Peptide Materials Research Articles

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.

Isolation and characterisation of recalcitrant organic components from an estuarine sediment core

The purpose of this research was to isolate humic substances and humin from an estuarine sediment core using a novel sequential extraction procedure to characterise the isolates, to determine their compositions and to understand how the organic matter (OM) changes with time. The sediments were exhaustively extracted using the following media: 0.1 M NaOH; 0.1 M NaOH + 6 M urea; and dimethylsulphoxide (DMSO) + H2SO4 (94:6 v/v). Pyrolysis gas chromatography mass spectroscopy (pyGC/MS), and nuclear magnetic resonance (NMR) spectroscopy were the analytical tools that gave the most significant data for the characterisation of the organic isolates. The results indicate subtle molecular compositional differences in relation to the alkaline and alkaline-urea isolates. The humic acids (HAs) and fulvic acids (FAs) are readily solvated in aqueous alkaline media compared to the aqueous insoluble hydrophobic humin (HU) components. In addition to aliphatic hydrocarbons, peptide materials make considerable contributions to the component structures of the isolates. Aryl and O-aryl C units characteristic of lignin, and of cutan structures from plants, indicate contributions from terrestrial OM to the organic components in the HU, especially from the base of the core. The evidence suggests that components of terrestrial plant materials and of microbial biomass are preferentially preserved with time and make the major contributions to the OM retained over long time scales. The data acquired provide detailed information about the origins, compositions, nature and the associations of the OM in the estuarine sediment core. Such information provides a greater understanding of the role of these organic components in the carbon (C) sink. Humic acids and FAs are mineralised with time whereas HU is highly recalcitrant and represents a long-term sink for organic C. Humin is a separate organic entity and does not conform to the definitions of a humic substance. Terrestrial OM is preserved over long time scales in the environment. Protein makes significant contributions to all the organic isolates and its preservation suggests encapsulation in hydrophobic domains, or interactions with clay minerals.

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.

Structure and stability of solid oligoglycines

Although peptides have been extensively studied within many disciplines, their solid state chemistry is not sufficiently explored. Crystalline peptide materials could present new opportunities in solid state organic synthesis as well as pharmaceutical, cosmetic and food industries. In order to facilitate these future applications, a better understanding of the relationship between the solid state structure and chemical reactivity in peptide crystals is required. In this work, a series of linear glycine oligomers was studied to see how their crystal structure and thermal reactivity changes with the chains growth. Single crystals of tetra- and pentaglycines were grown from aqueous solutions and investigated with XRD analysis (see Figure for pentaglycine). The newly collected data together with those reported previously reveal general trends likely present throughout all glycine oligomers. An antiparallel, hydrogen bound, β-sheet-like structure exists throughout the whole series and is the most stable polymorphic form in higher oligomers. Further, the thermal reactivity of this series was studied using gas chromatography – mass spectrometry (GC-MS), thermal gravimetric analysis (TGA) coupled with IR spectroscopy, differential scanning calorimetry (DSC) and bulk oven heating regimes. Finally 1H and 13C NMR and XRD analysis were used to identify key components of the thermal decomposition pathways as well as new products discovered during the thermal treatment. The results from all these studies suggest that the thermal stability of the oligomers increases with the chain length, but the decomposition pathways for the series are similar. In all cases, 2,5-diketopiperazine was formed through either condensation reactions (glycine and diglycine[1]) or depolymerisation of the peptide chain[2] in parallel with competing decomposition mechanisms. 2,5-Diketopiperazine and its derivatives are important biologically active molecules, and if this trend holds for other peptide oligomers, this solid state reaction could form a new, widely applicable synthetic method.

A structural analysis of N-glycans of neonatal porcine islet-like cell clusters (NPCC)

BackgroundN-glycans isolated from neonatal porcine islet-like cell clusters (NPCCs) were analyzed by a mapping technique, to examine the differences in glycosylation and antigenicity between adult pig islets (APIs) and NPCCs. MethodsNPCCs were isolated from 1-to-3day-old neonatal wild-type pigs and cultured for 9days, using the technique described by Korbutt et al. The extract was proteolyzed by treatment with a chymotrypsin and trypsin mixture and further digested with glycoamidase A to release the N-glycans. After the removal of the peptide materials, the reducing ends of the N-glycans were derivatized with 2-aminopyridine. This mixture was applied to DEAE, amide and ODS columns. PA-oligosaccharides were also subjected to MALDI TOF-MS analysis. ResultsThe NPCC glycans were comprised of 14 neutral, 5 mono-sialyl and 5 di-sialyl glycans. As a feature of the N-glycans of NPCC, NPCC contained large amounts of high mannose structures. On the other hand, all of the hybrid and complex types contained a Fucα1-6GlcNAc structure, but were not modified with sulfate residues. Among them, the NPCC preparation contained five neutral and two mono-sialyl glycans and two di-sialyl glycans that were not typically found in adult islets, and seven of these nine were not detected in human islets. Moreover, most of the structures could be clearly identified in this study. ConclusionsThe data herein will be helpful for future studies of the glycoantigen associated with NPCC.

Self-Assembling Peptide of d-Amino Acids Boosts Selectivity and Antitumor Efficacy of 10-Hydroxycamptothecin

D-peptides, which consist of D-amino acids and can resist the hydrolysis catalyzed by endogenous peptidases, are one of the promising candidates for construction of peptide materials with enhanced biostability in vivo. In this paper, we report on a self-assembling supramolecular nanostructure of D-amino acid-based peptide Nap-G(D)F(D)F(D)YGRGD (D-fiber, (D)F meant D-phenylalanine, (D)Y meant D-tyrosine), which were used as carriers for 10-hydroxycamptothecin (HCPT). Transmission electron microscopy observations demonstrated the filamentous morphology of the HCPT-loaded peptides (d-fiber-HCPT). The better selectivity and antitumor activity of D-fiber-HCPT than L-fiber-HCPT were found in the in vitro and in vivo antitumor studies. These results highlight that this model D-fiber system holds great promise as vehicles of hydrophobic drugs for cancer therapy.

Nucleobase peptide amphiphiles

A new class of peptide materials is introduced, integrating orthogonal aspects of peptide, nucleoside, and amphiphile chemistry.

Molecular Assembly of Zinc Chlorophyll Derivatives by Using Recombinant Light-Harvesting Polypeptides with His-tag and Immobilization on a Gold Electrode

LH1-α and -β polypeptides, which make up the light-harvesting 1 (LH1) complex of purple photosynthetic bacteria, along with bacteriochlorophylls, have unique binding properties even for various porphyrin analogs. Herein, we used the porphyrin analogs, Zn-Chlorin and the Zn-Chlorin dimer, and examined their binding behaviors to the LH1-α variant, which has a His-tag at the C-terminus (MBP-rubα-YH). Zn-Chlorin and the Zn-Chlorin dimer could bind to MBP-rubα-YH and form a subunit-type assembly, similar to that from the native LH1 complex. These complexes could be immobilized onto Ni-nitrilotriacetic acid-modified Au electrodes, and the cathodic photocurrent was successfully observed by photoirradiation. Since Zn-Chlorins in this complex are too far for direct electron transfer from the electrode, a contribution of polypeptide backbone for efficient electron transfer was implied. These findings not only show interesting properties of LH1-α polypeptides but also suggest a clue to construct artificial photosynthesis systems using these peptide materials.

Tripeptide Self-Assembled Hydrogels: Soft Nanomaterials for Biological Applications

Short peptide self-assembled hydrogels are a promising class of soft nanomaterials for drug delivery and regenerative medicine. Here we describe the gelation of tripeptides, consisting of d and l hydrophobic amino acids, in buffer, following a pH switch to 7.4 (i.e., physiological pH). Interestingly, tripeptide analogues consisting of l-only amino acids fail to form a gel under the same conditions. The nanostructure of these self-assembling peptides is investigated by a number of techniques (atomic force microscopy, transmission electron microscopy, and confocal light microscopy) to unveil their architectures. In addition, these self-assembled soft nanomaterials can be potentially used as vehicles to deliver bioactive molecules. For instance, we describe the incorporation into the gel of rhodamine dye as a model compound in a two-step procedure: firstly, the dye is dissolved in the gel precursor solution; secondly, dilution to a final pH of 7.4 triggers self-assembly and gelation of the system. We analyse the effect of dye incorporation within either precursor solution on the secondary structure, nanoarchitecture, and rheological properties of the resulting peptide materials. We also present data concerning dye release kinetics. Dye release is achieved within 48 h, and no burst release is observed. We anticipate that these systems will find biological applications for the delivery of bioactive compounds.

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.

Rational Molecular Design of Complementary Self‐Assembling Peptide Hydrogels

Rational molecular design of self- assembling peptide-based materials that spontaneously form self-supporting hydrogels shows potential in many healthcare applications. Binary peptides based on complementary charged sequences are developed, and the use of biophysical analysis and cell-based studies highlights that the charged interactions can influence the properties of peptide materials and ultimately affect biomaterial applications.

ChemInform Abstract: Self‐Assembling Peptide Scaffolds for Regenerative Medicine

AbstractReview: 93 refs.

Designer nanomaterials using chiral self-assembling peptide systems and their emerging benefit for society

Chirality is absolutely central in chemistry and biology. The recent findings of chiral self-assembling peptides' remarkable chemical complementarity and structural compatibility make it one of the most inspired designer materials and structures in nanobiotechnology. The emerging field of designer chemistry and biology further explores biological and medical applications of these simple D,L- amino acids through producing marvellous nanostructures under physiological conditions. These self-assembled structures include well-ordered nanofibers, nanotubes and nanovesicles. These structures have been used for 3-dimensional tissue cultures of primary cells and stem cells, sustained release of small molecules, growth factors and monoclonal antibodies, accelerated wound-healing in reparative and regenerative medicine as well as tissue engineering. Recent advances in molecular designs have also led to the development of 3D fine-tuned bioactive tissue culture scaffolds. They are also used to stabilize membrane proteins including difficult G-protein coupled receptors for designing nanobiodevices. One of the self-assembling peptides has been used in human clinical trials for accelerated wound-healings. It is our hope that these peptide materials will open doors for more and diverse clinical uses. The field of chiral self-assembling peptide nanobiotechnology is growing in a number of directions that has led to many surprises in areas of novel materials, synthetic biology, clinical medicine and beyond.

Self-assembling peptide scaffolds for regenerative medicine

Biomaterials made from self-assembling, short peptides and peptide derivatives have great potential to generate powerful new therapies in regenerative medicine. The high signaling capacity and therapeutic efficacy of peptidic scaffolds has been established in several animal models, and the development of more complex, hierarchical structures based on peptide materials is underway. This highlight discusses several classes of self-assembling peptide-based materials, including peptide amphiphiles, Fmoc-peptides, self-complementary ionic peptides, hairpin peptides, and others. The self-assembly designs, bioactive signalling strategies, and cell signalling capabilities of these bioactive materials are reported. The future challenges of the field are also discussed, including short-term goals such as integration with biopolymers and traditional implants, and long term goals, such as immune system programming, subcellular targeting, and the development of highly integrated scaffold systems.

Structural, thermal and enzymatic analysis of naturally occurring and D-amino acid substituted peptides

Designing of new peptide materials for biomedical and protein engineering applications are important. In the present work an attempt has been made to study the effect of D-Leu in collagen like tetra peptide on the structure and stability of peptide against enzymes and results are compared with its chiral counterpart L-form. Effect of replacement of L-Leu in Leu-Gly-Pro-Ala tetra peptide with D-Leu on structure has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, D-Leu substitution leads to conformational changes in Leu-Gly-Pro-Ala secondary structure from β-sheet to turns. L → D-Leu Configurational changes in Leu-Gly-Pro-Ala owes to enhanced thermal stability which has been substantiated through CD and differential scanning calorimetry. Change in chirality of the leucine inhibits collagenolytic activity, which enables to design selective inhibition of proteases with greater specificity.

The influence of peptide impurity profiles on functional tissue–organ bath response: The 11-mer peptide INSL6[151–161] case

Bioactive peptides have great pharmaceutical potential as nutraceuticals, diagnostics, and therapeutic drugs in several clinical areas. Thus, the search for novel lead peptides with a biological function has attracted renewed interest. Crude peptide material (i.e., ∼70% purity) of INSL6[151–161] (NH2-FRSLFWGNHSQ-COOH) was found to trigger a contractile response in guinea pig ileum longitudinal smooth muscle preparations using tissue–organ baths. However, the purified peptide (i.e., ⩾95% purity) had no effect on this model. Further investigation with crude materials from other suppliers, with purities ranging between 50% and 80%, indicated that the crude products gave a false-positive functional tissue–organ bath conclusion. These observations question the functionality conclusions when using crude-purity peptide materials; during the initial research or discovery phase, peptide quality is generally neglected, possibly leading to misinterpretation of biological results due to by-products from peptide synthesis and, thus, wrong fail/pass decisions. Therefore, we strongly recommend appropriate quality control testing before using any peptides for initial biomedical research or discovery purposes.

Materials from peptide assembly: towards the treatment of cancer and transmittable disease

As the prevalence of cancer and transmittable disease persists, the development of new and more advanced therapies remains a priority in medical research. An emerging platform for the treatment of these illnesses is the use of materials formed via peptide assembly where the bulk material itself acts as the therapeutic. Higher ordered peptide structures with defined chemistry are capable of cellular targeting, recognition, and internalization. Recent design efforts are being made to exploit the nanoscale definition of the materials formed by assembling peptides to target cancer and microbial cells and to function as vaccines. This review focuses on assembled peptide materials that actively participate in the biological processes important to cancer and transmittable diseases to exert an anticipated functional outcome.

Using small angle scattering (SAS) to structurally characterise peptide and protein self-assembled materials

In the past 20 years protein and peptide self-assembly has attracted material scientists' interest due to the possibility to exploit such molecular mechanism to create novel biomaterials including hydrogels. One of the main challenges when dealing with "soft" biological materials is their structural and morphological characterisation. Small angle scattering (SAS) can be a highly complementary tool to microscopy for the characterisation of such materials as it allows the investigation of samples in their wet-state without the need for any sample preparation such as drying and/or freezing. In this tutorial review we introduce briefly the SAS technique to the non-expert and through selected examples from the literature show how SAS can be readily used thanks to existing analytical approaches developed by a number of authors to extract structural information on the self-assembly of peptide and proteins.