Flattened and Wrinkled Encapsulated Droplets: Shape Morphing Induced by Gravity and Evaporation.

We report surprising morphological changes of suspension droplets (containing class II hydrophobin protein HFBI from Trichoderma reesei in water) as they evaporate with a contact line pinned on a rigid solid substrate. Both pendant and sessile droplets display the formation of an encapsulating elastic film as the bulk concentration of solute reaches a critical value during evaporation, but the morphology of the droplet varies significantly: for sessile droplets, the elastic film ultimately crumples in a nearly flattened area close to the apex while in pendant droplets, circumferential wrinkling occurs close to the contact line. These different morphologies are understood through a gravito-elastocapillary model that predicts the droplet morphology and the onset of shape changes, as well as showing that the influence of the direction of gravity remains crucial even for very small droplets (where the effect of gravity can normally be neglected). The results pave the way to control droplet shape in several engineering and biomedical applications.

Strong and Elastic Membranes via Hydrogen Bonding Directed Self-Assembly of Atomically Precise Nanoclusters.

2D nanomaterials have provided an extraordinary palette of mechanical, electrical, optical, and catalytic properties. Ultrathin 2D nanomaterials are classically produced via exfoliation, delamination, deposition, or advanced synthesis methods using a handful of starting materials. Thus, there is a need to explore more generic avenues to expand the feasibility to the next generation 2D materials beyond atomic and molecular-level covalent networks. In this context, self-assembly of atomically precise noble nanoclusters can, in principle, suggest modular approaches for new generation 2D materials, provided that the ligand engineering allows symmetry breaking and directional internanoparticle interactions. Here the self-assembly of silver nanoclusters (NCs) capped with p-mercaptobenzoic acid ligands (Na4 Ag44 -pMBA30 ) into large-area freestanding membranes by trapping the NCs in a transient solvent layer at air-solvent interfaces is demonstrated. The patchy distribution of ligand bundles facilitates symmetry breaking and preferential intralayer hydrogen bondings resulting in strong and elastic membranes. The membranes with Young's modulus of 14.5 ± 0.2 GPa can readily be transferred to different substrates. The assemblies allow detection of Raman active antibiotic molecules with high reproducibility without any need for substrate pretreatment.

Microalgae Chlorella vulgaris and kraft lignin stabilized cellulosic wet foams for camouflage.

Plants, animals, and humans use camouflage to blend in with their surroundings. The camouflage is achieved with different combinations of colors, patterns, and morphologies. In stealth applications, the simplest camouflage uses textiles colored similarly to the environment to create an illusion. However, often, visible light range camouflage is not enough since the multispectral detection technologies of today are readily utilized for identification. Foams can be created with a straightforward fabricating process, and lightweight material exhibits good thermal insulation properties, providing stealth in the infrared light region. Herein, we produce cellulosic wet foams from surfactant and bleached pulp or cellulose nanofibrils. The visible light camouflage is created with green microalgae, Chlorella vulgaris, and brown kraft lignin, which also stabilized the foams. The thermal and spectral camouflage performance of foams was influenced by the cellulose content as well as the stability and water content of foams. Overall, these results give insight into how stability impacts the thermal and spectral properties of wet foams and provide a solid base for further material development to improve camouflage performance. While there is plenty of data on dry foams, the functional behavior of wet foams is currently not well known. Our method, using plant-based components can be exploited in a variety of other applications where simplicity and scalability are important.

Dynamic Assembly of Class II Hydrophobins from T. reesei at the Air-Water Interface.

Class II hydrophobins are amphiphilic proteins produced by filamentous fungi. One of their typical features is the tendency to accumulate at the interface between an aqueous phase and a hydrophobic phase, such as the air-water interface. The kinetics of the interfacial self-assembly of wild-type hydrophobins HFBI and HFBII and some of their engineered variants at the air-water interface were measured by monitoring the accumulated mass at the interface via nondestructive ellipsometry measurements. The resulting mass vs time curves revealed unusual kinetics for a monolayer formation that did not follow a typical Langmuir-type of behavior but had a rather coverage-independent rate instead. Typically, the full surface coverage was obtained at masses corresponding to a monolayer. The formation of multilayers was not observed. Atomic force microscopy revealed formation and growth of non-fusing protein clusters at the interface. The mechanism of the adsorption was studied by varying the structure or charges of the protein or the ionic strength of the subphase, revealing that the lateral interactions between the hydrophobins play a role in their interfacial assembly. Additionally, a theoretical model was introduced to identify the underlying mechanism of the unconventional adsorption kinetics.

Binding Forces of Cellulose Binding Modules on Cellulosic Nanomaterials.

In this study, the interaction forces between different cellulosic nanomaterials and a protein domain belonging to cellulose binding modules family 1 (CBM1) were investigated at the molecular scale. Cellulose binding modules are protein domains found in carbohydrate active enzymes having an affinity toward cellulosic materials. Here, the binding force of a fusion protein containing a cellulose binding module (CBM1) produced recombinantly in E. coli was quantified on different cellulose nanocrystals immobilized on surfaces. Adhesion of the CBM on cellulose with different degrees of crystallinity as well as on chitin nanocrystals was examined. This study was carried out by single molecule force spectroscopy using an atomic force microscope, which enables the detection of binding force of individual molecules. The study contains a preliminary quantification of the interactions at the molecular level that sheds light on the development of new nanocellulose-based nanocomposites with improved strength and elasticity.

Adhesion Properties of Freestanding Hydrophobin Bilayers.

Hydrophobins are a family of small-sized proteins featuring a distinct hydrophobic patch on the protein's surface, rendering them amphiphilic. This particularity allows hydrophobins to self-assemble into monolayers at any hydrophilic/hydrophobic interface. Moreover, stable pure protein bilayers can be created from two interfacial hydrophobin monolayers by contacting either their hydrophobic or their hydrophilic sides. In this study, this is achieved via a microfluidic approach, in which also the bilayers' adhesion energy can be determined. This enables us to study the origin of the adhesion of hydrophobic and hydrophilic core bilayers made from the class II hydrophobins HFBI and HFBII. Using different fluid media in this setup and introducing genetically modified variants of the HFBI molecule, the different force contributions to the adhesion of the bilayer sheets are studied. It was found that in the hydrophilic contact situation, the adhesive interaction was higher than that in the hydrophobic contact situation and could be even enhanced by reducing the contributions of electrostatic interactions. This effect indicates that the van der Waals interaction is the dominant contribution that explains the stability of the observed bilayers.

Pectin and Mucin Enhance the Bioadhesion of Drug Loaded Nanofibrillated Cellulose Films.

PURPOSE: Bioadhesion is an important property of biological membranes, that can be utilized in pharmaceutical and biomedical applications. In this study, we have fabricated mucoadhesive drug releasing films with bio-based, non-toxic and biodegradable polymers that do not require chemical modifications. METHODS: Nanofibrillar cellulose and anionic type nanofibrillar cellulose were used as film forming materials with known mucoadhesive components mucin, pectin and chitosan as functional bioadhesion enhancers. Different polymer combinations were investigated to study the adhesiveness, solid state characteristics, film morphology, swelling, mechanical properties, drug release with the model compound metronidazole and in vitro cytotoxicity using TR146 cells to model buccal epithelium. RESULTS: SEM revealed lamellar structures within the films, which had a thickness ranging 40-240 µm depending on the film polymer composition. All bioadhesive components were non-toxic and showed high adhesiveness. Rapid drug release was observed, as 60-80% of the total amount of metronidazole was released in 30 min depending on the film formulation. CONCLUSIONS: The liquid molding used was a straightforward and simple method to produce drug releasing highly mucoadhesive films, which could be utilized in treating local oral diseases, such as periodontitis. All materials used were natural biodegradable polymers from renewable sources, which are generally regarded as safe.

Elastic and pH-Responsive Hybrid Interfaces Created with Engineered Resilin and Nanocellulose.

We investigated how a genetically engineered resilin fusion protein modifies cellulose surfaces. We characterized the pH-responsive behavior of a resilin-like polypeptide (RLP) having terminal cellulose binding modules (CBM) and showed its binding to cellulose nanofibrils (CNF). Characterization of the resilin fusion protein at different pHs revealed substantial conformational changes of the protein, which were observed as swelling and contraction of the protein layer bound to the nanocellulose surface. In addition, we showed that employment of the modified resilin in cellulose hydrogel and nanopaper increased their modulus of stiffness through a cross-linking effect.

Noncovalent Dispersion and Functionalization of Cellulose Nanocrystals with Proteins and Polysaccharides.

Native cellulose nanocrystals (CNCs) are valuable high quality materials with potential for many applications including the manufacture of high performance materials. In this work, a relatively effortless procedure was introduced for the production of CNCs, which gives a nearly 100% yield of crystalline cellulose. However, the processing of the native CNCs is hindered by the difficulty in dispersing them in water due to the absence of surface charges. To overcome these difficulties, we have developed a one-step procedure for dispersion and functionalization of CNCs with tailored cellulose binding proteins. The process is also applicable for polysaccharides. The tailored cellulose binding proteins are very efficient for the dispersion of CNCs due to the selective interaction with cellulose, and only small fraction of proteins (5-10 wt %, corresponds to about 3 µmol g(-1)) could stabilize the CNC suspension. Xyloglucan (XG) enhanced the CNC dispersion above a fraction of 10 wt %. For CNC suspension dispersed with carboxylmethyl cellulose (CMC) we observed the most long-lasting stability, up to 1 month. The cellulose binding proteins could not only enhance the dispersion of the CNCs, but also functionalize the surface. This we demonstrated by attaching gold nanoparticles (GNPs) to the proteins, thus, forming a monolayer of GNPs on the CNC surface. Cryo transmission electron microscopy (Cryo-TEM) imaging confirmed the attachment of the GNPs to CNC solution conditions.

Enhanced plastic deformations of nanofibrillated cellulose film by adsorbed moisture and protein-mediated interactions.

Biological composites are typically based on an adhesive matrix that interlocks rigid reinforcing elements in fiber composite or brick-and-mortar assemblies. In nature, the adhesive matrix is often made up of proteins, which are also interesting model systems, as they are unique among polymers in that we know how to engineer their structures with atomic detail and to select protein elements for specific interactions with other components. Here we studied how fusion proteins that consist of cellulose binding proteins linked to proteins that show a natural tendency to form multimer complexes act as an adhesive matrix in combination with nanofibrillated cellulose. We found that the fusion proteins are retained with the cellulose and that the proteins mainly affect the plastic yield behavior of the cellulose material as a function of water content. Interestingly, the proteins increased the moisture absorption of the composite, but the well-known plastifying effect of water was clearly decreased. The work helps to understand the functional basis of nanocellulose composites as materials and aims toward building model systems for molecular biomimetic materials.

The Effect of Hydrophobin Protein on Conductive Properties of Carbon Nanotube Field-Effect Transistors: First Study on Sensing Mechanism.

Hydrophobin is a surface active protein having both hydrophobic and hydrophilic functional domains which has previously been used for functionalization and solubilization of graphene and carbon nanotubes. In this work, field-effect transistors based on single nanotubes have been employed for electronic detection of hydrophobin protein in phosphate buffer solution. Individual nanotubes, single- and multiwalled, are characterized by atomic force microscopy after being immersed in protein solution, showing a relatively dense coverage with hydrophobin. We have studied aspects such as nanotube length (0.3-1.2 µm) and the hysteresis effect in the gate voltage dependent conduction. When measured in ambient condition after the exposure to hydrophobin, the resistance increase has a strong dependence on the nanotube length, which we ascribe to mobility degradation and localization effects. The change could be exceptionally large when measured in-situ in solution and at suitable gate voltage conditions, which is shown to relate to the different mechanism behind the hysteresis effect.

Modular architecture of protein binding units for designing properties of cellulose nanomaterials.

Molecular biomimetic models suggest that proteins in the soft matrix of nanocomposites have a multimodular architecture. Engineered proteins were used together with nanofibrillated cellulose (NFC) to show how this type of architecture leads to function. The proteins consist of two cellulose-binding modules (CBM) separated by 12-, 24-, or 48-mer linkers. Engineering the linkers has a considerable effects on the interaction between protein and NFC in both wet colloidal state and a dry film. The protein optionally incorporates a multimerizing hydrophobin (HFB) domain connected by another linker. The modular structure explains effects in the hydrated gel state, as well as the deformation of composite materials through stress distribution and crosslinking. Based on this work, strategies can be suggested for tuning the mechanical properties of materials through the coupling of protein modules and their interlinking architectures.

Intravenous delivery of hydrophobin-functionalized porous silicon nanoparticles: stability, plasma protein adsorption and biodistribution.

Rapid immune recognition and subsequent elimination from the circulation hampers the use of many nanomaterials as carriers to targeted drug delivery and controlled release in the intravenous route. Here, we report the effect of a functional self-assembled protein coating on the intravenous biodistribution of (18)F-labeled thermally hydrocarbonized porous silicon (THCPSi) nanoparticles in rats. (18)F-Radiolabeling enables the sensitive and easy quantification of nanoparticles in tissues using radiometric methods and allows imaging of the nanoparticle biodistribution with positron emission tomography. Coating with Trichoderma reesei HFBII altered the hydrophobicity of (18)F-THCPSi nanoparticles and resulted in a pronounced change in the degree of plasma protein adsorption to the nanoparticle surface in vitro. The HFBII-THCPSi nanoparticles were biocompatible in RAW 264.7 macrophages and HepG2 liver cells making their intravenous administration feasible. In vivo, the distribution of the nanoparticles between the liver and spleen, the major mononuclear phagocyte system organs in the body, was altered compared to that of uncoated (18)F-THCPSi. Identification of the adsorbed proteins revealed that certain opsonins and apolipoproteins are enriched in HFBII-functionalized nanoparticles, whereas the adsorption of abundant plasma components such as serum albumin and fibrinogen is decreased.

Self-assembly of class II hydrophobins on polar surfaces.

Hydrophobins are structural proteins produced by filamentous fungi that are amphiphilic and function through self-assembling into structures such as membranes. They have diverse roles in the growth and development of fungi, for example in adhesion to substrates, for reducing surface tension to allow aerial growth, in forming protective coatings on spores and other structures. Hydrophobin membranes at the air-water interface and on hydrophobic solids are well studied, but understanding how hydrophobins can bind to a polar surface to make it more hydrophobic has remained unresolved. Here we have studied different class II hydrophobins for their ability to bind to polar surfaces that were immersed in buffer solution. We show here that the binding under some conditions results in a significant increase of water contact angle (WCA) on some surfaces. The highest contact angles were obtained on cationic surfaces where the hydrophobin HFBI has an average WCA of 62.6° at pH 9.0, HFBII an average of 69.0° at pH 8.0, and HFBIII had an average WCA of 61.9° at pH 8.0. The binding of the hydrophobins to the positively charged surface was shown to depend on both pH and ionic strength. The results are significant for understanding the mechanism for formation of structures such as the surface of mycelia or fungal spore coatings as well as for possible technical applications.

Facile method for stiff, tough, and strong nanocomposites by direct exfoliation of multilayered graphene into native nanocellulose matrix.

Nanofibrillated cellulose (NFC) is a natural fibrillar material with exceptionally high mechanical properties. It has, however, been exceedingly difficult to achieve nanocomposites with drastically improved mechanical properties by dispersing NFC as random networks to polymer matrices, even using compatibilization. We show nanocomposites consisting of aligned assemblies of multilayered graphene and NFC with excellent tensile mechanical properties without any surface treatments. The optimum composition was found at 1.25 wt % graphene multilayers, giving a Young's modulus of 16.9 GPa, ultimate strength of 351 MPa, strain of 12%, and work-of-fracture of 22.3 MJ m(-3). This combines high strength with relatively high toughness and is obtained by direct exfoliation of graphite within aqueous hydrogels of NFC where an optimum sonication power is described. The results suggest the existence of an attractive interaction between multilayered graphene flakes and cellulose. Aligned assemblies are obtained by removal of water by filtration. The concept can be beneficial for applications because it results in high mechanical properties by a simple and environmentally green process.

Entangled and colloidally stable microcrystalline cellulose matrices in controlled drug release.

Drug release from a new type of matrix material consisting of partially fibrillated microcrystalline cellulose was investigated. A mechanical treatment of novel AaltoCell™ cellulose microcrystals caused partial opening of the nanofibrillary structure of the cellulose particles and entanglement of individual particles led into formation of an elastic network of microcrystalline cellulose. The rheological properties of the stable hydrogel-like materials were characterised by shear rheometry. Model compounds metronidazole and lysozyme were successfully employed in drug release experiments carried out by delignified (bleached) and lignin-containing matrices. The viscosity as well as the lignin-content played a role in the release dynamics of the drugs. Microcrystalline AaltoCell™ was proven as high-performing material for diffusion controlled release of the chosen model compounds and can be seen as a safe and economical alternative for novel matrix materials such as nanocellulose or cellulose derivatives.

Advanced Materials through Assembly of Nanocelluloses.

There is an emerging quest for lightweight materials with excellent mechanical properties and economic production, while still being sustainable and functionalizable. They could form the basis of the future bioeconomy for energy and material efficiency. Cellulose has long been recognized as an abundant polymer. Modified celluloses were, in fact, among the first polymers used in technical applications; however, they were later replaced by petroleum-based synthetic polymers. Currently, there is a resurgence of interest to utilize renewable resources, where cellulose is foreseen to make again a major impact, this time in the development of advanced materials. This is because of its availability and properties, as well as economic and sustainable production. Among cellulose-based structures, cellulose nanofibrils and nanocrystals display nanoscale lateral dimensions and lengths ranging from nanometers to micrometers. Their excellent mechanical properties are, in part, due to their crystalline assembly via hydrogen bonds. Owing to their abundant surface hydroxyl groups, they can be easily modified with nanoparticles, (bio)polymers, inorganics, or nanocarbons to form functional fibers, films, bulk matter, and porous aerogels and foams. Here, some of the recent progress in the development of advanced materials within this rapidly growing field is reviewed.

Coacervation of resilin fusion proteins containing terminal functionalities.

Liquid-liquid phase transition known as coacervation of resilin-like-peptide fusion proteins containing different terminal domains were investigated. Two different modular proteins were designed and produced and their behavior were compared to a resilin-like-peptide without terminal domains. The size of the particle-like coacervates was modulated by the protein concentration, pH and temperature. The morphology and three-dimensional (3D) structural details of the coacervate particles were investigated by cryogenic transmission electron microscopy (cryo-TEM) and tomography (cryo-ET) reconstruction. Selective adhesion of the coacervates on cellulose and graphene surfaces was demonstrated.

Pure Protein Bilayers and Vesicles from Native Fungal Hydrophobins.

Pure protein bilayers and vesicles are formed using the native, fungal hydrophobin HFBI. Bilayers with hydrophobic (red) and hydrophilic (blue) core are produced and, depending on the type of core, vesicles in water, oily media, and even in air can be created using microfluidic jetting. Vesicles in water are even able to incorporate functional gramicidin A pores.

Single-Molecule Force Spectroscopy Study on Modular Resilin Fusion Protein.

The adhesive and mechanical properties of a modular fusion protein consisting of two different types of binding units linked together via a flexible resilin-like-polypeptide domain are quantified. The adhesive domains have been constructed from fungal cellulose-binding modules (CBMs) and an amphiphilic hydrophobin HFBI. This study is carried out by single-molecule force spectroscopy, which enables stretching of single molecules. The fusion proteins are designed to self-assemble on the cellulose surface, leading into the submonolayer of proteins having the HFBI pointing away from the surface. A hydrophobic atomic force microscopy (AFM) tip can be employed for contacting and lifting the single fusion protein from the HFBI-functionalized terminus by the hydrophobic interaction between the tip surface and the hydrophobic patch of the HFBI. The work of rupture, contour length at rupture and the adhesion forces of the amphiphilic end domains are evaluated under aqueous environment at different pHs.