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.

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.

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.

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.

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.

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.