Non-fibril amyloid aggregation at the air/water interface: self-adaptive pathway resulting in a 2D Janus nanofilm.

The amyloid states of proteins are implicated in several neurodegenerative diseases and bioadhesion processes. However, the classical amyloid fibrillization mechanism fails to adequately explain the formation of polymorphic aggregates and their adhesion to various surfaces. Herein, we report a non-fibril amyloid aggregation pathway, with disulfide-bond-reduced lysozyme (R-Lyz) as a model protein under quasi-physiological conditions. Very different from classical fibrillization, this pathway begins with the air-water interface (AWI) accelerated oligomerization of unfolded full-length protein, resulting in unique plate-like oligomers with self-adaptive ability, which can adjust their conformations to match various interfaces such as the asymmetric AWI and amyloid-protein film surface. The pathway enables a stepwise packing of the plate-like oligomers into a 2D Janus nanofilm, exhibiting a divergent distribution of hydrophilic/hydrophobic residues on opposite sides of the nanofilm. The resulting Janus nanofilm possesses a top-level Young's modulus (8.3 ± 0.6 GPa) among amyloid-based materials and exhibits adhesive strength two times higher (145 ± 81 kPa) than that of barnacle cement. Furthermore, we found that such an interface-directed pathway exists in several amyloidogenic proteins with a similar self-adaptive 2D-aggregation process, including bovine serum albumin, insulin, fibrinogen, hemoglobin, lactoferrin, and ovalbumin. Thus, our findings on the non-fibril self-adaptive mechanism for amyloid aggregation may shed light on polymorphic amyloid assembly and their adhesions through an alternative pathway.

Synthesis and functionalization of scalable and versatile 2D protein films via amyloid-like aggregation.

Two-dimensional (2D) protein films can be used to modify the properties of surfaces, and find applications predominantly in the fields of biomaterials, lithography, optics and electronics. However, it is difficult to produce scalable homogeneous and robust protein films with an easy, low-cost, green and efficient method. Further challenges include encapsulating and releasing functional building blocks in the film without inactivating them, and maintaining or improving the bioactivities of proteins used for the formation of the films. Here we detail the process to prepare large 2D protein films with user-defined features and structures via the amyloid-like aggregation of commonly synthesized proteins. These films can be synthesized at meter scales, have high interface adhesion, high functional expansibility and tunable functional properties, obtained by controlling the position of the disulfide bond breakage. For example, we can retain or even enhance the natural antibacterial, biomineralization and antifouling activity of proteins involved in film formation, and the properties can also be expanded through the physical blending or chemical grafting of additional functional blocks on the surface of the film. A 2D protein film can be prepared in ~3 h using four alternative coating techniques: immersion, transfer, hydrogel stamping and spraying. The characterization process of the film requires ~5 d. The procedure can be carried out by users with basic expertise in materials science.

pH-Responsive Protein Conformation Transistor.

Analogous to electronic transistors, transistor-like responsive materials undergo sharp structural transitions in response to a very narrow range of microenvironment signals. This kind of material is typically limited to synthetic polymer-derived nanoscale assembly or disassembly and has profound implications for modern high-tech applications. Herein, we evolve this system from synthetic polymers to biopolymers and extend the corresponding assembly scale from the nanoscale to meso/macro-scale. We develop unique protein nanocrystals with core-shell structures through a two-step nucleation process. The protein nanocrystals exhibit exceptional transistor-like pH-responsive mesoscale assembly through the formation of inter-particle ß-sheet linkers. This allows ultrasensitive cross-linking behavior, such as self-coacervation at a water/water interface, ultrafast gelation in seconds, and ultrasensitive swelling for detection of basic vapors at extremely low concentrations. This breakthrough has great promise for broader applications such as drug encapsulation and delivery, biosensing, cytomimetic materials, and microfluidic chemistry.

Synthesis of robust underwater glues from common proteins via unfolding-aggregating strategy.

Underwater adhesive proteins secreted by organisms greatly inspires the development of underwater glue. However, except for specific proteins such as mussel adhesive protein, barnacle cement proteins, curli protein and their related recombinant proteins, it is believed that abundant common proteins cannot be converted into underwater glue. Here, we demonstrate that unfolded common proteins exhibit high affinity to surfaces and strong internal cohesion via amyloid-like aggregation in water. Using bovine serum albumin (BSA) as a model protein, we obtain a stable unfolded protein by cleaving the disulfide bonds and maintaining the unfolded state by means of stabilizing agents such as trifluoroethanol (TFE) and urea. The diffusion of stabilizing agents into water exposes the hydrophobic residues of an unfolded protein and initiates aggregation of the unfolded protein into a solid block. A robust and stable underwater glue can thus be prepared from tens of common proteins. This strategy deciphers a general code in common proteins to construct robust underwater glue from abundant biomass.

α-Helix-Mediated Protein Adhesion.

Proteins have been adopted by natural living organisms to create robust bioadhesive materials, such as biofilms and amyloid plaques formed in microbes and barnacles. In these cases, ß-sheet stacking is recognized as a key feature that is closely related to the interfacial adhesion of proteins. Herein, we challenge this well-known recognition by proposing an α-helix-mediated interfacial adhesion model for proteins. By using bovine serum albumin (BSA) as a model protein, it was discovered that the reduction of disulfide bonds in BSA results in random coils from unfolded BSA dragging α-helices to gather at the solid/liquid interface (SLI). The hydrophobic residues in the α-helix then expose and break through the hydration layer of the SLI, followed by the random deposition of hydrophilic and hydrophobic residues to achieve interfacial adhesion. As a result, the first assembled layer is enriched in the α-helix secondary structure, which is then strengthened by intermolecular disulfide bonds and further initiates stepwise layering protein assembly. In this process, ß-sheet stacking is transformed from the α-helix in a gradually evolving manner. This finding thus indicates a valuable clue that ß-sheet-featuring amyloid may form after the interfacial adhesion of proteins. Furthermore, the finding of the α-helix-mediated interfacial adhesion model of proteins affords a unique strategy to prepare protein nanofilms with a well-defined layer number, presenting robust and modulable adhesion on various substrates and exhibiting good resistance to acid, alkali, organic solvent, ultrasonic, and adhesive tape peeling.

Rectifying disorder of extracellular matrix to suppress urethral stricture by protein nanofilm-controlled drug delivery from urinary catheter.

Urethral stricture secondary to urethral injury, afflicting both patients and urologists, is initiated by excessive deposition of extracellular matrix in the submucosal and periurethral tissues. Although various anti-fibrotic drugs have been applied to urethral stricture by irrigation or submucosal injection, their clinical feasibility and effectiveness are limited. Here, to target the pathological state of the extracellular matrix, we design a protein-based nanofilm-controlled drug delivery system and assemble it on the catheter. This approach, which integrates excellent anti-biofilm properties with stable and controlled drug delivery for tens of days in one step, ensures optimal efficacy and negligible side effects while preventing biofilm-related infections. In a rabbit model of urethral injury, the anti-fibrotic catheter maintains extracellular matrix homeostasis by reducing fibroblast-derived collagen production and enhancing metalloproteinase 1-induced collagen degradation, resulting in a greater improvement in lumen stenosis than other topical therapies for urethral stricture prevention. Such facilely fabricated biocompatible coating with antibacterial contamination and sustained-drug-release functionality could not only benefit populations at high risk of urethral stricture but also serve as an advanced paradigm for a range of biomedical applications.

Amyloid-Mediated Nanoarchitectonics with Biomimetic Mineralization of Polyetheretherketone for Enhanced Osseointegration.

Polyetheretherketone (PEEK), a widely used implant material, has attracted the attention of scientific researchers because of its bone-matched elastic modulus, radiolucency, and chemical resistance. However, the bioinert chemical properties of PEEK do not promote bone apposition once implanted. In this study, using a phase-transitioned lysozyme (PTL) nanofilm as a sandwiched layer, a robust hydroxyapatite (HAp) coating on PEEK (HAp@PTL@PEEK) is constructed. The PTL nanofilm shows strong adhesion to the PEEK surface and induces biomimetic mineralization to form a compact HAp coating on PEEK in simulated body fluids. This HAp coating not only shares a higher adhesion strength and better stability but can also be applied to implants with complex 3D structures. HAp@PTL@PEEK showed significantly enhanced osteogenic capacity when cultured with rat bone marrow mesenchymal stem cells by promoting initial cell adhesion, proliferation, and osteogenic differentiation in vitro. In vivo evaluations utilizing models of femoral condyle defects and skull defects confirm that the HAp coating substantially augments bone remodeling and osseointegration ability. Compared with the traditional method, our modified method is simpler, more environmentally friendly, and uses less hazardous components. Furthermore, the obtained HAp coating shares a higher adhesion strength to PEEK and a better osteogenic capacity. The study offers a novel method to improve the osseointegration of PEEK-based implants in biointerfaces and tissue engineering.

Oral administration of octacosanol modulates the gut bacteria and protects the intestinal barrier in ulcerative colitis mice.

Octacosanol (Oct), a kind of long-chain fatty alcohol extracted from rice bran was applied to study its effects on alleviating ulcerative colitis (UC). Oct was orally administered at 10 mg/kg (Oct-L) and 30 mg/kg (Oct-H) to dextran sulfate sodium (DSS)-induced mice. Here, we reported that oral administration of 30 mg/kg Oct can significantly prevent the weight loss, colon shortening, and decrease the disease activity index (DAI) score. Oct-H supplementation modified the intestinal flora by lowering the Firmicutes/Bacteroidetes (F/B) ratio, increasing the abundance of Prevotellaceae, S24-7, Turicibacter, and meanwhile decreasing Enterococcus and Stenotrophomonas. Based on the PICRUSt2 analysis, Oct-H may exert effects by anti-inflammation and xenobiotics degradation. Furthermore, short-chain fatty acids (SCFAs) levels were raised and the integrity of the gut barrier was protected. In conclusion, Oct-H can relieve clinical symptoms, modulate the gut bacteria and protect the intestinal barrier in UC mice, suggesting the potential of Oct as a food supplementation in alleviating UC. PRACTICAL APPLICATIONS: Ulcerative colitis (UC) is a hard-to-cure disease, with increasing morbidity in recent years. Therefore, finding out a food supplement to alleviate UC is very meaningful. In this work, we showed that octacosanol significantly alleviated ulcerative colitis in mice. We revealed, for the first time, octacosanol's effects on protecting the integrity of the gut barrier, modulating the intestinal flora and its metabolism (SCFAs). Therefore, octacosanol was expected to prevent colitis in an all-round way. Our research might also lay the theoretical foundation for the further development of related functional foods.

Controlling the Structure and Function of Protein Thin Films through Amyloid-like Aggregation.

Protein thin films (PTFs) with tunable structure and function can offer multiple opportunities in various fields such as surface modification, biomaterials, packaging, optics, electronics, separation, energy, and environmental science. Although nature may offer a variety of examples of high-level control of structure and function, e.g., the S layer of cells, synthetic alternatives for large-area protein-based thin films with fine control over both biological function and material structure are a key challenge, especially when aiming for facile, low-cost, green, and large-scale preparation as well as a further extension of function, such as the encapsulation and release of functional building blocks.Therefore, regarding the structure and function of PTFs, we will first briefly comment on the problems associated with PTF fabrication, and then, regarding the basis of our long-term research on protein-based thin films, we will summarize the new strategies that we have developed in recent years to explore and control the structure and function of PTFs for frontier research and practical applications.Inspired by naturally occurring protein amyloid fibrillization, we proposed the amyloid-like protein aggregation strategy to assemble proteins into supramolecular 2D films with extremely large sizes and enduring interfacial adhesion stability. This approach opened a new window for PTF fabrication in which the spontaneous interfacial 2D aggregation of protein oligomers instead of traditional 1D protofibril elongation directs the assembly of proteins. As a result, the film morphology, thickness, porosity, and function can be tailored by simply tuning the interfacial aggregation pathways.We further modified amyloid-like protein aggregation to develop chemoselective reaction-induced protein aggregation (CRIPA). It is well known that chemoselective reactions have been employed for protein modification. However, the application of such reactions in PTF fabrication has been overlooked. We initiated this new strategy by employing thiol-disulfide exchange reactions. These reactions are chemoselective toward proteins containing specific disulfide bonds with high redox potentials, resulting in amyloid-like aggregation and thin film formation. Functional proteins with immunity to such reactions can be encapsulated in thin films and released on demand without a loss of activity, opening a new avenue for the development of functional PTFs and coatings.Finally, the resultant amyloid-inspired PTFs, as a new type of biomimetic materials, provide a good platform for integration with various biomedical functions. Here, the creation of bioactive surfaces on virtually arbitrary substrates by amyloid-like PTFs will be discussed, highlighting antimicrobial, antifouling, molecular separation, and interfacial biomineralization activities that exceed those of their native protein precursors and synthetic alternatives.

Amyloid-like protein aggregates combining antifouling with antibacterial activity.

Infections related to implanted medical devices have placed a heavy burden on public health and require feasible solutions. In this study, a simple approach is reported to fabricate an antifouling and antibacterial dual-functional coating. One-step aqueous supramolecular assembly of bovine serum albumin (BSA) is employed to immobilize ε-polylysine (ε-PL) and form a coating (PTB@ε-PL). Based on amyloid-like protein aggregation through the rapid reduction of the intramolecular disulfide bonds of BSA by tris(2-carboxyethyl) phosphine, a dense PTB@ε-PL nanofilm with controllable thickness and ε-PL loading density can be covered on virtually arbitrary material surfaces by simple aqueous dipping. In vitro and in vivo experiments show that this coating not only exhibits effective antibacterial activity against Gram-positive/Gram-negative bacteria, but also imparts excellent antifouling property to the surface. As a pure biopolymer coating, the PTB@ε-PL nanofilm shows negligible cytotoxicity and hemolysis. In addition, due to the various functional groups exposed on the surface of the nanofilm, the coating shows excellent interfacial bonding stability and can maintain bactericidal and antifouling properties under harsh conditions including ultrasound, autoclaving, organic solvents, and physiological body fluids.

Functional amyloid materials at surfaces/interfaces.

With the development of nanotechnology, functional amyloid materials are drawing increasing attention, and numerous remarkable applications are emerging. Amyloids, defined as a class of supramolecular assemblies of misfolded proteins or peptides into ß-sheet fibrils, have evolved in many new respects and offer abundant chemical/biological functions. These proteinaceous micro/nano-structures provide excellent biocompatibility, rich phase behaviours, strong mechanical properties, and stability at interfaces not only in nature but also in functional materials, displaying versatile interactions with surfaces/interfaces that have been widely adopted in bioadhesion, synthetic biology, and composites. Overall, functional amyloids at surfaces/interfaces have excellent potential applications in next-generation biotechnology and biomaterials.