Self-assembling Peptide Materials Research Articles

Preparation, structural and morphological characterization of cartilage type II collagen peptide assemblies from sturgeon head.

Sturgeon cartilage type II collagen peptides (SHCPs) can self-assemble and be used to prepare collagen peptide assemblies. Self-assembled peptides have great potential for applications in the food industry. In the present study, self-assembled peptides were prepared from sturgeon cartilage and then characterized. The SHCPs self-assembled and formed collagen peptide assemblies. After response surface experiment optimization, the optimal enzyme digestion process comprised 43.1 °C, 3.37 h and 0.96% enzyme addition, and the peptide yield was 78.46%. Physicochemical analysis showed that the SHCPs were amphiphilic, with an average molecular weight of 1081 Da, and were rich in hydrophobic amino acids. Peptide sequence identification showed that the peptides of SHCPs with polar amino acids followed by hydrophobic amino acids could be self-assembled through hydrogen bonding and hydrophobic interaction. Through turbidity experiments, Fourier transform infrared spectroscopy and scanning electron microscopy, we demonstrated that SHCPs can self-assemble into reticular and tubular structures under specific conditions. Furthermore, both the SHCPs-Ca and SHCPs-Mg assemblies were stabilized within a pH range consistent with that of the human gastrointestinal tract. The present study provides a simple and safe method for preparing novel self-assembled peptide materials from sturgeon by-products, providing a scientific basis for the exploitation of sturgeon cartilage and potentially reducing resource wastage. © 2024 Society of Chemical Industry.

Computational Design of Peptide Assemblies.

With the ongoing development of peptide self-assembling materials, there is growing interest in exploring novel functional peptide sequences. From short peptides to long polypeptides, as the functionality increases, the sequence space is also expanding exponentially. Consequently, attempting to explore all functional sequences comprehensively through experience and experiments alone has become impractical. By utilizing computational methods, especially artificial intelligence enhanced molecular dynamics (MD) simulation and de novo peptide design, there has been a significant expansion in the exploration of sequence space. Through these methods, a variety of supramolecular functional materials, including fibers, two-dimensional arrays, nanocages, etc., have been designed by meticulously controlling the inter- and intramolecular interactions. In this review, we first provide a brief overview of the current main computational methods and then focus on the computational design methods for various self-assembled peptide materials. Additionally, we introduce some representative protein self-assemblies to offer guidance for the design of self-assembling peptides.

Peptide-based bioactivated in vivo assembly nanomaterials and its biomedical applications: a review

Based on the self-assembly process occurring in the human body all the time, self-assembled nanomaterials were designed by the researchers. The self-assembled nanomaterials have controllability, biocompatibility and functional advantages in vivo. The self-assembled nanomaterials constructed in situ under a physiological environment display various biological characteristics which can be used for imaging, therapy, and broad clinical applications. In situ self-assembled nanomaterials can boost drug function, reduce toxic and side effects, prolong imaging time and enlarge signal-to-noise ratio. By using pathological conditions to trigger specific responses in vivo, well-ordered nanoaggregates can be spontaneously formed by multiple weak bonding interactions. The assembly shows higher accumulation and longer retention in situ. Endogenous triggers for in situ assembly, such as enzymes, pH, reactive oxygen species and ligand receptor interaction, can be used to transform the materials into a variety of controllable nanostructures including nanoparticles, nanofibers and gels through bioactivated in vivo assembly (BIVA) strategies. BIVA strategies can be applied for treatment, imaging or participate in the physiological activities of cells at the lesion site. This review summarized and prospected the design of self-assembled peptide materials based on BIVA technology and their biomedical applications. The nanostructures of the self-assembly enable some beneficial biological effects, such as assembly induced retention (AIR) effect, enhanced targeting effect, multivalent bond effect, and membrane disturbance. Thus, the BIVA nanotechnology is promising for efficient drug delivery, enhancement of targeting and treatment, as well as optimization of the biological distribution of drugs.

De novo designed peptides form a highly catalytic ordered nanoarchitecture on a graphite surface.

Here we demonstrate that short peptides, de novo designed from first principles, self-assemble on the surface of graphite to produce a highly robust and catalytic nanoarchitecture, which promotes peroxidation reactions with activities that rival those of natural enzymes in both single and multi-substrate reactions. These designable peptides recapitulate the symmetry of the underlying graphite surface and act as molecular scaffolds to immobilize hemin molecules on the electrode in a hierarchical self-assembly manner. The highly ordered and uniform hybrid graphite–peptide–hemin nanoarchitecture shows the highest faradaic efficiency of any hybrid electrode reported. Given the explosive growth of the types of chemical reactions promoted by self-assembled peptide materials, this new approach to creating complex electrocatalytic assemblies will yield highly efficient and practically applicable electrocatalysts.

Self-Assembly of Peptide Hierarchical Helical Arrays with Sequence-Encoded Circularly Polarized Luminescence.

Self-assembled peptide materials with sequence-encoded properties have attracted great interest. Despite their intrinsic chirality, the generation of circularly polarized luminescence (CPL) based on the self-assembly of simple peptides has been rarely reported. Here, we report the self-assembly of peptides into hierarchical helical arrays (HHAs) with controlled supramolecular handedness. The HHAs can emit full-color CPL signals after the incorporation of various achiral fluorescent molecules, and the glum value is 40 times higher than that of the CPL signal from the solutions. By simply changing the amino acid sequence of the peptides, CPL signals with opposite handedness can be generated within the HHAs. The peptide HHAs can provide hydrophobic pockets to accommodate the fluorescent molecules with helical arrangement through strong aromatic stacking interactions, which are responsible for the CPL signals. This work provides a pathway to construct highly ordered chiral materials, which have broad applications in the chiroptical field.

Self-Assembled Peptide Nanofibrils Designed to Release Membrane-Lysing Antimicrobial Peptides.

Membrane-disrupting antimicrobial peptides continue to attract increasing attention due to their potential to combat multidrug-resistant bacteria. However, some limitations are found in the success of clinical setting-based antimicrobial peptide agents, for instance, the poor stability of antimicrobial peptides in vivo and their short-term activity. Self-assembled peptide materials can improve the stability of antimicrobial peptides, but the biosafety of peptide-based materials is the main concern, although they are considered to be biocompatible, because some peptide aggregates would possibly induce protein misfolding, which could be related to amyloid-related diseases. Therefore, in this work, we designed two peptides and constructed peptide-based nanofibrils by self-assembly before its utilization. It is found that the fibrils could release the antimicrobial peptide by disassembly for microbial membrane lysis in the presence of bacteria. The designed peptide-based fibrils presented a good and long-term antimicrobial activity with bacterial membrane disruption and the efflux of calcium from bacteria. Furthermore, it could be used to construct hybrid macrofilms displaying low cytotoxicity, low hemolytic activity, and good biocompatibility. The innovative design strategy could be beneficial for the development of smart antimicrobial nanomaterials.

Self-Assembly-Assisted Kinetically Controlled Papain-Catalyzed Formation of mPEG-b-Phe(Leu)x.

Self-assembling peptide materials are promising next-generation materials with applications that include tissue engineering scaffolds, drug delivery, bionanomedicine, and enviro-responsive materials. Despite these advances, synthetic methods to form peptides and peptide-polymer conjugates still largely rely on solid-phase peptide synthesis (SPPS) and N-carboxyanhydride ring-opening polymerization (NCA-ROP), while green methods remain largely undeveloped. This work demonstrates a protease-catalyzed peptide synthesis (PCPS) capable of directly grafting leucine ethyl ester (Leu-OEt) from the C-terminus of a methoxy poly(ethylene glycol)-phenylalanine ethyl ester macroinitiator in a one-pot, aqueous reaction. By using the natural tendency of the growing hydrophobic peptide segment to self-assemble, a large narrowing of the (Leu)x distributions for both mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts, relative to oligo(Leu)x synthesized in the absence of a macroinitiator (mPEG45-Phe-OEt), was achieved. A mechanism is described where in situ β-sheet coassembly of mPEG45-b-Phe(Leu)x and oligo(Leu)x coproducts during polymerization prevents peptide hydrolysis, providing a means to control the degree of polymerization (DP) and dispersity of diblock (Leu)x segments (matrix-assisted laser desorption time-of-flight (MALDI-TOF) x = 5.1, dispersity ≤ 1.02). The use of self-assembly to control the uniformity of peptides synthesized by PCPS paves the way for precise peptide block copolymer architectures with various polymer backbones and amino acid compositions synthesized by a green process.

Towards Developing Bioresponsive, Self-Assembled Peptide Materials: Dynamic Morphology and Fractal Nature of Nanostructured Matrices.

(Arginine-alanine-aspartic acid-alanine)4 ((RADA)4) nanoscaffolds are excellent candidates for use as peptide delivery vehicles: they are relatively easy to synthesize with custom bio-functionality, and assemble in situ to allow a focal point of release. This enables (RADA)4 to be utilized in multiple release strategies by embedding a variety of bioactive molecules in an all-in-one “construct”. One novel strategy focuses on the local, on-demand release of peptides triggered via proteolysis of tethered peptide sequences. However, the spatial-temporal morphology of self-assembling nanoscaffolds may greatly influence the ability of enzymes to both diffuse into as well as actively cleave substrates. Fine structure and its impact on the overall effect on peptide release is poorly understood. In addition, fractal networks observed in nanoscaffolds are linked to the fractal nature of diffusion in these systems. Therefore, matrix morphology and fractal dimension of virgin (RADA)4 and mixtures of (RADA)4 and matrix metalloproteinase 2 (MMP-2) cleavable substrate modified (RADA)4 were characterized over time. Sites of high (glycine-proline-glutamine-glycine+isoleucine-alanine-serine-glutamine (GPQG+IASQ), CP1) and low (glycine-proline-glutamine-glycine+proline-alanine-glycine-glutamine (GPQG+PAGQ), CP2) cleavage activity were chosen. Fine structure was visualized using transmission electron microscopy. After 2 h of incubation, nanofiber networks showed an established fractal nature; however, nanofibers continued to bundle in all cases as incubation times increased. It was observed that despite extensive nanofiber bundling after 24 h of incubation time, the CP1 and CP2 nanoscaffolds were susceptible to MMP-2 cleavage. The properties of these engineered nanoscaffolds characterized herein illustrate that they are an excellent candidate as an enzymatically initiated peptide delivery platform.

Practical Considerations in the Design and Use of Immunologically Active Fibrillar Peptide Assemblies.

The design, formulation, and immunological evaluation of self-assembling peptide materials is relatively straightforward. Indeed, one of the advantages of synthetic self-assembling peptides is that one can progress from initial concept to in vivo testing in a matter of days. However, because these materials are supramolecular, working with them is not without some practical challenges, and subtle changes in design, synthesis, handling, and formulation can affect the materials' immunogenicity. This chapter is intended to communicate some of these practical aspects of working with these materials that are not always enumerated in conventional research papers. Epitope considerations, peptide synthesis, purification, storage, nanofiber formation, quality control, immunological evaluation, and the overall phenotypic characteristics of the immune responses to be expected from these materials are discussed.

Control of Heme Coordination and Catalytic Activity by Conformational Changes in Peptide-Amphiphile Assemblies.

Self-assembling peptide materials have gained significant attention, due to well-demonstrated applications, but they are functionally underutilized. To advance their utility, we use noncovalent interactions to incorporate the biological cofactor heme-B for catalysis. Heme-proteins achieve differing functions through structural and coordinative variations. Here, we replicate this phenomenon by highlighting changes in heme reactivity as a function of coordination, sequence, and morphology (micelles versus fibers) in a series of simple peptide amphiphiles with the sequence c16-xyL3K3-CO2H where c16 is a palmitoyl moiety and xy represents the heme binding region: AA, AH, HH, and MH. The morphology of this peptide series is characterized using transmission electron and atomic force microscopies as well as dynamic light scattering. Within this small library of peptide constructs, we show that three spectroscopically (UV/visible and electron paramagnetic resonance) distinct heme environments were generated: noncoordinated/embedded high-spin, five-coordinate high-spin, and six-coordinate low-spin. The resulting material's functional dependence on sequence and supramolecular morphology is highlighted 2-fold. First, the heme active site binds carbon monoxide in both micelles and fibers, demonstrating that the heme active site in both morphologies is accessible to small molecules for catalysis. Second, peroxidase activity was observed in heme-containing micelles yet was significantly reduced in heme-containing fibers. We briefly discuss the implications these findings have in the production of functional, self-assembling peptide materials.

Peptide self-assembly for nanomaterials: the old new kid on the block.

Peptide self-assembly is an increasingly attractive tool for nanomaterials. Perfected in biology peptide self-assembling systems have impacted on nearly any conceivable nanomaterial type. However, with all the information available to us commercialisation of peptide materials remains in its infancy. In an attempt to better understand the reasons behind this shortcoming we categorise peptide self-assembled materials in relation to their non-peptide counterparts. A particular emphasis is placed on the versatility of peptide self-assembly in terms of modularity, responsiveness and functional diversity, which enables direct comparisons with more traditional material chemistries.

Probing peptide amphiphile self-assembly in blood serum.

There has been recent interest in designing smart diagnostic or therapeutic self-assembling peptide or polymeric materials that can selectively undergo morphological transitions to accumulate at a disease site in response to specific stimuli. Developing approaches to probe these self-assembly transitions in environments that accurately amalgamate the diverse plethora of proteins, biomolecules, and salts of blood is essential for creating systems that function in vivo. Here, we have developed a fluorescence anisotropy approach to probe the pH-dependent self-assembly transition of peptide amphiphile (PA) molecules that transform from spherical micelles at pH 7.4 to nanofibers under more acidic pH's in blood serum. By mixing small concentrations of a Ru(bipy)3(2+)-tagged PA with a Gd(DO3A)-tagged PA having the same lipid-peptide sequence, we showed that the pH dependence of self-assembly is minimally affected and can be monitored in mouse blood serum. These PA vehicles can be designed to transition from spherical micelles to nanofibers in the pH range 7.0-7.4 in pure serum. In contrast to the typical notion of serum albumin absorbing isolated surfactant molecules and disrupting self-assembly, our experiments showed that albumin does not bind these anionic PAs and instead promotes nanofibers due to a molecular crowding effect. Finally, we created a medium that replicates the transition pH in serum to within 0.08 pH units and allows probing self-assembly behavior using conventional spectroscopic techniques without conflicting protein signals, thus simplifying the development pathway from test tube to in vivo experimentation for stimuli-responsive materials.

Photoinitiated [corrected] charge separation in a hybrid titanium dioxide metalloporphyrin peptide material.

In natural systems, electron flow is mediated by proteins that spatially organize donor and acceptor molecules with great precision. Achieving this guided, directional flow of information is a desirable feature in photovoltaic media. Here, we design self-assembled peptide materials that organize multiple electronic components capable of performing photoinduced charge separation. Two peptides, c16-AHL3K3-CO2H and c16-AHL3K9-CO2H, self-assemble into fibres and provide a scaffold capable of binding a metalloporphyrin via histidine axial ligation and mineralize titanium dioxide (TiO2) on the lysine-rich surface of the resulting fibrous structures. Electron paramagnetic resonance studies of this self-assembled material under continuous light excitation demonstrate charge separation induced by excitation of the metalloporphyrin and mediated by the peptide assembly structure. This approach to dye-sensitized semiconducting materials offers a means to spatially control the dye molecule with respect to the semiconducting material through careful, strategic peptide design.

Controllably degradable β-sheet nanofibers and gels from self-assembling depsipeptides

Self-assembled peptide materials have received considerable interest for a range of applications, including 3D cell culture, tissue engineering, and the delivery of cells and drugs. One challenge in applying such materials within these areas has been the extreme stability of β-sheet fibrillized peptides, which are resistant to proteolysis, degradation, and turnover in biological environments. In this study, we designed self-assembling depsipeptides containing ester bonds within the peptide backbone. Beta-sheet fibrillized nanofibers were formed in physiologic conditions, and two of these nanofiber-forming depsipeptides produced hydrogels that degraded controllably over the course of days-to-weeks via ester hydrolysis. With HPLC, TEM, and oscillating rheometry, we show that the rate of hydrolysis can be controlled in a straightforward manner by specifying the amino acid residues surrounding the ester bond. In 3D cell cultures, depsipeptide gels softened over the course of several days and permitted considerably more proliferation and spreading of C3H10T1/2 pluripotent stem cells than non-degradable analogs. This approach now provides a reliable and reproducible means to soften or clear β-sheet fibrillized peptide materials from biological environments.

Lysine functionalised amyloid fibrils: the design and assembly of a TTR1-based peptide

Non-core residues can affect the formation and protofilament packing of fibrils assembled from short peptide sequences. These residues are of interest in understanding amyloid diseases and in the design of self-assembling peptide materials with a cross-β core, where the assembly process should be reproducible and functional groups accessible on the fibril surface. In this study, the well characterised TTR1 peptide, also known as TTR105–115, was functionalised with glycine and lysine residues forming the peptide TTR1-GGK, with the aim of producing a self-assembling fibril scaffold that can be functionalised following assembly. A second aim was to develop a sequence capable of fibril assembly under a wide range of solution conditions. The lysine residue was found accessible on the surface of TTR1-GGK fibrils and the C-terminal residues influenced the mature fibril width and rate of fibril assembly, as observed for other TTR1-based fibrils. The assembly of TTR1-GGK fibrils was examined for conditions of varying ionic strength (NaCl, 0–0.5 M), solution pH or in the presence of anions (NaCl, NaI, NaNO3 and NaSO4) or cations (NaCl, CaCl2, MgCl2, LiCl and KCl). The addition of salt increased the rate of TTR1-GGK fibril nucleation but decreased the rate of elongation at high salt concentrations. A combination of electrostatic and hydrophobic interactions was found to promote initial contacts between peptides. Specific ion effects were seen with chaotropic anions, which promoted fibril nucleation. The cross-β core structure, secondary structure and morphology of TTR1-GGK fibrils were largely unaltered by the presence of salt or a range of solution pH. The length of fibrils was also maintained at the high ionic strengths tested, indicating that these fibrils may make suitable scaffolds for fibril functionalisation under a range of conditions.

Photodynamic Control of Bioactivity in a Nanofiber Matrix

Self-assembling peptide materials have been used extensively to mimic natural extracellular matrices (ECMs) by presenting bioactive epitopes on a synthetic matrix. Although this approach can facilitate a desired response from cells grown in the matrix, it lacks the capacity for spatial or temporal regulation of the presented signals. We describe here a photoresponsive, synthetic ECM using a supramolecular platform composed of peptide amphiphiles (PAs) that self-assemble into cylindrical nanofibers. A photocleavable nitrobenzyl ester group was included in the peptide backbone using a novel Fmoc-amino acid that is compatible with microwave-assisted solid-phase peptide synthesis. The placement of the photolabile group on the peptide backbone enabled efficient removal of the ECM-derived cell adhesion epitope RGDS from PA molecules upon exposure to light (half-life of photolysis ~1.9 min) without affecting the nanofiber assembly. Fibroblasts cultured on RGDS-presenting PA nanofiber substrates demonstrated increased cell spreading and more mature focal adhesions compared with unfunctionalized and control (RGES-presenting) surfaces, as determined by immunostaining and cell morphological analysis. Furthermore, we observed an arrest in fibroblast spreading on substrates containing a cleavable RGDS epitope when the culture was exposed to light; in contrast, this dynamic shift in cell response was absent when the RGDS epitope was attached to the PA molecule by a light-insensitive control linker. Light-responsive bioactive materials can contribute to the development of synthetic systems that more closely mimic the dynamic nature of native ECM.

Designer Self‐Assembling Peptide Materials for Diverse Applications

AbstractSummary: Macromolecuar chemistry and polymer science have had an enormous impact in many areas of science, engineering, medicine and our daily life. It has not only changed our way of life forever, but also continuously to improve our living standard. Macromolecuar chemistry now also encompasses bioengineering, biomimetics, designer biological materials and nanobiotechnology. Here we summarize a few classes of short peptides that we discovered and invented with broad applications including 3D tissue cell culture, reparative and regenerative medicine, tissue engineering, slow drug release, stabilization of membrane proteins for develop nanobiotechnology and molecular devices. Molecular design using short peptides as new materials will play increasingly important role in biomedical research, nanobiotechnology, clinical science and medicine.

Modulating the mechanical properties of self-assembled peptide hydrogels via native chemical ligation

Hydrogels produced from self-assembling peptides and peptide derivatives are being investigated as synthetic extracellular matrices for defined cell culture substrates and scaffolds for regenerative medicine. In many cases, however, they are less stiff than the tissues and extracellular matrices they are intended to mimic, and they are prone to cohesive failure. We employed native chemical ligation to produce peptide bonds between the termini of fibrillized β-sheet peptides to increase gel stiffness in a chemically specific manner while maintaining the morphology of the self-assembled fibrils. Polymerization, fibril structure, and mechanical properties were measured by SDS–PAGE, mass spectrometry, TEM, circular dichroism, and oscillating rheometry; and cellular responses to matrix stiffening were investigated in cultures of human umbilical vein endothelial cells (HUVECs). Ligation led to a fivefold increase in storage modulus and a significant enhancement of HUVEC proliferation and expression of CD31 on the surface of the gels. The approach was also orthogonal to the inclusion of unprotected RGD-functionalized self-assembling peptides, which further increased proliferation. This strategy broadens the utility of self-assembled peptide materials for applications that require enhancement or modulation of matrix mechanical properties by providing a chemoselective means for doing so without significantly disrupting the gels' fibrillar structure.

Dynamic Behaviors of Lipid-Like Self-Assembling Peptide A<SUB>6</SUB>D and A<SUB>6</SUB>K Nanotubes

Nanoscience and nanotechnology require development of nanomaterials that are amiable for molecular design from bottom up. Molecular designer self-assembling peptides are one of such nanomaterials that will become increasingly important for the endeavor. Peptides have not only been used in all aspects of biomedical and pharmaceutical research and medical products, but also have had enormous impact in nascent field of designed biological materials. We here report the dynamic structures of lipid-like designer peptide A6D (AAAAAAD) and A6K (AAAAAAK) that undergo self-assembly into nanotubes in water and salt solution. We not only analyzed their self-assemblies using dynamic light scattering to determine the critical aggregation concentration (CAC), but also use atomic force microscope to observe their nanostructures. We also propose a simple scheme by which these lipid-like peptides self-assemble into dynamic nanostructures. Since the knowledge of CAC is important for uses of these peptides for a variety of applications, these findings may have significant implications in the study of molecular self-assembly and for a wide range of utilities of designer self-assembling peptide materials.