Proteins for Applied and Functional Materials.

Shifting from a petroleum-based plastic society to a newer one built on circular economy principles requires maximizing the use of renewable resources and resolving the challenges that come with their use. Biopolymers have taken an important role in the design of biobased materials with functional properties, especially those derived from biomass available at a large scale. A number of recent studies have shown how proteins have a new dimension in developing functional materials, taking a step forward from their traditional use in food and biomedicine. Correlating the amino acidic profile of proteins at the nanoscale with their thermomechanical properties at the macroscale enables us to translate these precision polymers into a versatile design of materials, targeting large-scale applications such as foams and food packaging. Moreover, the advances in understanding proteins from a bottom-up perspective reached promising achievements for their use in applications that were not foreseen before, including biosensors, optoelectronics, and semiconductors.

From Soy Waste to Bioplastics: Industrial Proof of Concept.

The global plastic waste problem is pushing for the development of sustainable alternatives, encouraged by stringent regulations combined with increased environmental consciousness. In response, this study presents an industrial-scale proof of concept to produce self-standing, transparent, and flexible bioplastic films, offering a possible solution to plastic pollution and resource valorization. We achieve this by combining amyloid fibrils self-assembled from food waste with methylcellulose and glycerol. Specifically, soy whey and okara, two pivotal protein-rich byproducts of tofu manufacturing, emerge as sustainable and versatile precursors for amyloid fibril formation and bioplastic development. An exhaustive industrial-scale feasibility study involving the transformation of 500 L of soy whey into ∼1 km (27 kg) of bioplastic films underscores the potential of this technology. To extend the practicality of our approach, we further processed a running kilometer of film at the industrial scale into transparent windows for paper-based packaging. The mechanical properties and the water interactions of the novel film are tested and compared with those of commercially used plastic films. By pioneering the large-scale production of biodegradable bioplastics sourced from food byproducts, this work not only simultaneously addresses the dual challenges of plastic pollution and food waste but also practically demonstrates the feasibility of biopolymeric building block valorization for the development of sustainable materials in real-world scenarios.

Water-processable, biodegradable and coatable aquaplastic from engineered biofilms.

Petrochemical-based plastics have not only contaminated all parts of the globe, but are also causing potentially irreversible damage to our ecosystem because of their non-biodegradability. As bioplastics are limited in number, there is an urgent need to design and develop more biodegradable alternatives to mitigate the plastic menace. In this regard, we report aquaplastic, a new class of microbial biofilm-based biodegradable bioplastic that is water-processable, robust, templatable and coatable. Here, Escherichia coli was genetically engineered to produce protein-based hydrogels, which are cast and dried under ambient conditions to produce aquaplastic, which can withstand strong acid/base and organic solvents. In addition, aquaplastic can be healed and welded to form three-dimensional architectures using water. The combination of straightforward microbial fabrication, water processability and biodegradability makes aquaplastic a unique material worthy of further exploration for packaging and coating applications.

Highly Adhesive Amyloid-Polyphenol Hydrogels for Cell Scaffolding.

Rationally designing microstructures of soft hydrogels for specific biological functionalization is a challenge in tissue engineering applications. A novel and affordable soft hydrogel scaffold is constructed here by incorporating polyphenol modules with lysozyme amyloid fibrils (Lys AFs) via non-covalent self-assembly. Embedded polyphenols not only trigger hydrogel formation but also determine gel behavior by regulating the polyphenol gallol density and complex ratio. The feasibility of using a polyphenol-Lys AF hydrogel as a biocompatible cell scaffold, which is conducive to cell proliferation and spreading, is also shown. Notably, introducing polyphenols imparts the corresponding hydrogels a superior cell bioadhesive efficiency without further biofunctional decoration and thus may be successfully employed in both healthy and cancer cell lines. Confocal laser scanning microscopy also reveals that the highly expressed integrin-mediated focal adhesions form due to stimulation of the polyphenol-AF composite hydrogel, direct cell adhesion, proliferation, and spreading. Overall, this work constitutes a significant step forward in creating highly adhesive tissue culture platforms for in vitro culture of different cell types and may greatly expand prospects for future biomaterial design and development.

Sustainable Removal of Microplastics and Natural Organic Matter from Water by Coagulation-Flocculation with Protein Amyloid Fibrils.

Water contamination is a global threat due to its damaging effects on the environment and human health. Water pollution by microplastics (MPs), dissolved natural organic matter (NOM), and other turbid particles is ubiquitous in water treatment. Here, we introduce lysozyme amyloid fibrils as a novel natural bio-flocculant and explore their ability to flocculate and precipitate the abovementioned undesired colloidal objects. Thanks to their positively charged surface in a very broad range of pH, lysozyme amyloid fibrils show an excellent turbidity removal efficiency of 98.2 and 97.9% for dispersed polystyrene MPs and humic acid (HA), respectively. Additionally, total organic carbon measurements confirm these results by exhibiting removal efficiencies of 93.4 and 61.9% for purifying water from dispersed MPs and dissolved HA, respectively. The comparison among amyloid fibrils, commercial flocculants (FeCl3 and polyaluminumchloride), and native lysozyme monomers points to the superiority of amyloid fibrils at the same dosage and sedimentation time. Furthermore, the turbidity of pristine and MP-spiked wastewater and lake water decreased after the treatment by amyloid fibrils, validating their coagulation-flocculation performance under natural conditions. All these results demonstrate lysozyme amyloid fibrils as an appropriate natural bio-flocculant for removing dispersed MPs, NOM, and turbid particles from water.

Designing Cellulose Nanofibrils for Stabilization of Fluid Interfaces.

Particles of biological origin are of increasing interest for the Pickering stabilization of biocompatible and environmentally friendly foams and emulsions. Cellulose nanofibrils (CNFs) are readily employed in that respect; however, the underlying mechanisms of interfacial stabilization remain widely unknown. For instance, it has not been resolved why CNFs are unable to stabilize foams while efficiently stabilizing emulsions. Here, we produce CNFs with varying contour lengths and charge densities to investigate their behavior at the air-water phase boundary. CNFs adsorbing at the air-water interface reduce surface tension and form interfacial layers with high viscoelasticity, which are attributed to the thermodynamic and kinetic stability of CNF-stabilized colloids, respectively. CNF adsorption is accelerated and higher surface pressures are attained at lower charge densities, indicating that CNF surface charges limit both adsorption and surface coverage. CNFs form monolayers with ∼40% coverage and are primarily wetted by the aqueous phase indicating a contact angle <90°, as demonstrated by neutron reflectometry. The low contact angle at the air-water interface is energetically unfavorable for adsorbed CNFs, which is proposed as a potential explanation why CNFs show poor foaming capacity.

Nanostructural Properties and Twist Periodicity of Cellulose Nanofibrils with Variable Charge Density.

Cellulose nanofibrils (CNFs) are a renewable and facile to produce nanomaterial that recently gained a lot of attention in soft material research. The nanostructural properties of the fibrils largely determine their self-organizing functionalities, and the ability to tune the CNF nanostructure through control of the processing parameters is therefore crucial for developing new applications. In this study, we systematically altered the CNF production parameters (i.e., variation in cellulose source, chemical, and mechanical treatment) to observe their impact on the nanostructural properties of the resulting fibrils. Atomic force microscopy (AFM) allowed detailed topological examination of individual CNFs to elucidate fibril properties such as contour length, kink distribution and the right-handed twist periodicity of individual fibrils. Statistical analysis revealed a large dependency of the fibril properties on the industrial treatment of the cellulose source material. Our results furthermore confirm that the average charge density of the fibrils regulates both contour length and twist periodicity and, thus, has a very strong impact on the final morphology of CNFs. These results provide a route to tune the detailed nanostructure of CNFs with potential impact on the self-organization of these biological colloids and their optimal use in new nanomaterials.

Creating gradients of amyloid fibrils from the liquid-liquid interface.

We report a method to deposit amyloid fibrils on a substrate creating gradients in orientation and coverage on demand. For this purpose, we adapt a colloidal self-assembly method at liquid-liquid interfaces to deposit amyloid fibrils on a substrate from the water-hexane interface, while simultaneously compressing it. The amyloid fibril layers orient perpendicularly to the compression, ranging from isotropic to nematic distributions. We furthermore observe reproducible transitions from a monolayer to a bilayer and from a bilayer to multilayers with increasing surface pressures. The creation of each new layer is accompanied by a systematic drop in the structural order of the system, which is however regained upon further compression. This method shows great potential for overcoming the thin-film engineering challenges associated with the manipulation of sticky amyloid fibrils, and allows their ex situ visualisation under compression at the fluid-fluid interface, a situation relevant to understand the propagation of amyloid-related diseases, their functional role in biological systems, and their potential for technological applications.

Selective and Efficient Removal of Fluoride from Water: In Situ Engineered Amyloid Fibril/ZrO2 Hybrid Membranes.

We report a new strategy for efficient removal of F- from contaminated water streams, and it relies on carbon hybrid membranes made of amyloid fibril/ZrO2 nanoparticles (<10 nm). These membranes exhibit superior selectivity for F- against various competitive ions, with a distribution coefficient (Kd ) as high as 6820 mL g-1 , exceeding commercial ion-exchange resins (IRA-900) by 180 times and outdoing the performance of most commercial carbon-activated aluminum membranes. At both low and high (ca. 200 mg L-1 ) F- concentrations, the membrane efficiency exceeds 99.5 % removal. For real untreated municipal tap water (ca. 2.8 mg L-1 ) under continuous operating mode, data indicates that about 1750 kg water m-2 membrane can be treated while maintaining drinking water quality, and the saturated membranes can be regenerated and reused several times without decrease in performance. This technology is promising for mitigating the problem of fluoride water contamination worldwide.

Squid Suckerin Biomimetic Peptides Form Amyloid-like Crystals with Robust Mechanical Properties.

We present the self-assembly of fibers formed from a peptide sequence (A1H1) derived from suckerin proteins of squid sucker ring teeth (SRT). SRT are protein-only biopolymers with an unconventional set of physicochemical and mechanical properties including high elastic modulus coupled with thermoplastic behavior. We have identified a conserved peptide building block from suckerins that possess the ability to assemble into materials with similar mechanical properties as the native SRT. A1H1 displays amphiphilic characteristics and self-assembles from the bottom-up into mm-scale fibers initiated by the addition of a polar aprotic solvent. A1H1 fibers are thermally resistant up to 239 °C, coupled with an elastic modulus of ∼7.7 GPa, which can be explained by the tight packing of ß-sheet-enriched crystalline building blocks as identified by wide-angle X-ray scattering (WAXS), with intersheet and interstrand distances of 5.37 and 4.38 Å, respectively. A compact packing of the peptides at their Ala-rich terminals within the fibers was confirmed from molecular dynamics simulations, and we propose a hierarchical model of fiber assembly of the mature peptide fiber.

Hybrid Amyloid Membranes for Continuous Flow Catalysis.

Amyloid fibrils are promising nanomaterials for technological applications such as biosensors, tissue engineering, drug delivery, and optoelectronics. Here we show that amyloid-metal nanoparticle hybrids can be used both as efficient active materials for wet catalysis and as membranes for continuous flow catalysis applications. Initially, amyloid fibrils generated in vitro from the nontoxic ß-lactoglobulin protein act as templates for the synthesis of gold and palladium metal nanoparticles from salt precursors. The resulting hybrids possess catalytic features as demonstrated by evaluating their activity in a model catalytic reaction in water, e.g., the reduction of 4-nitrophenol into 4-aminophenol, with the rate constant of the reduction increasing with the concentration of amyloid-nanoparticle hybrids. Importantly, the same nanoparticles adsorbed onto fibrils surface show improved catalytic efficiency compared to the same unattached particles, pointing at the important role played by the amyloid fibril templates. Then, filter membranes are prepared from the metal nanoparticle-decorated amyloid fibrils by vacuum filtration. The resulting membranes serve as efficient flow catalysis active materials, with a complete catalytic conversion achieved within a single flow passage of a feeding solution through the membrane.

Oil transfer converts phosphatidylcholine vesicles into nonlamellar lyotropic liquid crystalline particles.

There is a need for the development of low-energy dispersion methods tailored to the formation of phospholipid-based nonlamellar lyotropic liquid crystalline (LLC) particles for delivery system applications. Here, facile formation of nonlamellar LLC particles was obtained by simple mixing of a phosphatidylcholine (PC) liposome solution and an oil-in-water emulsion, with limonene or isooctane as an oil. The internal structure of the particles was controlled by the PC-to-oil ratio, consistently with the sequence observed in bulk phase. For the first time, reverse micellar cubosomes with Fm3̅m inner structure were produced. The size, morphology, and inner structure of the particles were characterized by small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and freeze-fracture cryo scanning electron microscopy (cryo-SEM). These findings pave the way to new strategies in low-energy formulation of LLC delivery systems.

Isolation and Characterization of Monodisperse Core-Shell Nanoparticle Fractions.

Monodispersity is a key property to control the self-assembly of colloidal particles, and is typically reached after fine-tuning of the synthesis conditions. Monodisperse particle fractions can also be separated from polydisperse suspensions via ultracentrifugation. This paper demonstrates the capability of isolating and characterizing suspensions of core-shell iron oxide-polymer nanoparticles with extremely low polydispersity (p < 0.01) and, thus, of complementing nanoparticle synthetic approaches in the pursuit of highly monodisperse materials.

Polynuclear iron(II)-aminotriazole spincrossover complexes (polymers) in solution.

Polynuclear spincrossover (SCO) complexes prepared by the combination of [Fe(DMF)6](2+) and NH2trz (NH2trz = 4-amino-1,2,4-triazole) were studied (2ns(-) = counterion 2-naphthalenesulfonate). It is demonstrated that these [Fe(NH2trz)3](2ns)2 complexes can be dissolved-contrary to common reported experience-in N,N-dimethylformamide (DMF) and, therefore, can be conveniently processed by simple means. The resulting solutions were examined with UV/vis and X-ray absorption spectroscopy (XANES and EXAFS) as well as with small-angle X-ray scattering (SAXS). At a molar NH2trz/Fe(2+) ratio of 3/1, corresponding to the stoichiometric ratio of the ideal coordination compound, [Fe(NH2trz)3](2+) in the low-spin state was found to be in equilibrium with polynuclear species in the high-spin state. The equilibrium can be shifted virtually completely to the side of low-spin Fe(2+) by an excess of the ligand. The polymer therewith formed contains 100 or more Fe(2+) ions and is of a pronounced rigid-rod structure, with Fe-Fe distances around 3.32 Å (in comparison to 3.94 Å of the polynuclear species in the high-spin state). Reversible spin crossover takes place in solution upon a temperature increase to around 60 °C; this process is associated with a shift in equilibrium toward species shorter than the initial polynuclear species.

Unravelling secondary structure changes on individual anionic polysaccharide chains by atomic force microscopy.

The structural conformations of the anionic carrageenan polysaccharides in the presence of monovalent salt close to physiological conditions are studied by atomic force microscopy. Iota-carrageenan undergoes a coil-helix transition at high ionic strength, whereas lambda-carrageenan remains in the coiled state. Polymer statistical analysis reveals an increase in persistence length from 22.6±0.2 nm in the random coil, to 26.4±0.2 nm in the ordered helical conformation, indicating an increased rigidity of the helical iota-carrageenan chains. The many decades-long debated issue on whether the ordered state can exist as single or double helix, is conclusively resolved by demonstrating the existence of a unimeric helix formed intramolecularly by a single polymer chain.

Protein Nanotubes as Advanced Material Platforms and Delivery Systems.

Protein nanotubes (PNTs) as state-of-the-art nanocarriers are promising for various potential applications both in the food and pharmaceutical industries. Derived from edible starting sources like α-lactalbumin, lysozyme, and ovalbumin, PNTs bear properties of biocompatibility and biodegradability. Their large specific surface area and hydrophobic core facilitate chemical modification and loading of bioactive substances, respectively. Moreover, their enhanced permeability and penetration ability across biological barriers such as intestinal mucus, extracellular matrix, and thrombus clot, make it promising platforms for health-related applications. Most importantly, their simple preparation processes enable large-scale production, supporting applications in the biomedical and nanotechnological fields. Understanding the self-assembly principles is crucial for controlling their morphology, size, and shape, and thus provides the ground to a multitude of applications. Here, the current state-of-the-art of PNTs including their building materials, physicochemical properties, and self-assembly mechanisms are comprehensively reviewed. The advantages and limitations, as well as challenges and prospects for their successful applications in biomaterial and pharmaceutical sectors are then discussed and highlighted. Potential cytotoxicity of PNTs and the need of regulations as critical factors for enabling in vivo applications are also highlighted. In the end, a brief summary and future prospects for PNTs as advanced platforms and delivery systems are included.

Structure and enzymatic properties of molecular dendronized polymer-enzyme conjugates and their entrapment inside giant vesicles.

Macromolecular hybrid structures were prepared in which two types of enzymes, horseradish peroxidase (HRP) and bovine erythrocytes Cu,Zn-superoxide dismutase (SOD), were linked to a fluorescently labeled, polycationic, dendronized polymer (denpol). Two homologous denpols of first and second generation were used and compared, and the activities of HRP and SOD of the conjugates were measured in aqueous solution separately and in combination. In the latter case the efficiency of the two enzymes in catalyzing a two-step cascade reaction was evaluated. Both enzymes in the two types of conjugates were highly active and comparable to free enzymes, although the efficiency of the enzymes bound to the second-generation denpol was significantly lower (up to a factor of 2) than the efficiency of HRP and SOD linked to the first-generation denpol. Both conjugates were analyzed by atomic force microscopy (AFM), confirming the expected increase in object size compared to free denpols and demonstrating the presence of enzyme molecules localized along the denpol chains. Finally, giant phospholipid vesicles with diameters of up to about 20 µm containing in their aqueous interior pool a first-generation denpol-HRP conjugate were prepared. The HRP of the entrapped conjugate was shown to remain active toward externally added, membrane-permeable substrates, an important prerequisite for the development of vesicular multienzyme reaction systems.

Nanotopographic surfaces with defined surface chemistries from amyloid fibril networks can control cell attachment.

We show for the first time the possibility of using networks of amyloid fibrils, adsorbed to solid supports and with plasma polymer coatings, for the fabrication of chemically homogeneous surfaces with well-defined nanoscale surface features reminiscent of the topography of the extracellular matrix. The robust nature of the fibrils allows them to withstand the plasma polymer deposition conditions used with no obvious deleterious effect, thus enabling the underlying fibril topography to be replicated at the polymer surface. This effect was seen despite the polymer coating thickness being an order of magnitude greater than the fibril network. The in vitro culture of fibroblast cells on these surfaces resulted in increased attachment and spreading compared to flat plasma polymer films with the same chemical composition. The demonstrated technique allows for the rapid and reproducible fabrication of substrates with nanoscale fibrous topography that we believe will have applications in the development of new biomaterials allowing, for example, the investigation of the effect of extracellular matrix mimicking nanoscale morphology on cellular phenotype.

Functionalization of multiwalled carbon nanotubes and their pH-responsive hydrogels with amyloid fibrils.

New biocompatible, pH-responsive, and fully fibrous hydrogels have been prepared based on amyloid fibrils hybridized and gelled by functionalized multiwalled carbon nanotubes (MWNTs) far below the gelling concentration of amyloid fibrils. Sulfonic functional groups were introduced on the surfaces of MWNTs either by a covalent diazonium reaction or by physical π-π interactions. The presence of the isoelectric point of amyloid fibrils allows a reversible gelling behavior through ionic interactions with functionalized MWNTs.

Effect of Polysaccharide Conformation on Ultrafiltration Separation Performance.

The manifold array of saccharide linkages leads to a great variety of polysaccharide architectures, comprising three conformations in aqueous solution: compact sphere, random coil, and rigid rod. This conformational variation limits the suitability of the commonly applied molecular weight cut-off (MWCO) as selection criteria for polysaccharide ultrafiltration membranes, as it is based on globular marker proteins with narrow Mw and hydrodynamic volume relation. Here we show the effect of conformation on ultrafiltration performance using randomly coiled pullulan and rigid rod-like scleroglucan as model polysaccharides for membrane rejection and molecular weight distribution. Ultrafiltration with a 10 kDa polyethersulfone membrane yielded significant different recoveries for pullulan and scleroglucan showing 1% and 71%, respectively. We found deviations greater than 77-fold between nominal MWCO and apparent Mw of pullulan and scleroglucan, while recovering over 90% polysaccharide with unchanged Mw. We anticipate our work as starting point towards an optimized membrane selection for polysaccharide applications.