Search

Wearable pressure sensors have become increasingly popular for personal healthcare and motion detection applications due to recent advances in materials science and functional nanomaterials. In this study, a novel composite hydrogel is presented as a sensitive piezoresistive sensor that can be utilized for various biomedical applications, such as wearable skin patches and integrated artificial skin that can measure pulse and blood pressure, as well as monitor sound as a self-powered microphone. The hydrogel is composed of self-assembled short peptides containing aromatic, positively- or negatively charged amino acids combined with 2D Ti 3 C 2 T z MXene nanosheets. This material is low-cost, facile, reliable, and scalable for large areas while maintaining high sensitivity, a wide detection range, durability, oxidation stability, and biocompatibility. The bioinspired nanostructure, strong mechanical stability, and ease of functionalization make the assembled peptide-based composite MXene-hydrogel a promising and widely applicable material for use in bio-related wearable electronics., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some xenobiotics, several enzymatic steps are needed to obtain nontoxic products. Another challenge is the low durability and stability of many native enzymes in their purified form. Herein, we coupled peptide-based encapsulation of bacterial phosphotriesterase with soil-originated bacteria, Arthrobacter sp. 4Hβ as an efficient system capable of biodegradation of paraoxon, a neurotoxin pesticide. Specifically, recombinantly expressed and purified methyl parathion hydrolase (MPH), with high hydrolytic activity toward paraoxon, was encapsulated within peptide nanofibrils, resulting in increased shelf life and retaining ∼50% activity after 132 days since purification. Next, the addition of Arthrobacter sp. 4Hβ, capable of degrading para-nitrophenol (PNP), the hydrolysis product of paraoxon, which is still toxic, resulted in nondetectable levels of PNP. These results present an efficient one-pot system that can be further developed as an environmentally friendly solution, coupling purified enzymes and native bacteria, for pesticide bioremediation. We further suggest that this system can be tailored for different xenobiotics by encapsulating the rate-limiting key enzymes followed by their combination with environmental bacteria that can use the enzymatic step products for full degradation without the need to engineer synthetic bacteria.

Phenylketonuria is characterized by the accumulation of phenylalanine, resulting in severe cognitive and neurological disorders if not treated by a remarkably strict diet. There are two approved drugs today, yet both provide only a partial solution. We have previously demonstrated the formation of amyloid-like toxic assemblies by aggregation of phenylalanine, suggesting a new therapeutic target to be further pursued. Moreover, we showed that compounds that halt the formation of these assemblies also prevent their resulting toxicity. Here, we performed high-throughput screening, searching for compounds with inhibitory effects on phenylalanine aggregation. Morin hydrate, one of the most promising hits revealed during the screen, was chosen to be tested in vivo using a phenylketonuria mouse model. Morin hydrate significantly improved cognitive and motor function with a reduction in the number of phenylalanine brain deposits. Moreover, while phenylalanine levels remained high, we observed a recovery in dopaminergic, adrenergic, and neuronal markers. To conclude, the ability of Morin hydrate to halt phenylalanine aggregation without reducing phenylalanine levels implies the toxic role of the phenylalanine assemblies in phenylketonuria and opens new avenues for disease-modifying treatment., Competing Interests: Declaration of competing interest Shira Shaham-Niv, Assaf Ezra, Dor Zaguri, Lihi Adler-Abramovich, and Ehud Gazit have a pending patent application filed to Ramot at Tel Aviv University. No other authors have known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ehud Gazit reports financial support was provided by Israel Science Foundation The Nancy Stephen Grand Israel National Center for Personalized Medicine (grant no. 236115 E.G.). Ehud Gazit reports financial support was provided by The Colton Foundation (grant no. 590046 E.G.). Ehud Gazit reports financial support was provided by March of Dimes Foundation (grant no. 23-FY20–36 E.G.). Shira Shaham-Niv, Assaf Ezra, Dor Zaguri, Adler-Abramovich, Lihi, Ehud Gazit has patent Not filed yet pending to Ramot at Tel Aviv University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The cornerstone of structural biology is the unique relationship between protein sequence and the 3D structure at equilibrium. Although intrinsically disordered proteins (IDPs) do not fold into a specific 3D structure, breaking this paradigm, some IDPs exhibit large-scale organization, such as liquid-liquid phase separation. In such cases, the structural plasticity has the potential to form numerous self-assembled structures out of thermal equilibrium. Here, we report that high-temperature incubation time is a defining parameter for micro and nanoscale self-assembly of resilin-like IDPs. Interestingly, high-resolution scanning electron microscopy micrographs reveal that an extended incubation time leads to the formation of micron-size rods and ellipsoids that depend on the amino acid sequence. More surprisingly, a prolonged incubation time also induces amino acid composition-dependent formation of short-range nanoscale order, such as periodic lamellar nanostructures. We, therefore, suggest that regulating the period of high-temperature incubation, in the one-phase regime, can serve as a unique method of controlling the hierarchical self-assembly mechanism of structurally disordered proteins., (© 2023. The Author(s).)

Diabetes is a global epidemic accompanied by impaired wound healing and increased risk of persistent infections and resistance to standard treatments. Therefore, there is an immense need to develop novel methods to specifically target therapeutics to affected tissues and improve treatment efficacy. This study aims to use enzyme-responsive nanoparticles for the targeted delivery of an anti-inflammatory drug, dexamethasone, to treat inflammation in diabetes. These nanoparticles are assembled from fluorescently-labeled, dexamethasone-loaded peptide-polymer amphiphiles. The nanoparticles are injected in vivo, adjacent to labeled collagen membranes sub-periosteally implanted on the calvaria of diabetic rats. Following their implantation, collagen membrane resorption is linked to inflammation, especially in hyperglycemic individuals. The nanoparticles show strong and prolonged accumulation in inflamed tissue after undergoing a morphological switch into microscale aggregates. Significantly higher remaining collagen membrane area and less inflammatory cell infiltration are observed in responsive nanoparticles-treated rats, compared to control groups injected with free dexamethasone and non-responsive nanoparticles. These factors indicate improved therapeutic efficacy in inflammation reduction. These results demonstrate the potential use of enzyme-responsive nanoparticles as targeted delivery vehicles for the treatment of diabetic and other inflammatory wounds., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

Di- and triblock amphiphiles can form different mesophases ranging from micelles to hydrogels depending on their chemical structures, hydrophilic to hydrophobic ratios, and their ratio in the mixture. In addition, their different architectures dictate their exchange rate between the assembled and unimer states and consequently affect their responsiveness toward enzymatic degradation. Here we report the utilization of the different reactivities of di- and triblock amphiphiles, having exactly the same hydrophilic to lipophilic balance, toward enzymatic degradation as a tool for programming formulations to undergo sequential enzymatically induced transitions from (i) micelles to (ii) hydrogel and finally to (iii) dissolved polymers. We show that the rate of transition between the mesophases can be programmed by changing the ratio of the amphiphiles in the formulation, and that the hydrogels can maintain encapsulated cargo, which was loaded into the micelles. The reported results demonstrate the ability of molecular architecture to serve as a tool for programming smart formulations to adopt different structures and functions.

Collagen is the most abundant protein in various mammalian tissues and has an essential role in various cellular processes. Collagen is necessary for food-related biotechnological applications such as cultivated meat, medical engineering, and cosmetics. High-yield expression of natural collagen from mammalian cells is challenging and not cost-effective. Thus, external collagen is obtained primarily from animal tissues. Under cellular hypoxia, overactivation of the transcription factor hypoxia-inducible factor (HIF) was shown to correlate with enhanced accumulation of collagen. Herein, we showed that the small molecule ML228, a known molecular activator of HIF, enhances the accumulation of collagen type-I in human fibroblast cells. We report an increase in collagen levels by 2.33 ± 0.33 when fibroblasts were incubated with 5 μM of ML228. Our experimental results demonstrated, for the first time, that external modulation of the hypoxia biological pathway can boost collagen levels in mammalian cells. Our findings pave the way for enhancing natural collagen production in mammals by altering cellular signaling pathways.

9-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF), has been has been extensively explored due to its ultrafast self-assembly kinetics, inherent biocompatibility, tunable physicochemical properties, and especially, the capability of forming self-sustained gels under physiological conditions. Consequently, various methodologies to develop Fmoc-FF gels and their corresponding applications in biomedical and industrial fields have been extensively studied. Herein, we systemically summarize the mechanisms underlying Fmoc-FF self-assembly, discuss the preparation methodologies of Fmoc-FF hydrogels, and then deliberate the properties as well as the diverse applications of Fmoc-FF self-assemblies. Finally, the contemporary shortcomings which limit the development of Fmoc-FF self-assembly are raised and the alternative solutions are proposed, along with future research perspectives., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Aim: To investigate the potential of an ultrashort aromatic peptide hydrogelator integrated with hyaluronic acid (HA) to serve as a scaffold for bone regeneration., Materials and Methods: Fluorenylmethyloxycarbonyl-diphenylalanine (FmocFF)/HA hydrogel was prepared and characterized using microscopy and rheology. Osteogenic differentiation of MC3T3-E1 preosteoblasts was investigated using Alizarin red, alkaline phosphatase and calcium deposition assays. In vivo, 5-mm-diameter calvarial critical-sized defects were prepared in 20 Sprague-Dawley rats and filled with either FmocFF/HA hydrogel, deproteinized bovine bone mineral, FmocFF/Alginate hydrogel or left unfilled. Eight weeks after implantation, histology and micro-computed tomography analyses were performed. Immunohistochemistry was performed in six rats to assess the hydrogel's immunomodulatory effect., Results: A nanofibrous FmocFF/HA hydrogel with a high storage modulus of 46 KPa was prepared. It supported osteogenic differentiation of MC3T3-E1 preosteoblasts and facilitated calcium deposition. In vivo, the hydrogel implantation resulted in approximately 93% bone restoration. It induced bone deposition not only around the margins, but also generated bony islets along the defect. Elongated M2 macrophages lining at the periosteum-hydrogel interface were observed 1 week after implantation. After 3 weeks, these macrophages were dispersed through the regenerating tissue surrounding the newly formed bone., Conclusions: FmocFF/HA hydrogel can serve as a cell-free, biomimetic, immunomodulatory scaffold for bone regeneration., (© 2022 The Authors. Journal of Clinical Periodontology published by John Wiley & Sons Ltd.)

Defrost sensors are a crucial element for proper functioning of the pharmaceutical cold chain. In this paper, the self-assembled peptide-based hydrogels were used to construct a sensitive defrost sensor for the transportation and storage of medications and biomaterials. The turbidity of the peptide hydrogel was employed as a marker of the temperature regime. The gelation kinetics under different conditions was studied to detect various stages of hydrogel structural transitions aimed at tuning the system properties. The developed sensor can be stored at room temperature for a long period, irreversibly indicates whether the product has been thawed, and can be adjusted to a specific temperature range and detection time.

Peptide-based hydrogels were shown to serve as good matrices for 3D cell culture and to be applied in the field of regenerative medicine. The study of the cell-matrix interaction is important for the understanding of cell attachment, proliferation, and migration, as well as for the improvement of the matrix. Here, we used scanning ion conductance microscopy (SICM) to study the growth of cells on self-assembled peptide-based hydrogels. The hydrogel surface topography, which changes during its formation in an aqueous solution, were studied at nanoscale resolution and compared with fluorescence lifetime imaging microscopy (FLIM). Moreover, SICM demonstrated the ability to map living cells inside the hydrogel. A zwitterionic label-free pH nanoprobe with a sensitivity > 0.01 units was applied for the investigation of pH mapping in the hydrogel to estimate the hydrogel applicability for cell growth. The SICM technique that was applied here to evaluate the cell growth on the peptide-based hydrogel can be used as a tool to study functional living cells.

Major stress has systemic effects on the body that can have adverse consequences for physical and mental health. However, the molecular basis of these damaging effects remains incompletely understood. Here we use a longitudinal approach to characterise the acute systemic impact of major psychological stress in a pig model. We perform untargeted metabolomics on non-invasively obtained saliva samples from pigs before and 24 h after transfer to the novel physical and social environment of a slaughterhouse. The main molecular changes occurring include decreases in amino acids, B-vitamins, and amino acid-derived metabolites synthesized in B-vitamin-dependent reactions, as well as yet-unidentified metabolite features. Decreased levels of several of the identified metabolites are implicated in the pathology of human psychological disorders and neurodegenerative disease, suggesting a possible neuroprotective function. Our results provide a fingerprint of the acute effect of psychological stress on the metabolome and suggest candidate biomarkers with potential roles in stress-related disorders., (© 2022. The Author(s).)

The potential of ultra-short peptides to self-assemble into well-ordered functional nanostructures makes them promising minimal components for mimicking the basic ingredient of nature and diverse biomaterials. However, selection and modular design of perfect de novo sequences are extremely tricky due to their vast possible combinatorial space. Moreover, a single amino acid substitution can drastically alter the supramolecular packing structure of short peptide assemblies. Here, we report the design of rigid hybrid hydrogels produced by sequence engineering of a new series of ultra-short collagen-mimicking tripeptides. Connecting glycine with different combinations of proline and its post-translational product 4-hydroxyproline, the single triplet motif, displays the natural collagen-helix-like structure. Improved mechanical rigidity is obtained via co-assembly with the non-collagenous hydrogelator, fluorenylmethoxycarbonyl (Fmoc) diphenylalanine. Characterizations of the supramolecular interactions that promote the self-supporting and self-healing properties of the co-assemblies are performed by physicochemical experiments and atomistic models. Our results clearly demonstrate the significance of sequence engineering to design functional peptide motifs with desired physicochemical and electromechanical properties and reveal co-assembly as a promising strategy for the utilization of small, readily accessible biomimetic building blocks to generate hybrid biomolecular assemblies with structural heterogeneity and functionality of natural materials.

Ordered supramolecular hydrogels assembled by modified aromatic amino acids often exhibit low mechanical rigidity. Aiming to stabilize the hydrogel and understand the impact of conformational freedom and hydrophobicity on the self-assembly process, two building blocks based on 9-fluorenyl-methoxycarbonyl-phenylalanine (Fmoc-Phe) gelator which contain two extra methylene units in the backbone, generating Fmoc-γPhe and Fmoc-(3-hydroxy)-γPhe are designed. Fmoc-γPhe spontaneously assembled in aqueous media forming a hydrogel with exceptional mechanical and thermal stability. Moreover, Fmoc-(3-hydroxy)-γPhe, with an extra backbone hydroxyl group decreasing its hydrophobicity while maintaining some molecular flexibility, self-assembled into a transient fibrillar hydrogel, that later formed microcrystalline aggregates through a phase transition. Molecular dynamics simulations and single crystal X-ray analyses reveal the mechanism underlying the two residues' distinct self-assembly behaviors. Finally, Fmoc-γPhe and Fmoc-(3-OH)-γPhe co-assembly to form a supramolecular hydrogel with notable mechanical properties are demonstrated. It has been believed that the understanding of the structure-assembly relationship will enable the design of new functional amino acid-based hydrogels., (© 2022 The Authors. Macromolecular Rapid Communications published by Wiley-VCH GmbH.)

Over the last decade, three-dimensional (3D) printing technologies have attracted the interest of researchers due to the possibility of fabricating tissue- and organ-like structures with similarities to the organ of interest. One of the most widely used materials for the fabrication of bioinks is gelatin (Gel) due to its excellent biocompatibility properties. However, in order to fabricate stable scaffolds under physiological conditions, the most common approach is to use gelatin methacrylate (GelMA) that allows the crosslinking and therefore the stabilization of the hydrogel through UV crosslinking. The crosslinking process can be harmful to cells thus decreasing total cell viability. To overcome the need for post-printing crosslinking, a new approach of bioink formulation was studied, incorporating the Fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) peptide into the Gel bioink. However, although Fmoc-FF possesses excellent mechanical properties, the lack of elasticity and viscosity makes it unsuitable for 3D-printing. Here, we demonstrate that covalent conjugation of two different ethylene glycol (EG) motifs to the Fmoc-FF peptide increases the hydrophilicity and elasticity properties, which are essential for 3D-printing. This new approach for bioink formulation avoids the need for any post-printing manufacturing processes, such as chemical or UV crosslinking.

Sulfated polysaccharides of red marine microalgae have recently gained much attention for biomedical applications due to their anti-inflammatory and antioxidant properties. However, their low mechanical properties limit their use in tissue engineering. Herein, to enhance the mechanical properties of the sulfated polysaccharide produced by the red marine microalga, Porphyridium sp. (PS) , it was integrated with the fluorenylmethoxycarbonyl diphenylalanine (FmocFF) peptide hydrogelator. Transparent, stable hydrogels were formed when mixing the two components at a 1:1 ratio in three different concentrations. Electron microscopy showed that all hydrogels exhibited a nanofibrous structure, mimicking the extracellular matrix. Furthermore, the hydrogels were injectable, and tunable mechanical properties were obtained by changing the hydrogel concentration. The composite hydrogels allowed the sustained release of curcumin which was controlled by the change in the hydrogel concentration. Finally, the hydrogels supported MC3T3-E1 preosteoblasts viability and calcium deposition. The synergy between the sulfated polysaccharide, with its unique bioactivities, and FmocFF peptide, with its structural and mechanical properties, bears a promising potential for developing novel tunable scaffolds for tissue engineering that may allow cell differentiation into various lineages.

The occurrence of sequential multiple aromatic residues in a helical sequence is rare compared to the β-sheet rich structure. Here, using helix promoting α-aminoisobutyric acid (Aib) residues, we unravel atomistic details of the helical secondary structure formation and the super helical assembly of two heptapeptides composed of sequential five and six phenylalanine (Phe) residues.

Engineering ordered nanostructures through molecular self-assembly of simple building blocks constitutes the essence of modern nanotechnology to develop functional supramolecular biomaterials. However, the lack of adequate chemical and functional diversity often hinders the utilization of unimolecular self-assemblies for practical applications. Co-assembly of two different building blocks can essentially harness both of their attributes and produce nanostructured macro-scale objects with improved physical properties and desired functional complexity. Herein, the authors report the co-operative co-assembly of a modified amino acid, fluorenylmethoxycarbonyl-pentafluoro-phenylalanine (Fmoc-F 5 -Phe), and a peptide, Fmoc-Lys(Fmoc)-Arg-Gly-Asp [Fmoc-K(Fmoc)-RGD] into a functional supramolecular hydrogel. A change in the morphology and fluorescence emission, as well as improvement of the mechanical properties in the mixed hydrogels compared to the pristine hydrogels, demonstrate the signature of co-operative co-assembly mechanism. Intriguingly, this approach harnesses the advantages of both components in a synergistic way, resulting in a single homogeneous biomaterial possessing the antimicrobial property of Fmoc-F 5 -Phe and the biocompatibility and cell adhesive characteristics of Fmoc-K(Fmoc)-RGD. This work exemplifies the importance of the co-assembly process in nanotechnology and lays the foundation for future developments in supramolecular chemistry by harnessing the advantages of diverse functional building blocks into a mechanically stable functional biomaterial., (© 2022 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.)

Molecular self-assembly is a spontaneous natural process resulting in highly ordered nano to microarchitectures. We report temperature-independent formation of robust stable membranes obtained by the spontaneous interaction of intrinsically disordered elastin-like polypeptides (ELPs) with short aromatic peptides at temperatures both below and above the conformational transition temperature of the ELPs. The membranes are stable over time and display durability over a wide range of parameters including temperature, pH, and ultrasound energy. The morphology and composition of the membranes were analyzed using microscopy. These robust structures support preosteoblast cell adhesion and proliferation as well as pH-dependent cargo release. Simple noncovalent interactions with short aromatic peptides can overcome conformational restrictions due to the phase transition to facilitate the formation of complex bioactive scaffolds that are stable over a wide range of environmental parameters. This approach offers novel possibilities for controlling the conformational restriction of intrinsically disordered proteins and using them in the design of new materials.

The self-assembly of peptides is a key direction for fabrication of advanced materials. Novel approaches for fine tuning of macroscopic and microscopic properties of peptide self-assemblies are of a high demand for constructing biomaterials with desired properties. In this work, while studying the kinetics of the Fmoc-Diphenylalanine (Fmoc-FF) dipeptide self-assembly using the Thioflavin T (ThT) dye, we observed that the presence of ThT strongly modifies structural and mechanical properties of the Fmoc-FF hydrogel. Notably, the presence of ThT resulted in a tenfold increase of the gelation time and in the formation of short and dense fibers in the hydrogel. As a result of these morphological alteration higher thermal stability, and most important, tenfold increase of the hydrogel rigidity was achieved. Hence, ThT not only slowed the kinetics of the Fmoc-FF hydrogel formation, but also strongly enhanced its mechanical properties. In this study, we provide a detailed description of the ThT effect on the hydrogel properties and suggest the mechanisms for this phenomenon, paving the way for the novel approach to the control of the peptide hydrogels' micro- and macroscale properties., (© 2021 Wiley-VCH GmbH.)

In this paper, we report the in situ growth of FF nanotubes examined via liquid-cell transmission electron microscopy (LCTEM). This direct, high spatial, and temporal resolution imaging approach allowed us to observe the growth of peptide-based nanofibrillar structures through directional elongation. Furthermore, the radial growth profile of FF nanotubes through the addition of monomers perpendicular to the tube axis has been observed in real-time with sufficient resolution to directly observe the increase in diameter. Our study demonstrates that the kinetics, dynamics, structure formation, and assembly mechanism of these supramolecular assemblies can be directly monitored using LCTEM. The performance of the peptides and the assemblies they form can be verified and evaluated using post-mortem techniques including time-of-flight secondary ion mass spectrometry (ToF-SIMS).

Hydrogel scaffolds have attracted much interest in the last few years for applications in the field of bone and cartilage tissue engineering. These scaffolds serve as a convenient three-dimensional structure on which cells can grow while sensing the native environment. Natural polymer-based hydrogels are an interesting choice for such purposes, but they lack the required mechanical properties. In contrast, composite hydrogels formed by biopolymers and short peptide hydrogelators possess mechanical characteristics suitable for osteogenesis. Here, we describe how combining the short peptide hydrogelator, Pyrene-Lysine-Cysteine (PyKC), with other biopolymers, can produce materials that are suitable for tissue engineering purposes. The presence of PyKC considerably enhances the strength and water content of the composite hydrogels, and confers thixotropic behavior. The hyaluronic acid-PyKC composite hydrogels were shown to be biocompatible, with the ability to support osteogenesis, since MC3 T3-E1 osteoblast progenitor cells grown on the materials displayed matrix calcification and osteogenic differentiation. The osteogenesis results and the injectability of these composite hydrogels hold promise for their future utilization in tissue engineering.

Supramolecular hydrogels formed by the self-assembly of amino-acid based gelators are receiving increasing attention from the fields of biomedicine and material science. Self-assembled systems exhibit well-ordered functional architectures and unique physicochemical properties. However, the control over the kinetics and mechanical properties of the end-products remains puzzling. A minimal alteration of the chemical environment could cause a significant impact. In this context, we report the effects of modifying the position of a single atom on the properties and kinetics of the self-assembly process. A combination of experimental and computational methods, used to investigate double-fluorinated Fmoc-Phe derivatives, Fmoc-3,4F-Phe and Fmoc-3,5F-Phe, reveals the unique effects of modifying the position of a single fluorine on the self-assembly process, and the physical properties of the product. The presence of significant physical and morphological differences between the two derivatives was verified by molecular-dynamics simulations. Analysis of the spontaneous phase-transition of both building blocks, as well as crystal X-ray diffraction to determine the molecular structure of Fmoc-3,4F-Phe, are in good agreement with known changes in the Phe fluorination pattern and highlight the effect of a single atom position on the self-assembly process. These findings prove that fluorination is an effective strategy to influence supramolecular organization on the nanoscale. Moreover, we believe that a deep understanding of the self-assembly process may provide fundamental insights that will facilitate the development of optimal amino-acid-based low-molecular-weight hydrogelators for a wide range of applications.

The engineering of biological molecules is the fundamental concept behind the design of complex materials with desirable functions. Over the last few decades, peptides and proteins have emerged as useful building blocks for well-defined nanostructures with controlled size and dimensions. Short peptides in particular have received much attention due to their inherent biocompatibility, lower synthetic cost, and ease of tunability. In addition to the diverse self-assembling properties of short peptides comprising coded amino acids and their emerging applications in nanotechnology, there is now growing interest in the properties of peptides composed of non-canonical amino acids. Such non-natural oligomers have been shown in recent years to form well-defined secondary structures similar to natural proteins, with the ability to self-assemble to generate a wide variety of nanostructures with excellent biostability. This review describes recent events in the development of supramolecular assemblies of peptides composed completely of non-coded amino acids and their hybrid analogues. Special attention is paid to understanding the supramolecular assemblies at the atomic level and to considering their potential applications in nanotechnology., (© 2021 Wiley-VCH GmbH.)

Molecular oxygen (O 2 ) is a highly reactive oxidizing agent and is harmful to many biological and industrial systems. Although O 2 often interacts via metals or reducing agents, a binding mechanism involving an organic supramolecular structure has not been described to date. In this work, the prominent dipeptide hydrogelator fluorenylmethyloxycarbonyl-diphenylalanine is shown to encage O 2 and significantly limit its diffusion and penetration through the hydrogel. Molecular dynamics simulations suggested that the O 2 binding mechanism is governed by pockets formed between the aromatic rings in the supramolecular structure of the gel, which bind O 2 through hydrophobic interactions. This phenomenon is harnessed to maintain the activity of the O 2 -hypersensitive enzyme [FeFe]-hydrogenase, which holds promising potential for utilizing hydrogen gas for sustainable energy applications. Hydrogenase encapsulation within the gel allows hydrogen production following exposure to ambient O 2 . This phenomenon may lead to utilization of this low molecular weight gelator in a wide range of O 2 -sensitive applications.

Bone tissue engineering is a rapidly developing, minimally invasive technique for regenerating lost bone with the aid of biomaterial scaffolds that mimic the structure and function of the extracellular matrix (ECM). Recently, scaffolds made of electrospun fibers have aroused interest due to their similarity to the ECM, and high porosity. Hyaluronic acid (HA) is an abundant component of the ECM and an attractive material for use in regenerative medicine; however, its processability by electrospinning is poor, and it must be used in combination with another polymer. Here, we used electrospinning to fabricate a composite scaffold with a core/shell morphology composed of polycaprolactone (PCL) polymer and HA and incorporating a short self-assembling peptide. The peptide includes the arginine-glycine-aspartic acid (RGD) motif and supports cellular attachment based on molecular recognition. Electron microscopy imaging demonstrated that the fibrous network of the scaffold resembles the ECM structure. In vitro biocompatibility assays revealed that MC3T3-E1 preosteoblasts adhered well to the scaffold and proliferated, with significant osteogenic differentiation and calcium mineralization. Our work emphasizes the potential of this multi-component approach by which electrospinning, molecular self-assembly, and molecular recognition motifs are combined, to generate a leading candidate to serve as a scaffold for bone tissue engineering.

The need to increase agricultural yield has led to an extensive use of antibiotics against plant pathogens, which has resulted in the emergence of resistant strains. Therefore, there is an increasing demand for new methods, preferably with lower chances of developing resistant strains and a lower risk to the environment or public health. Many Gram-negative bacterial pathogens use quorum sensing, a population-density-dependent regulatory mechanism, to monitor the secretion of N -acyl-homoserine lactones (AHLs) and pathogenicity. Therefore, quorum sensing represents an attractive antivirulence target. AHL lactonases hydrolyze AHLs and have potential antibacterial properties; however, their use is limited by thermal instability and durability, or low activity. Here, we demonstrate that an AHL lactonase from the phosphotriesterase-like lactonase family exhibits high activity with the AHL secreted from the plant pathogen Erwinia amylovora and attenuates infection in planta . Using directed enzyme evolution, we were able to increase the enzyme's temperature resistance ( T 50 , the temperature at which 50% of the activity is retained) by 8 °C. Then, by performing enzyme encapsulation in nanospherical capsules composed of tertbutoxycarbonyl-Phe-Phe-OH peptide, the shelf life was extended for more than 5 weeks. Furthermore, the encapsulated and free mutant were able to significantly inhibit up to 70% blossom's infection in the field, achieving the same efficacy as seen with antibiotics commonly used today to treat the plant pathogen. We conclude that specific AHL lactonase can inhibit E. amylovora infection in the field, as it degrades the AHL secreted by this plant pathogen. The combination of directed enzyme evolution and peptide nanostructure encapsulation significantly improved the thermal resistance and shelf life of the enzyme, respectively, increasing its potential in future development as antibacterial treatment.

Amyloid-β (Aβ), reported as a significant constituent of drusen, was implicated in the pathophysiology of age-related macular degeneration (AMD), yet the identity of the major pathogenic Aβ species in the retina has remained hitherto unclear. Here, we examined the in-vivo retinal impact of distinct supramolecular assemblies of Aβ. Fibrillar (Aβ40, Aβ42) and oligomeric (Aβ42) preparations showed clear biophysical hallmarks of amyloid assemblies. Measures of retinal structure and function were studied longitudinally following intravitreal administration of the various Aβ assemblies in rats. Electroretinography (ERG) delineated differential retinal neurotoxicity of Aβ species. Oligomeric Aβ42 inflicted the major toxic effect, exerting diminished ERG responses through 30 days post injection. A lesser degree of retinal dysfunction was noted following treatment with fibrillar Aβ42, whereas no retinal compromise was recorded in response to Aβ40 fibrils. The toxic effect of Aβ42 architectures was further reflected by retinal glial response. Fluorescence labelling of Aβ42 species was used to detect their accumulation into the retinal tissue. These results provide conceptual evidence of the differential toxicity of particular Aβ species in-vivo, and promote the mechanistic understanding of their retinal pathogenicity. Stratifying the impact of pathological Aβ aggregation in the retina may merit further investigation to decipher the pathophysiological relevance of processes of molecular self-assembly in retinal disorders.

Bacterial biofilm formation on wet surfaces represents a significant problem in medicine and environmental sciences. One of the strategies to prevent or eliminate surface adhesion of organisms is surface modification and coating. However, the current coating technologies possess several drawbacks, including limited durability, low biocompatibility and high cost. Here, we present a simple antibacterial modification of titanium, mica and glass surfaces using self-assembling nano-structures. We have designed two different nano-structure coatings composed of fluorinated phenylalanine via the drop-cast coating technique. We investigated and characterized the modified surfaces by scanning electron microscopy, X-ray diffraction and wettability analyses. Exploiting the antimicrobial property of the nano-structures, we successfully hindered the viability of Streptococcus mutans and Enterococcus faecalis on the coated surfaces in both aerobic and anaerobic conditions. Notably, we found lower bacteria adherence to the coated surfaces and a reduction of 86-99% in the total metabolic activity of the bacteria. Our results emphasize the interplay between self-assembly and antimicrobial activity of small self-assembling molecules, thus highlighting a new approach of biofilm control for implementation in biomedicine and other fields.

The well-studied dipeptide fluorenylmethyloxycarbonyl-di-phenylalanine (FmocFF) forms a rigid hydrogel upon dissolving in dimethylsulfoxide (DMSO) and dilution in H2O. Here, we explored the pre-aggregation of the peptide in pure DMSO by vibrational spectroscopies, X-ray powder diffraction and dynamic light scattering. Our results show an equilibrium between a dominant population of amorphous oligomers (on a length scale of 2 nm) and a small number of protofibrils/fibrils (on a length scale of 30 nm in the centimolar and of 200 nm in the sub-molar region). To probe the mechanism underlying the formation of these protofilaments, we measured the 1H-NMR, IR and visible Raman spectra of DMSO containing different FmocFF concentrations, ranging between 10 and 300 mM. Our data reveal that interpeptide hydrogen bonding leads to the self-assembly of FmocFF in the centimolar region, while π-π stacking between Fmoc-groups is observed above 100 mM. The high 3J(HNHCα) coupling constant of the N-terminal amide proton indicates that the Fmoc end-cap of the peptide locks the N-terminal residue into a conformational ensemble centered at a φ-value of ca. -120°, which corresponds to a parallel β-sheet type conformation. The 3J(HNHCα) coupling constant of the C-terminal residue is indicative of a polyproline II (pPII)/βt mixture. Our results suggest that the gelation of FmocFF caused by the addition of a small amount of water to DMSO mixtures is facilitated by the formation of disordered protofibrils in pure DMSO.

Collagen, the most abundant protein in mammals, possesses notable cohesion and elasticity properties and efficiently induces tissue regeneration. The Gly-Pro-Hyp canonical tripeptide repeating unit of the collagen superhelix has been well-characterized. However, to date, the shortest tripeptide repeat demonstrated to attain a helical conformation contained 3-10 peptide repeats. Here, taking a minimalistic approach, we studied a single repeating unit of collagen in its protected form, Fmoc-Gly-Pro-Hyp. The peptide formed single crystals displaying left-handed polyproline II superhelical packing, as in the native collagen single strand. The crystalline assemblies also display head-to-tail H-bond interactions and an "aromatic zipper" arrangement at the molecular interface. The coassembly of this tripeptide, with Fmoc-Phe-Phe, a well-studied dipeptide hydrogelator, produced twisted helical fibrils with a polyproline II conformation and improved hydrogel mechanical rigidity. The design of these peptides illustrates the possibility to assemble superhelical nanostructures from minimal collagen-inspired peptides with their potential use as functional motifs to introduce a polyproline II conformation into hybrid hydrogel assemblies.

Over the last few years, hydrogels have been proposed for many biomedical applications, including drug delivery systems and scaffolds for tissue engineering. In particular, peptides have been envisioned as excellent candidates for the development of hydrogel materials, due to their intrinsic biocompatibility, ease of handling, and intrinsic biodegradability. Recently, we developed a novel hybrid polymer-peptide conjugate, PEG8-(FY)3, which is able to self-assemble into a self-supporting soft hydrogel over dry and wet surfaces as demonstrated by molecular dynamics simulation. Here, we describe the synthesis and supramolecular organization of six novel hexapeptides rationally designed by punctual chemical modification of the primary peptide sequence of the ancestor peptide (FY)3. Non-coded amino acids were incorporated by replacing the phenylalanine residue with naphthylalanine (Nal) and tyrosine with dopamine (Dopa). We also studied the effect of the modification of the side chain and the corresponding PEGylated peptide analogues, on the structural and mechanical properties of the hydrogel. Secondary structure, morphology and rheological properties of all the peptide-based materials were assessed by various biophysical tools. The in vitro biocompatibility of the supramolecular nanostructures was also evaluated on fibroblast cell lines. We conclude that the PEG8-(Nal-Dopa)3 hydrogel possesses the right properties to serve as a scaffold and support cell growth.

The formation of metabolite fibrillar assemblies represents a paradigm shift in the study of human metabolic disorders. Yet, direct clinical relevance has been attributed only to metabolite crystals. A notable example for metabolite crystallization is calcium oxalate crystals observed in various diseases, including primary hyperoxaluria. We unexpectedly observed retinal damage among young hyperoxaluria patients in the absence of crystals. Exploring the possible formation of alternative supramolecular organizations and their biological role, here we show that oxalate can form ordered fibrils with no associated calcium. These fibrils inflict intense retinal cytotoxicity in cultured cells. A rat model injected with oxalate fibrils recaptures patterns of retinal dysfunction observed in patients. Antibodies purified from hyperoxaluria patient sera recognize oxalate fibrils regardless of the presence of calcium. These findings highlight a new molecular basis for oxalate-associated disease, and to our knowledge provide the first direct clinical indication for the pathogenic role of metabolite fibrillar assemblies., (© 2020. The Author(s).)

Peptide-based supramolecular hydrogels are utilized as functional materials in tissue engineering, axonal regeneration, and controlled drug delivery. The Arg-Gly-Asp (RGD) ligand based supramolecular gels have immense potential in this respect, as this tripeptide is known to promote cell adhesion. Although several RGD-based supramolecular hydrogels have been reported, most of them are devoid of adequate resilience and long-range stability for in vitro cell culture. In a quest to improve the mechanical properties of these tripeptide-based gels and their durability in cell culture media, the Fmoc-RGD hydrogelator is non-covalently functionalized with a biocompatible and biodegradable polymer, chitosan, resulting in a composite hydrogel with enhanced gelation rate, mechanical properties and cell media durability. Interestingly, both Fmoc-RGD and Fmoc-RGD/chitosan composite hydrogels exhibit thixotropic properties. The utilization of the Fmoc-RGD/chitosan composite hydrogel as a scaffold for 2D and 3D cell cultures is demonstrated. The composite hydrogel is found to have notable antibacterial activity, which stems from the inherent antibacterial properties of chitosan. Furthermore, the composite hydrogels are able to produce ultra-small, mono-dispersed, silver nanoparticles (AgNPs) arranged on the fiber axis. Therefore, the authors' approach harnesses the attributes of both the supramolecular-polymer (Fmoc-RGD) and the covalent-polymer (chitosan) component, resulting in a composite hydrogel with excellent potential., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The rapid advancement of peptide- and amino-acid-based nanotechnology offers new approaches for the development of biomedical materials. The utilization of fluorenylmethyloxycarbonyl (Fmoc)-decorated self-assembling building blocks for antibacterial and anti-inflammatory purposes represents promising advancements in this field. Here, we present the antibacterial capabilities of the nanoassemblies formed by Fmoc-pentafluoro-l-phenylalanine-OH, their substantial effect on bacterial morphology, as well as new methods developed for the functional incorporation of these nanoassemblies within resin-based composites. These amalgamated materials inhibit and hinder bacterial growth and viability and are not cytotoxic toward mammalian cell lines. Importantly, due to the low dosage required to confer antibacterial activity, the integration of the nanoassemblies does not affect their mechanical and optical properties. This approach expands on the growing number of accounts on the intrinsic antibacterial capabilities of self-assembling building blocks and serves as a basis for further design and development of enhanced composite materials for biomedical applications.

The high demand for tissue engineering scaffolds capable of inducing bone regeneration using minimally invasive techniques prompts the need for the development of new biomaterials. Herein, we investigate the ability of Alginate incorporated with the fluorenylmethoxycarbonyl-diphenylalanine (FmocFF) peptide composite hydrogel to serve as a potential biomaterial for bone regeneration. We demonstrate that the incorporation of the self-assembling peptide, FmocFF, in sodium alginate leads to the production of a rigid, yet injectable, hydrogel without the addition of cross-linking agents. Scanning electron microscopy reveals a nanofibrous structure which mimics the natural bone extracellular matrix. The formed composite hydrogel exhibits thixotropic behavior and a high storage modulus of approximately 10 kPA, as observed in rheological measurements. The in vitro biocompatibility tests carried out with MC3T3-E1 preosteoblast cells demonstrate good cell viability and adhesion to the hydrogel fibers. This composite scaffold can induce osteogenic differentiation and facilitate calcium mineralization, as shown by Alizarin red staining, alkaline phosphatase activity and RT-PCR analysis. The high biocompatibility, excellent mechanical properties and similarity to the native extracellular matrix suggest the utilization of this hydrogel as a temporary three-dimensional cellular microenvironment promoting bone regeneration.

Conducting polymer hydrogels (CPHs) emerge as excellent functional materials, as they harness the advantages of conducting polymers with the mechanical properties and continuous 3D nanostructures of hydrogels. This bicomponent organization results in soft, all-organic, conducting micro-/nanostructures with multifarious material applications. However, the application of CPHs as functional materials for biomedical applications is currently limited due to the necessity to combine the features of biocompatibility, self-healing, and fine-tuning of the mechanical properties. To overcome this issue, we choose to combine a protected dipeptide as the supramolecular gelator, owing to its intrinsic biocompatibility and excellent gelation ability, with the conductive polymer polyaniline (PAni), which was polymerized in situ. Thus, a two-component, all-organic, conducting hydrogel was formed. Spectroscopic evidence reveals the formation of the emeraldine salt form of PAni by intrinsic doping. The composite hydrogel is mechanically rigid with a very high storage modulus ( G') value of ∼2 MPa, and the rigidity was tuned by changing the peptide concentration. The hydrogel exhibits ohmic conductivity, pressure sensitivity, and, importantly, self-healing features. By virtue of its self-healing property, the polymeric nonmetallic hydrogel can reinstate its intrinsic conductivity when two of its macroscopically separated blocks are rejoined. High cell viability of cardiomyocytes grown on the composite hydrogel demonstrates its noncytotoxicity. These combined attributes of the hydrogel allowed its utilization for dynamic range pressure sensing and as a conductive interface for electrogenic cardiac cells. The composite hydrogel supports cardiomyocyte organization into a spontaneously contracting system. The composite hydrogel thus has considerable potential for various applications.

Short peptides or single amino acids are interesting building blocks for fabrication of hydrogels, frequently used as extracellular matrix-mimicking scaffolds for cell growth in tissue engineering. The combination of two or more peptide hydrogelators could allow obtaining different materials exhibiting new architectures, tunable mechanical properties, high stability and improved biofunctionality. Here we report on the synthesis, formulation and multi-scale characterization of peptide-based mixed hydrogels formed by the low molecular weight Fmoc-FF (N α -fluorenylmethyloxycarbonyl diphenylalanine) hydrogelator and of the PEG 8 -(FY)3 hexapeptide, containing three repetitions of the Phe-Tyr motif and a PEG moiety at its N-terminus. Mixed hydrogels were also prepared by replacing PEG 8 -(FY)3 with its analogue (FY)3, without the PEG moiety. Rheology analysis confirmed the improved mechanical features of the multicomponent gels prepared at two different ratios (2/1 or 1/1, v/v). However, the presence of the hydrophilic PEG polymeric moiety causes a slowing down of the gel kinetic formation (from 42 to 18 minutes) and a decrease of the gel rigidity (G' from 9 to 6 kPa). Preliminary in vitro biocompatibility and cell adhesion assays performed on Chinese hamster ovarian (CHO) cells suggest a potential employment of these multicomponent hydrogels as exogenous scaffold materials for tissue engineering.

The ensemble of native, folded state was once considered to represent the global energy minimum of a given protein sequence. More recently, the discovery of the cross-β amyloid state revealed that deeper energy minima exist, often associated with pathogenic, fibrillar deposits, when the concentration of proteins reaches a critical value. Fortunately, a sizable energy barrier impedes the conversion from native to pathogenic states. However, little is known about the structure of the related transition state. In addition, there are indications of polymorphism in the amyloidogenic process. Here, we report the first evidence of the conversion of metastable cross-α-helical crystals to thermodynamically stable cross-β-sheet-like fibrils by a de novo designed heptapeptide. Furthermore, for the first time, we demonstrate at atomic resolution that the flip of a peptide plane from a type I to a type II' turn facilitates transformation to cross-β structure and assembly of a dry steric zipper. This study establishes the potential of a peptide turn, a common protein secondary structure, to serve as a principal gatekeeper between a native metastable folded state and the amyloid state.

Biomaterials play a critical role in regenerative strategies such as stem cell-based therapies and tissue engineering, aiming to replace, remodel, regenerate, or support damaged tissues and organs. The design of appropriate three-dimensional (3D) scaffolds is crucial for generating bio-inspired replacement tissues. These scaffolds are primarily composed of degradable or non-degradable biomaterials and can be employed as cells, growth factors, or drug carriers. Naturally derived and synthetic biomaterials have been widely used for these purposes, but the ideal biomaterial remains to be found. Researchers from diversified fields have attempted to design and fabricate novel biomaterials, aiming to find novel theranostic approaches for tissue engineering and regenerative medicine. Since no single biomaterial has been found to possess all the necessary characteristics for an ideal performance, over the years scientists have tried to develop composite biomaterials that complement and combine the beneficial properties of multiple materials into a superior matrix. Herein, we highlight the structural features and performance of various biomaterials and their application in regenerative medicine and for enhanced tissue engineering approaches.

Hyaluronic acid (HA), a major component of the extracellular matrix, is an attractive material for various medical applications. Yet, its low mechanical rigidity and fast in vivo degradation hinder its utilization. Here, we demonstrate the reinforcement of HA by its integration with a low-molecular-weight peptide hydrogelator to produce a composite hydrogel. The formulation of HA with the fluorenylmethoxycarbonyl diphenylalanine (FmocFF) peptide, one of the most studied self-assembling hydrogel-forming building blocks, showing notable mechanical properties, resulted in the formation of stable, homogeneous hydrogels. Electron microscopy analysis demonstrated a uniform distribution of the two matrices in the composite forms. The composite hydrogels showed improved mechanical properties and stability to enzymatic degradation while maintaining their biocompatibility. Moreover, the storage modulus of the FmocFF/HA composite hydrogels reached up to 25 kPa. The composite hydrogels allowed sustained release of curcumin, a hydrophobic polyphenol showing antioxidant, anti-inflammatory, and antitumor activities. Importantly, the rate of curcumin release was modulated as a function of the concentration of the FmocFF peptide within the hydrogel matrix. This work provides a new approach for conferring mechanical rigidity and stability to HA without the need of cross-linking, thus potentially facilitating its utilization in different clinical applications, such as sustained drug release.

Low-molecular-weight self-assembled peptides may serve as promising hydrogelators for drug delivery applications by changing their structural network in response to external stimuli. Herein, inspired by the well-studied low-molecular-weight peptide hydrogelator, fluorenyl-methoxycarbonyl-diphenylalanine (Fmoc-FF), a novel peptide is designed and synthesized to include an ultraviolet (UV)-sensitive phototrigger. Similar to Fmoc-FF, 6-nitroveratryloxycarbonyl-diphenylalanine (Nvoc-FF) self-assembles to form a 3D, self-supporting, nanofibrous hydrogel. The Nvoc-FF hydrogel exhibits good mechanical properties with a storage modulus of 40 kPa. UV irradiation of the Nvoc-FF hydrogel encapsulating insulin-fluorescein isothiocyanate (insulin-FITC) results in the cleavage of Nvoc-FF peptide to produce unmasked FF, thereby facilitating the degradation of the hydrogel and the release of insulin-FITC. This release is in linear correlation to the irradiation time. In the present study, a first insight into this rigid, fibrous, light-responsive hydrogel is provided, allowing the fabrication of a novel drug delivery system for controlled release of large molecules., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rationally designed two-component supramolecular organogels based on multiple chemical interactions between percarboxylato- and peramino-pillararenes are described. Mixing low concentration solutions (<1 % w/v) of decacarboxylato-pillar[5]arene (1) with decaamino-pillar[5]arenes (2 b-d) affords, rapidly and without heating, organogels displaying an exceptional combination of properties. These supramolecular organogels, the characteristics of which are tunable, were found to be thixotropic and thermally stable, with T gel values in some cases exceeding the boiling point of the embedded solvent. It is demonstrated that both structural complementarity and multivalency are important determinants in the gelation process of these attractive soft materials., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The release of nanoscale structures from microcapsules, triggered by changes in the capsule in response to external stimuli, has significant potential for active component delivery. Here, we describe an orthogonal strategy for controlling molecular species' release across oil/water interfaces by modulating their intrinsic self-assembly state. We show that although the soluble peptide Boc-FF can be stably encapsulated for days, its self-assembly into nanostructures triggers jet-like release within seconds. Moreover, we exploit this self-assembly-mediated release to deliver other molecular species that are transported as cargo. These results demonstrate the role of self-assembly in modulating the transport of peptides across interfaces.