Hydrogel-Stiffening and Non-Cell Adhesive Properties of Amphiphilic Peptides with Central Alkylene Chains

Amphiphilic peptides bearing terminal alkyl tails form supramolecular nanofibers that are increasingly used as biomaterials with multiple functionalities. Insertion of alkylene chains in peptides can be designed as another type of amphiphilic peptide, yet the influence of the internal alkylene chains on self-assembly and biological properties remains poorly defined. Unlike the terminal alkyl tails, the internal alkylene chains can affect not only the hydrophobicity but also the flexibility and packing of the peptides. Herein, we demonstrate the supramolecular and biological effects of the central alkylene chain length inserted in a peptide. Insertion of the alkylene chain at the center of the peptide allowed for strengthened β-sheet hydrogen bonds and modulation of the packing order, and consequently the amphiphilic peptide bearing C2 alkylene chain formed a hydrogel with the highest stiffness. Interestingly, the amphiphilic peptides bearing internal alkylene chains longer than C2 showed a diminished cell-adhesive property. This study offers a novel molecular design to tune mechanical and biological properties of peptide materials.     

Shape-specific nanofibers via self-assembly of three-branched peptide

A novel amphiphilic branched peptide (1), in which three β-sheet formable peptides (L(4)K(8)L(4)) were connected by Lys residue, was newly prepared as a building block for self-assembly. A detailed analysis of the conformation and self-assembling property of 1 in water at various pH conditions was performed by using circular dichroism, FTIR, atomic force and transmission electron microscopies. The experimental results revealed that the branched peptide showed a pH-dependent conformation forming a shape-specific β-sheet-based nanofiber with morphologically kinked structures under specific pH conditions. Exploring a novel peptide building unit that has the ability to self-assemble into designed and complicated nano-objects is valuable to facilitate a bottom-up nanotechnology.     

The interactions between doxorubicin and amphiphilic and acidic β-sheet peptides towards drug delivery hydrogels

Amphiphilic β-sheet peptides decorated by acidic amino acids may spontaneously assemble into ordered monolayers at interfaces as well as form hydrogels at near physiological pH values. Here we monitored interactions between the peptide Pro-Asp-(Phe-Asp)(5)-Pro and the mildly amphiphilic chemotherapeutic drug doxorubicin (Dox). The peptide in the form of monolayers at the air water interface was found to enhance Dox adsorption, pointing to favorable interactions between the amphiphilic peptide and Dox. In solutions the fluorescence emission of Dox which was fitted to the Stern-Volmer quenching models suggested the formation of Dox associated forms >25 μM and larger forms at >100 μM. In presence of the peptide these larger associated forms appeared already at Dox concentrations >50 μM, indicating enhanced interactions between Dox and the peptide in the β-sheet conformation. Peptide hydrogels loaded with the drug showed sustained release profiles over several days. Smaller fractions of the drug were released with increase in either peptide or initially loaded drug concentrations. The released Dox was found to retain its cytotoxic activity in vitro. This study provides insights on the interactions between the amphiphilic and acidic peptide and Dox that are useful for the bottom up development of Dox-loaded peptide hydrogels for local drug delivery applications.     

The Introduction of a Cysteine Residue Modulates The Mechanical Properties of Aromatic-Based Solid Aggregates and Self-Supporting Hydrogels

Peptide-based hydrogels, originated by multiscale self-assembling phenomenon, have been proposed as multivalent tools in different technological areas. Structural studies and molecular dynamics simulations pointed out the capability of completely aromatic peptides to gelificate if hydrophilic and hydrophobic forces are opportunely balanced. Here, the effect produced by the introduction of a Cys residue in the heteroaromatic sequence of (FY)3 and in its PEGylated variant was evaluated. The physicochemical characterization indicates that both FYFCFYF and PEG8-FYFCFYF are able to self-assemble in supramolecular nanostructures whose basic cross-β motif resembles the one detected in the ancestor (FY)3 assemblies. However, gelification occurs only for FYFCFYF at a concentration of 1.5 wt%. After cross-linking of cysteine residues, the hydrogel undergoes to an improvement of the rigidity compared to the parent (FY)3 assemblies as suggested by the storage modulus (G’) that increases from 970 to 3360 Pa. The mechanical properties of FYFCFYF are compatible with its potential application in bone tissue regeneration. Moreover, the avalaibility of a Cys residue in the middle of the peptide sequence could allow the hydrogel derivatization with targeting moieties or with biologically relevant molecules.     

Heterotypic Supramolecular Hydrogels Formed by Noncovalent Interactions in Inflammasomes

The advance of structural biology has revealed numerous noncovalent interactions between peptide sequences in protein structures, but such information is less explored for developing peptide materials. Here we report the formation of heterotypic peptide hydrogels by the two binding motifs revealed by the structures of an inflammasome. Specifically, conjugating a self-assembling motif to the positively or negatively charged peptide sequence from the ASCPYD filaments of inflammasome produces the solutions of the peptides. The addition of the peptides of the oppositely charged and complementary peptides to the corresponding peptide solution produces the heterotypic hydrogels. Rheology measurement shows that ratios of the complementary peptides affect the viscoelasticity of the resulted hydrogel. Circular dichroism indicates that the addition of the complementary peptides results in electrostatic interactions that modulate self-assembly. Transmission electron microscopy reveals that the ratio of the complementary peptides controls the morphology of the heterotypic peptide assemblies. This work illustrates a rational, biomimetic approach that uses the structural information from the protein data base (PDB) for developing heterotypic peptide materials via self-assembly.     

Supramolecular hydrogels of self-assembled zwitterionic-peptides

Zwitterionic polymer materials have been extensively studied, but zwitterionic peptides supramolecular hydrogel materials are rarely studied. In this study, the preparation of two zwitterionic hydrogels using self-assembled peptides were reported. The hydrogels could be fabricated easily by changing the temperature or enzyme catalysis in a short time. And the differences in structure and function of the zwitterion peptide hydrogels caused by the two preparation methods were also be compared. We found that the hydrogel prepared by enzyme induced self-assembly has better solubility and lower cytotoxicity than that prepared by the heating-cooling process. The result showed the enzyme induced self-assembly way to form zwitterionic peptides supramolecular hydrogel materials could have further biomedical applications.     

Parallel Versus Antiparallel β-Sheet Structure in Cyclic Peptide Hybrids Containing γ- or δ-Cyclic Amino Acids

Cyclic peptides with disc-shaped structures have emerged as potent building blocks for the preparation of new biomaterials in fields ranging from biological to material science. In this work, we analyze in depth the self-assembling properties of a new type of cyclic peptides based on the alternation of α-residues and cyclic δ-amino acids (α,δ-CPs). To examine the preferred stacking properties adopted by cyclic peptides bearing this type of amino acids, we carried out a synergistic in vitro/in silico approximation by using simple dimeric models and then extended to nanotubes. Although these new cyclic peptides (α,δ-CPs) can interact either in a parallel or antiparallel fashion, our results confirm that although the parallel β-sheet is more stable, it can be switched to the antiparallel stacking by choosing residues that can establish favorable cross-strand interactions. Moreover, the subsequent comparison by using the same methodology but applied to α,γ-CPs models, up to the moment assumed as antiparallel-like d,l -α-CPs, led to unforeseen conclusions that put into question preliminary conjectures about these systems. Surprisingly, they tend to adopt a parallel β-sheet directed by the skeleton interactions. These results imply a change of paradigm with respect to cyclic peptide designs that should be considered for dimers and nanotubes.     

Impact of C-Terminal Chemistry on Self-Assembled Morphology of Guanosine Containing Nucleopeptides

Herein, we report the design and characterization of guanosine-containing self-assembling nucleopeptides that form nanosheets and nanofibers. Through spectroscopy and microscopy analysis, we propose that the peptide component of the nucleopeptide drives the assembly into β-sheet structures with hydrogen-bonded guanosine forming additional secondary structures cooperatively within the peptide framework. Interestingly, the distinct supramolecular morphologies are driven not by metal cation responsiveness common to guanine-based materials, but by the C-terminal peptide chemistry. This work highlights the structural diversity of self-assembling nucleopeptides and will help advance the development of applications for these supramolecular guanosine-containing nucleopeptides.     

From fibers to micelles using point-mutated amphiphilic peptides

Biocompatible, self-assembled nanostructures are attracting ever more attention, in particular in aqueous media for biomedical applications. Here, we present the successful, solid-phase peptide synthesis (SPPS) and characterization of short amino acid sequences with amphiphilic character with the aim of gaining insight into their self-assembled, supramolecular structures. The peptide design includes three parts: (a) a charged lysine part, (b) an acetylated lysine part, and (c) a constant hydrophobic rodlike helix, based on gramicidin A (gA). By stepwise replacement of free lysine (K) with acetylated lysine (X) we generated a library of a total of 10 peptides, Ac-X(8)-gA and K(m)X(8-m)-gA (m ranging from 0 to 8). By using point mutations, we adjusted the degree of acetylation (DA) and thus the overall amphiphilicity of the peptides, which led to a change in the secondary structure in the aqueous environment from a β-sheet to an α-helix. This transition generated a significant change in the morphology of the self-assembled structures from fibers to micelles. Two different regions were observed for the conformation of the hydrophilic part of the peptide: one region, a β-sheet-like secondary structure, inducing fiber formation (high DA), the other an α-helical-like secondary structure, generating micelle formation (moderate and low DA). The micellar structures depended on the degree of acetylation, which influenced their critical micelle concentration (cmc). These morphology regions were determined by a combination of circular dichroism, dynamic light scattering, surface tension, and transmission electron microscopy, which allowed us to correlate the generated supramolecular architectures with the fine changes obtained by means of the point mutation strategy.     

Enhanced mechanical rigidity of hydrogels formed from enantiomeric peptide assemblies

Chirality can be used as a design tool to control the mechanical rigidity of hydrogels formed from self-assembling peptides. Hydrogels prepared from enantiomeric mixtures of self-assembling β-hairpins show nonadditive, synergistic, enhancement in material rigidity compared to gels prepared from either pure enantiomer, with the racemic hydrogel showing the greatest effect. CD spectroscopy, TEM, and AFM indicate that this enhancement is defined by nanoscale interactions between enantiomers in the self-assembled state.     

Role of alkylated residues in the tetrapeptide self-assembly—A molecular dynamics study

Designing peptide sequences that self-assemble into well-defined nanostructures can open a new venue for the development of novel drug carriers and molecular contrast agents. Current approaches are often based on a linear block-design of amphiphilic peptides where a hydrophilic peptide chain is terminated by a hydrophobic tail. Here, a new template for a self-assembling tetrapeptide (YXKX, Y = tyrosine, X = alkylated tyrosine, K = lysine) is proposed with two distinct sides relative to the peptide's backbone: alkylated hydrophobic residues on one side and hydrophilic residues on the other side. Using all-atom molecular dynamics simulations, the self-assembly pathway of the tetrapeptide is analyzed for two different concentrations. At both concentrations, tetrapeptides self-assembled into a nanosphere structure. The alkylated tyrosines initialize the self-assembly process via a strong hydrophobic effect and to reduce exposure to the aqueous solvent, they formed a hydrophobic core. The hydrophilic residues occupied the surface of the self-assembled nanosphere. Ordered arrangement of tetrapeptides within the nanosphere with the backbone hydrogen bonding led to a beta sheet formation. Alkyl chain length constrained the size and shape of the nanosphere. This study provides foundation for further exploration of self-assembling structures that are based on peptides with hydrophobic and hydrophilic moieties located on the opposite sides of a peptide backbone.     

Programming supramolecular peptide materials by modulating the intermediate steps in the complex assembly pathway: Implications for biomedical applications

Self-assembling peptides form a prominent class of supramolecular materials with in general good biocompatibility. To afford better control over the material properties, tremendous progress has been made in studying the supramolecular organization of the peptide assemblies. This knowledge has helped us to understand the correlation between the molecular structure of the peptide building blocks and the properties of the supramolecular products. However, peptide self-assembly consists of a complex pathway rather than a spontaneous thermodynamic process. This implies that the outcome of the self-assembly is critically governed by the assembly pathway. Here, we are going to discuss how peptide self-assembly can be modulated at the intermediate steps in the self-assembly pathway. The focus will be to demonstrate this engineering approach on the example of zero-dimensional/one-dimensional nanostructure selectivity over the β-sheet assembly pathway. In addition, we provide examples of biomedical applications of such steered peptide assemblies in the field of drug delivery and tissue engineering.     

Computational approaches for understanding and predicting the self-assembled peptide hydrogels

Self-assembled peptide hydrogel is a promising biomaterial and has been widely applied in many fields. As a typical self-assembly material, peptide hydrogel exhibits properties different from traditional polymer hydrogel, and has unique features in molecular design, structural elements of hydrogel, and control strategies. With the desire to apply the principles of self-assembly to the design and prediction of peptide hydrogels, there has more and more emphasis on understanding the driving forces and microscopic behaviors involved in the self-assembly process. Computational methods have played an increasingly important role in recent research in helping to reveal the relationship between molecular chemical structure and self-assembly processes as well as assembled morphologies, thus determining the ability of supramolecular gelation. This review aims to summarize the application of computational tools to obtain a better fundamental understanding of the multi-scale structural details of self-assembled peptide hydrogels and to predict the gelation behavior of supramolecular nanofibers. It is expected that researchers will consider using these computational tools when investigating and designing novel peptide hydrogel materials.     

Cation‐Tuned Stimuli‐Responsive and Optical Properties of Supramolecular Hydrogels

Hierarchical self‐assembly of an amphiphilic tris‐urea in aqueous media is shown. A mixture of the amphiphilic tris‐urea and an alkaline solution gave a viscous solution composed of fibrous aggregates. This viscous solution transformed into supramolecular hydrogels, which are capable of hierarchically organizing into higher‐order aggregates in response to several cationic triggers. The resulting supramolecular hydrogels were relatively stiff and their storage moduli attained over 10³ Pa. The stimuli‐responsive and optical properties of the resulting hydrogels were influenced by the cationic trigger. Proton and calcium ion triggers gave pH‐ and chemical stimuli‐responsive hydrogels, respectively. A terbium ion trigger also provided a highly luminescent hydrogel through energy transfer from the tris‐urea to terbium.     

Supramolecular parallel β-sheet and amyloid-like fibril forming peptides using δ-aminovaleric acid residue

Four terminally blocked tripeptides containing δ-aminovaleric acid residue self-assemble to form supramolecular β-sheet structures as are revealed from their FT-IR data. Single crystal X-ray diffraction studies of two representative peptides also show that they form parallel β-sheet structures. Self-aggregation of these β-sheet forming peptides leads to the formation of fibrillar structures, as is evident from scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images. These peptide fibrils bind to a physiological dye, Congo red and exhibit a typical green-gold birefringence under polarized light, showing close resemblance to neurodegenerative disease causing amyloid fibrils.     

Amyloid-β peptide structure in aqueous solution varies with fragment size

Various fragment sizes of the amyloid-β (Aβ) peptide have been utilized to mimic the properties of the full-length Aβ peptide in solution. Among these smaller fragments, Aβ16 and Aβ28 have been investigated extensively. In this work, we report the structural and thermodynamic properties of the Aβ16, Aβ28, and Aβ42 peptides in an aqueous solution environment. We performed replica exchange molecular dynamics simulations along with thermodynamic calculations for investigating the conformational free energies, secondary and tertiary structures of the Aβ16, Aβ28, and Aβ42 peptides. The results show that the thermodynamic properties vary from each other for these peptides. Furthermore, the secondary structures in the Asp1-Lys16 and Asp1-Lys28 regions of Aβ42 cannot be completely captured by the Aβ16 and Aβ28 fragments. For example, the β-sheet structures in the N-terminal region of Aβ16 and Aβ28 are either not present or the abundance is significantly decreased in Aβ42. The α-helix and β-sheet abundances in Aβ28 and Aβ42 show trends--to some extent--with the potential of mean forces but no such trend could be obtained for Aβ16. Interestingly, Arg5 forms salt bridges with large abundances in all three peptides. The formation of a salt bridge between Asp23-Lys28 is more preferred over the Glu22-Lys28 salt bridge in Aβ28 but this trend is vice versa for Aβ42. This study shows that the Asp1-Lys16 and Asp1-Lys28 regions of the full length Aβ42 peptide cannot be completely mimicked by studying the Aβ16 and Aβ28 peptides.     

Self-assembly of β-turn forming synthetic tripeptides into supramolecular β-sheets and amyloid-like fibrils in the solid state

We have described here the self-assembling properties of the synthetic tripeptides Boc-Ala(1)-Aib(2)-Val(3)-OMe 1, Boc-Ala(1)-Aib(2)-Ile(3)-OMe 2 and Boc-Ala(1)-Gly(2)-Val(3)-OMe 3 (Aib=α-amino isobutyric acid, β-Ala=β-alanine) which have distorted β-turn conformations in their respective crystals. These turn-forming tripeptides self-assemble to form supramolecular β-sheet structures through intermolecular hydrogen bonding and other noncovalent interactions. The scanning electron micrographs of these peptides revealed that these compounds form amyloid-like fibrils, the causative factor for many neurodegenerative diseases including Alzheimer's disease, Huntington's disease and Prion-related encephalopathies.     

Supramolecular Peptide/Polymer Hybrid Hydrogels for Biomedical Applications

Peptides and polymers are the “elite” building blocks in hydrogel fabrication where the typical approach consists of coupling specific peptide sequences (cell adhesive and/or enzymatically cleavable) to polymer chains aiming to obtain controlled cell responses (adhesion, migration, differentiation). However, the use of polymers and peptides as structural components for fabricating supramolecular hydrogels is less well established. Here, the literature on the design of peptide/polymer systems for self‐assembly into hybrid hydrogels, as either peptide‐polymer conjugates or combining both components individually, is reviewed. The properties (stiffness, mesh structure, responsiveness, and biocompatibility) of the hydrogels are then discussed from the viewpoint of their potential biomedical applications.     

Formation of macroporous self-assembled hydrogels through cryogelation of Fmoc-Phe-Phe

In this study, it was found that macroporous hydrogels were formed when self-assembly of fluorenyl-9-methoxycarbonyl (Fmoc)-diphenylalanine (Phe-Phe) peptides was induced using glucono-δ-lactone (GdL) in apparently frozen samples. Formed cryogels exhibited a heterogeneous structure with pore walls of densely packed fibres of assembled dipeptides and pores in the range 10-100 μm. Hydrogels formed from the same composition above the freezing point exhibited a homogenous structure without any apparent porosity. The formed gels were characterised using microscopy techniques, CD-spectroscopy and stress sweeps. The cryogels exhibited less mechanical strength than the hydrogels that might be due to the heterogeneous structure of the former. It appeared that the self-assembled peptide both in the cryo- and hydrogel maintained the β-sheet structure commonly attributed to these.     

Template-directed self-assembly of a designed amphiphilic hexapeptide on mica surface

Self-assembly of small molecules into highly ordered nanostructures offers many important potential applications in science research and industry. Precise self-assembling with the assistance of inorganic substrate is considered as an ideal strategy. In this experiment, the highly ordered mica surface was used to template the assembling of a novel designed amphiphilic hexapeptide to form orderly parallel fibers. The nanostructure and the self-assembly mechanism were investigated by atomic force microscopy (AFM), transmission electron microscopy, Fourier transform infrared spectroscopy, and circular dichroism techniques. By the experimental results, a dramatic conformation transition from random coil and/or α-helix into β-sheet was found after the peptide assembled on the mica surface under certain conditions, which was considered as a key factor for the ordered nanostructure. Finally, according to the AFM images and the simulated length of peptide molecules, a trilaminar β-sheet structure model was proposed to explain the hierarchical self-assembly mechanism.