nanocrystals (ChNCs), isolated from shrimp shells, and hen egg white lysozyme (HEWL) used in its monomeric or amyloid forms. Consolidation of the bicomponent suspensions, placed between glass substrates, results in long-range ordered superstructures. The formation of these structures is evaluated by surface energy considerations, followed by scanning electron, atomic force, and polarized microscopies of the consolidated materials. For 0.8 mg of bio-adhesive (lysozyme, ChNCs or their composites), lap shear loads of over 300 N are reached. Such remarkable adhesion reaches maximum values at protein-to-ChNC ratios below 1 : 4, reflecting the synergy established between the components (ca. 25% higher load compared to ChNCs, the strongest single component). We put the observed adhesive performance in perspective by comparing the lap-shear performance with current research on green supramolecular adhesives using natural biopolymers. The results are discussed in the context of current efforts to standardize the measurement of adhesive strength and bond preparation. The latter is key to formalizing the metrology and materials chemistry of bio-based adhesives. The proposed all-green system is expected to expand current developments in the design of bio-based adhesives.

Terahertz Birefringent Biomimetic Aerogels Based on Cellulose Nanofibers and Conductive Nanomaterials.

Biomimetic, lamellar, and highly porous transition-metal carbide (MXene) embedded cellulose nanofiber (CNF) aerogels are assembled by a facile bidirectional freeze-drying approach. The biopolymer aerogels have large-scale, parallel-oriented micrometer-sized pores and show excellent mechanical strength and flexibility, tunable electrical properties, and low densities (2.7-20 mg/cm3). The CNF, MXene, and lamellar pores are efficiently utilized to endow the aerogels with exceptionally high birefringence in the terahertz (THz) regime. Birefringence values as high as 0.09-0.27 at 0.4 THz are achieved, which is comparable to most commercial THz birefringent materials such as liquid crystals, which suffer from fast disintegration, high cost, and complicated preparation processes. Empirical modeling for different MXene contents and an experimental comparison with silver nanowire or carbon nanotube embedded CNF aerogels suggest that the intrinsic conductivity and content of embedded nanomaterials, the aerogel porosity, and the lamellar cell walls can affect the optical properties such as the THz birefringence and absorption. The determination of optical anisotropy in the biopolymer aerogels lays a foundation for further exploration of ultralight, freestanding, and low-cost biomimetic porous architecture-based THz devices.

Globular protein assembly and network formation at fluid interfaces: effect of oil.

The formation of viscoelastic networks at fluid interfaces by globular proteins is essential in many industries, scientific disciplines, and biological processes. However, the effect of the oil phase on the structural transitions of proteins, network formation, and layer strength at fluid interfaces has received little attention. Herein, we present a comprehensive study on the effect of oil polarity on globular protein networks. The formation dynamics and mechanical properties of the interfacial networks of three different globular proteins (lysozyme, ß-lactoglobulin, and bovine serum albumin) were studied with interfacial shear and dilatational rheometry. Furthermore, the degree of protein unfolding at the interfaces was evaluated by subsequent injection of disulfide bonds reducing dithiothreitol. Finally, we measured the interfacial layer thickness and protein immersion into the oil phase with neutron reflectometry. We found that oil polarity significantly affects the network formation, the degree of interfacial protein unfolding, interfacial protein location, and the resulting network strength. These results allow predicting emulsion stabilization of proteins, tailoring interfacial layers with desired mechanical properties, and retaining the protein structure and functionality upon adsorption.

Nanocellulose-lysozyme colloidal gels via electrostatic complexation.

Biohybrid colloids were fabricated based on electrostatic complexation between anionic TEMPO-oxidized cellulose nanofibrils (TO-CNF) and cationic hen egg white lysozyme (HEWL). By altering the loading of HEWL, physical colloidal complexes can be obtained at a relatively low concentration of TO-CNF (0.1 wt%). At neutral pH, increasing the HEWL loading induces an increase in charge screening, as probed by zeta-potential, resulting in enhanced TO-CNF aggregation and colloidal gel formation. Systematic rheological testing shows that mechanical reinforcement of the prepared biohybrid gels is easily achieved by increasing the loading of HEWL. However, due to the relatively weak nature of electrostatic complexation, the formed colloidal gels exhibit partial destruction when subjected to cyclic shear stresses. Still, they resist thermo-cycling up to 90 °C. Finally, the pH responsiveness of the colloidal complex gels was demonstrated by adjusting pH to above and below the isoelectric point of HEWL, representing a facile mechanism to tune the gelation of TO-CNF/HEWL complexes. This work highlights the potential of using electrostatic complexation between HEWL and TO-CNF to form hybrid colloids, and demonstrates the tunability of the colloidal morphology and rheology by adjusting the ratio between the two components and the pH.

Assembly of Cellulose Nanocrystal-Lysozyme Composite Films with Varied Lysozyme Morphology.

In modern society, there is a constant need for developing reliable, sustainable, and cost-effective antibacterial materials. Here, we investigate the preparation of cellulose nanocrystal (CNC)-lysozyme composite films via the well-established method of evaporation-induced self-assembly. We consider the effects of lysozyme concentration and aggregation state (native lysozyme, lysozyme amyloid fibers, and sonicated lysozyme amyloid fibers) on suspension aggregation and film-forming ability. Although at higher lysozyme loading levels (ca. 10 wt %), composite films lost their characteristic chiral nematic structuring, these films demonstrated improved mechanical properties and antibacterial activity with respect to CNC-only films, regardless of lysozyme aggregation state. We anticipate that the results presented herein could also contribute to the preparation of other CNC-protein-based materials, including films, hydrogels, and aerogels, with improved mechanical performance and antibacterial activity.