The biochemical and biophysical properties of the extracellular matrix (ECM) play a pivotal role in regulating cellular behaviors such as proliferation, migration, and differentiation. Engineered protein-based hydrogels, with highly tunable multifunctional properties, have the potential to replicate key features of the native ECM. Formed by self-assembly or crosslinking, engineered protein-based hydrogels can induce a range of cell behaviors through bioactive and functional domains incorporated into the polymer backbone. Using recombinant techniques, the amino acid sequence of the protein backbone can be designed with precise control over the chain-length, folded structure, and cell-interaction sites. In this review, the modular design of engineered protein-based hydrogels from both a molecular- and network-level perspective are discussed, and summarize recent progress and case studies to highlight the diverse strategies used to construct biomimetic scaffolds. This review focuses on amino acid sequences that form structural blocks, bioactive blocks, and stimuli-responsive blocks designed into the protein backbone for highly precise and tunable control of scaffold properties. Both physical and chemical methods to stabilize dynamic protein networks with defined structure and bioactivity for cell culture applications are discussed. Finally, a discussion of future directions of engineered protein-based hydrogels as biomimetic cellular scaffolds is concluded.

Self-assembled peptide-based hydrogels have attracted considerable interest from the research community. Particularly, low molecular weight gelators (LMWGs) consisting of amino acids and short peptides are highly suitable for biological applications owing to their facile synthesis and scalability, as well as their biocompatibility, biodegradability, and stability in physiological conditions. However, challenges in understanding the structure-property relationship and lack of design rules hinder the development of new gelators with the required properties for several applications. Hereby, in the plethora of peptide-based gelators, this review discusses the mechanical properties of single amino acid and dipeptide-based hydrogels. A mutual analysis of these systems allows us to highlight the relationship between the gel mechanical properties and amino acid sequence, preparation methods, or N capping groups. Additionally, recent advancements in the tuning of the gels\' rheological properties are reviewed. In this way, the present review aims to help bridge the knowledge gap between structure and mechanical properties, easing the selection or design of peptides with the required properties for biological applications.

Peptide-based self-assembly has been used to produce a wide range of nanostructures. While most of these systems involve self-assembly of α-peptides, more recently β-peptides have also been shown to undergo supramolecular self-assembly, and have been used to produce materials for applications in tissue engineering, cell culture and drug delivery. In order to engineer new materials with specific structure and function, theoretical molecular modelling can provide significant insights into the collective balance of non-covalent interactions that drive the self-assembly and determine the structure of the resultant supramolecular materials under different conditions. However, this approach has only recently become feasible for peptide-based self-assembled nanomaterials, particularly those that incorporate non α-amino acids. This perspective provides an overview of the challenges associated with computational modelling of the self-assembly of β-peptides and the recent success using a combination of experimental and computational techniques to provide insights into the self-assembly mechanisms and fully atomistic models of these new biocompatible materials.

Fmoc-diphenylalanine (Fmoc-FF) is a low-molecular-weight peptide hydrogelator. This simple all-aromatic peptide can generate self-supporting hydrogel materials, which have been proposed as novel materials for diagnostic and pharmaceutical applications. Our knowledge of the molecular determinants of Fmoc-FF aggregation is used as a guide to design new peptide-based gelators, with features for the development of improved tools. Here, we enlarge the plethora of Fmoc-FF-based hydrogelated matrices by studying the properties of the Fmoc-FFK tripeptide, alone or in combination with Fmoc-FF. For multicomponent matrices, the relative weight ratios between Fmoc-FFK and Fmoc-FF (specifically, 1/1, 1/5, 1/10, and 1/20 w/w) are evaluated. All the systems and their multiscale organization are studied using different experimental techniques, including rheology, circular dichroism, Fourier transform infrared spectroscopy, and scanning electron microscopy (SEM). Preliminary profiles of biocompatibility for the studied systems are also described by testing them in vitro on HaCaT and 3T3-L1 cell lines. Additionally, the lysine (K) residue at the C-terminus of the Fmoc-FF moiety introduces into the supramolecular material chemical functions (amino groups) which may be useful for modification/derivatization with bioactive molecules of interest, including diagnostic probes, chelating agents, active pharmaceutical ingredients, or peptide nucleic acids.

Multicomponent hydrogels (HGs) based on ultrashort aromatic peptides have been exploited as biocompatible matrices for tissue engineering applications, the delivery of therapeutic and diagnostic agents, and the development of biosensors. Due to its capability to gel under physiological conditions of pH and ionic strength, the low molecular-weight Fmoc-FF (Nα-fluorenylmethoxycarbonyl-diphenylalanine) homodimer is one of the most studied hydrogelators. The introduction into the Fmoc-FF hydrogel of additional molecules like protein, organic compounds, or other peptide sequences often allows the generation of novel hydrogels with improved mechanical and functional properties. In this perspective, here we studied a library of novel multicomponent Fmoc-FF based hydrogels doped with different amounts of the tripeptide Fmoc-FFX (in which X= Cys, Ser, or Thr). The insertion of these tripeptides allows to obtain hydrogels functionalized with thiol or alcohol groups that can be used for their chemical post-derivatization with bioactive molecules of interest like diagnostic or biosensing agents. These novel multicomponent hydrogels share a similar peptide organization in their supramolecular matrix. The hydrogels\' biocompatibility, and their propensity to support adhesion, proliferation, and even cell differentiation, assessed in vitro on fibroblast cell lines, allows us to conclude that the hybrid hydrogels are not toxic and can potentially act as a scaffold and support for cell culture growth.

Short and ultra-short peptides have been recently envisioned as excellent building blocks for the formulation of hydrogels with appealing properties. Due to its simplicity and capability to gel under physiological conditions, Fmoc-FF (Nα -fluorenylmethoxycarbonyl-diphenylalanine), remains one of the most studied low molecular-weight hydrogelators. Since its first identification in 2006, a plethora of its analogues were synthetized and investigated for the fabrication of novel supramolecular materials. Here we report a description of the Fmoc-FF analogues in which the aromatic Fmoc group is replaced with other substituents. These analogues are distinguished into five different classes including derivatives: i) customized with solid phase peptide synthesis protecting groups; ii) containing non-aromatic groups, iii) containing aromatic groups, iv) derivatized with metal complexes and v) containing stimuli-responsive groups. The morphological, mechanical, and functional effects caused by this modification on the resulting material are also pointed out.

Generated by a hierarchical and multiscale self-assembling phenomenon, peptide-based hydrogels (HGs) are soft materials useful for a variety of applications. Short and ultra-short peptides are intriguing building blocks for hydrogel fabrication. These matrices can also be obtained by mixing low-molecular-weight peptides with other chemical entities (e.g., polymers, other peptides). The combination of two or more constituents opens the door to the development of hybrid systems with tunable mechanical properties and unexpected biofunctionalities or morphologies. For this scope, the formulation, the multiscale analysis, and the supramolecular characterization of novel hybrid peptide-polymer hydrogels are herein described. The proposed matrices contain the Fmoc-FF (Nα-fluorenylmethyloxycarbonyl diphenylalanine) hydrogelator at a concentration of 0.5 wt% (5.0 mg/mL) and a diacrylate α-/ω-substituted polyethylene-glycol derivative (PEGDA). Two PEGDA derivatives, PEGDA 1 and PEGDA2 (mean molecular weights of 575 and 250 Da, respectively), are mixed with Fmoc-FF at different ratios (Fmoc-FF/PEGDA at 1/1, 1/2, 1/5, 1/10 mol/mol). All the multicomponent hybrid peptide-polymer hydrogels are scrutinized with a large panel of analytical techniques (including proton relaxometry, FTIR, WAXS, rheometry, and scanning electronic microscopy). The matrices were found to be able to generate mechanical responses in the 2-8 kPa range, producing a panel of tunable materials with the same chemical composition. The release of a model drug (Naphthol Yellow S) is reported too. The tunable features, the different topologies, and the versatility of the proposed materials open the door to the development of tools for different applicative areas, including diagnostics, liquid biopsies and responsive materials. The incorporation of a diacrylate function also suggests the possible development of interpenetrating networks upon cross-linking reactions. All the collected data allow a mutual comparison between the different matrices, thus confirming the significance of the hybrid peptide/polymer-based methodology as a strategy for the design of innovative materials.

Hydrogels (HGs) are tri-dimensional materials with a non-Newtonian flow behaviour formed by networks able to encapsulate high amounts of water or other biological fluids. They can be prepared using both synthetic or natural polymers and their mechanical and functional properties may change according to the preparation method, the solvent, the pH, and to others experimental parameters. Recently, many short and ultra-short peptides have been investigated as building blocks for the formulation of biocompatible hydrogels suitable for different biomedical applications. Due to its simplicity and capability to gel in physiological conditions, Fmoc-FF dipeptide is one of the most studied peptide hydrogelators. Although its identification dates to 15 ago, its behaviour is currently studied because of the observation that the final material obtained is deeply dependent on the preparation method. To collect information about their formulation, here are reported some different strategies adopted until now for the Fmoc-FF HG preparation, noting the changes in the structural arrangement and behaviour in terms of stiffness, matrix porosity, and stability induced by the different formulation strategy on the final material.

Aggregation-induced emissive materials are gaining particular attention in the last decades due to their wide application in different fields, from optical devices to biomedicine. In this work, compounds having these kinds of properties, composed of tetraphenylethylene scaffold combined with fatty acids of different lengths, were synthesized and characterized. These molecules were found able to self-assemble into different supramolecular emissive structures depending on the chemical composition and water content. Furthermore, they were used as N-terminus capping agents in the development of peptide-based materials. The functionalization of a 5-mer laminin-derived peptide led to the obtainment of luminescent fibrillary materials that were not cytotoxic and were able to form supramolecular gels in aqueous environment.

Self-assembled peptides are an emerging family of biomaterials that show great promise for a range of biomedical and biotechnological applications. Introducing and tuning the pH-responsiveness of the assembly is highly desirable for improving their biological activities. Inspired by proteins with internal ionizable residues, we report a simple but effective approach to constructing pH-responsive peptide assembly containing unnatural ionic amino acids with an aliphatic tertiary amine side chain. Through a combined experimental and computational investigation, we demonstrate that these residues can be accommodated and stabilized within the internal hydrophobic compartment of the peptide assembly. The hydrophobic microenvironment shifts their pKa significantly from a basic pH typically found for free amines to a more biologically relevant pH in the weakly acidic range. The pH-induced ionization and ionization-dependent self-assembly and disassembly are thoroughly investigated and correlated with the biological activity of the assembly. This new approach has unique advantages in tuning the pH-responsiveness of self-assembled peptides across a large pH range in a complex biological environment. We anticipate the ionizable amino acids developed here can be widely applicable to the synthesis and self-assembly of many amphiphilic peptides with endowed pH-responsive properties to enhance their biological activities toward applications ranging from targeted therapeutic delivery to proton transport.