Alanine-rich amphiphilic peptide containing the RGD cell adhesion motif: a coating material for human fibroblast attachment and culture

We studied the self-assembly of peptide A6RGD (A: alanine, R: arginine, G: glycine, D: aspartic acid) in water, and the use of A6RGD substrates as coatings to promote the attachment of human cornea stromal fibroblasts (hCSFs). The self-assembled motif of A6RGD was shown to depend on the peptide concentration in water, where both vesicle and fibril formation were observed. Oligomers were detected for 0.7 wt% A6RGD, which evolved into short peptide fibres at 1.0 wt% A6RGD, while a co-existence of vesicles and long peptide fibres was revealed for 2–15 wt% A6RGD. A6RGD vesicle walls were shown to have a multilayer structure built out of highly interdigitated A6 units, while A6RGD fibres were based on β-sheet assemblies. Changes in the self-assembly motif with concentration were reflected in the cell culture assay results. Films dried from 0.1–1.0 wt% A6RGD solutions allowed hCSFs to attach and significantly enhanced cell proliferation relative to the control. In contrast, films dried from 2.5 wt% A6RGD solutions were toxic to hCSFs.

Influence of elastase on alanine-rich peptide hydrogels

The self-assembly of the alanine-rich amphiphilic peptides Lys(Ala)6Lys (KA6K) and Lys(Ala)6Glu (KA6E)with homotelechelic or heterotelechelic charged termini respectively has been investigated in aqueous solution. These peptides contain hexa-alanine sequences designed to serve as substrates for the enzyme elastase. Electrostatic repulsion of the lysine termini in KA6K prevents self-assembly, whereas in contrast KA6E is observed, through electron microscopy, to form tape-like fibrils, which based on X-ray scattering contain layers of thickness equal to the molecular length. The alanine residues enable efficient packing of the side-chains in a beta-sheet structure, as revealed by circular dichroism, FTIR and X-ray diffraction experiments. In buffer, KA6E is able to form hydrogels at sufficiently high concentration. These were used as substrates for elastase, and enzyme-induced de-gelation was observed due to the disruption of the beta-sheet fibrillar network. We propose that hydrogels of the simple designed amphiphilic peptide KA6E may serve as model substrates for elastase and this could ultimately lead to applications in biomedicine and regenerative medicine.

PEG−peptide conjugates

The remarkable diversity of the self-assembly behavior of PEG−peptides is reviewed, including self-assemblies formed by PEG−peptides with β-sheet and α-helical (coiled-coil) peptide sequences. The modes of self-assembly in solution and in the solid state are discussed. Additionally, applications in bionanotechnology and synthetic materials science are summarized.

Hybrid proton and electron transport in peptide fibrils

Protons and electrons are being exploited in different natural charge transfer processes. Both types of charge carriers could be, therefore, responsible for charge transport in biomimetic self-assembled peptide nanostructures. The relative contribution of each type of charge carrier is studied in the present work for fi brils self-assembled from amyloid- β derived peptide molecules, in which two non-natural thiophene-based amino acids are included. It is shown that under low humidity conditions both electrons and protons contribute to the conduction, with current ratio of 1:2 respectively, while at higher relative humidity proton transport dominates the conductance. This hybrid conduction behavior leads to a bimodal exponential dependence of the conductance on the relative humidity. Furthermore, in both cases the conductance is shown to be affected by the peptide folding state under the entire relative humidity range. This unique hybrid conductivity behavior makes self-assembled peptide nanostructures powerful building blocks for the construction of electric devices that could use either or both types of charge carriers for their function.

Tuning self-assembled nanostructures through enzymatic degradation of a peptide amphiphile

The enzymatic cleavage of a peptide amphiphile (PA) is investigated. The self-assembly of the cleaved products is distinct from that of the PA substrate. The PA C16-KKFFVLK is cleaved by α-chymotrypsin at two sites leading to products C16-KKF with FVLK and C16-KKFF with VLK. The PA C16-KKFFVLK forms nanotubes and helical ribbons at room temperature. Both PAs C16-KKF and C16-KKFF corresponding to cleavage products instead self-assemble into 5-6 nm diameter spherical micelles, while peptides FVLK and VLK do not adopt well-defined aggregate structures. The secondary structures of the PAs and peptides are examined by FTIR and circular dichroism spectroscopy and X-ray diffraction. Only C16-KKFFVLK shows substantial β-sheet secondary structure, consistent with its self-assembly into extended aggregates, based on PA layers containing hydrogen-bonded peptide headgroups. This PA also exhibits a thermoreversible transition to twisted tapes on heating.

Self-assembly of a Model Peptide incorporating a Hexa-Histidine sequence attached to an Oligo-Alanine sequence, and binding to Gold NTA/Nickel nanoparticles

Amyloid fibrils are formed by a model surfactant-like peptide (Ala)10-(His)6 containing a hexahistidine tag. This peptide undergoes a remarkable two-step self-assembly process with two distinct critical aggregation concentrations (cac’s), probed by fluorescence techniques. A micromolar range cac is ascribed to the formation of prefibrillar structures, whereas a millimolar range cac is associated with the formation of well-defined but more compact fibrils. We examine the labeling of these model tagged amyloid fibrils using Ni-NTA functionalized gold nanoparticles (Nanogold). Successful labeling is demonstrated via electron microscopy imaging. The specificity of tagging does not disrupt the β-sheet structure of the peptide fibrils. Binding of fibrils and Nanogold is found to influence the circular dichroism associated with the gold nanoparticle plasmon absorption band. These results highlight a new approach to the fabrication of functionalized amyloid fibrils and the creation of peptide/nanoparticle hybrid materials.

Self-assembly of a dual functional bioactive peptide amphiphile incorporating both matrix metalloprotease substrate and cell adhesion motifs

We describe a bioactive lipopeptide that combines the capacity to promote the adhesion and subsequent self-detachment of live cells, using template-cell-environment feedback interactions. This self-assembling peptide amphiphile comprises a diene-containing hexadecyl lipid chain (C16e) linked to a matrix metalloprotease-cleavable sequence, Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, and contiguous with a cell-attachment and signalling motif, Arg-Gly-Asp-Ser. Biophysical characterisation revealed that the PA self-assembles into 3 nm diameter spherical micelles above a critical aggregation concentration (cac). In addition, when used in solution at 5–150 nM (well below the cac), the PA is capable of forming film coatings that provide a stable surface for human corneal fibroblasts to attach and grow. Furthermore, these coatings were demonstrated to be sensitive to metalloproteases expressed endogenously by the attached cells, and consequently to elicit the controlled detachment of cells without compromising their viability. As such, this material constitutes a novel class of multi-functional coating for both fundamental and clinical applications in tissue engineering.

Time-dependent gel to gel transformation of a peptide based supramolecular gelator

A dipeptide with a long fatty acid chain at its N-terminus gives hydrogels in phosphate buffer in the pH range 7.0–8.5. The hydrogel with a gelator concentration of 0.45% (w/v) at pH 7.46 (physiological pH) provides a very good platform to study dynamic changes within a supramolecular framework as it exhibits remarkable change in its appearance with time. Interestingly, the first formed transparent hydrogel gradually transforms into a turbid gel within 2 days. These two forms of the hydrogel have been thoroughly investigated by using small angle X-ray scattering (SAXS), powder X-ray diffraction (PXRD), field emission scanning electron microscopic (FE-SEM) and high-resolution transmission electron microscopic (HR-TEM) imaging, FT-IR and rheometric analyses. The SAXS and low angle PXRD studies substantiate different packing arrangements for the gelator molecules for these two different gel states (the freshly prepared and the aged hydrogel). Moreover, rheological studies of these two gels reveal that the aged gel is stiffer than the freshly prepared gel.

Molecular basis of platelet activation by an alphaIIbbeta3-CHAMPS peptide.

The present study demonstrates that the alphaIIb-CHAMPS peptide induces platelet activation through integrin alphaIIbbeta3-dependent and independent pathways with the former mediating tyrosine phosphorylation of FcR gamma-chain and Syk. The use of the alphaIIb-CHAMPS peptide to study integrin alphaIIbbeta3 function is compromised by non-integrin-mediated effects.

Bio-fabrication and physiological self-release of tissue equivalents using smart peptide amphiphile templates

In this study we applied a smart biomaterial formed from a self-assembling, multi-functional synthetic peptide amphiphile (PA) to coat substrates with various surface chemistries. The combination of PA coating and alignment-inducing functionalised substrates provided a template to instruct human corneal stromal fibroblasts to adhere, become aligned and then bio-fabricate a highlyordered, multi-layered, three-dimensional tissue by depositing an aligned, native-like extracellular matrix. The newly-formed corneal tissue equivalent was subsequently able to eliminate the adhesive properties of the template and govern its own complete release via the action of endogenous proteases. Tissues recovered through this method were structurally stable, easily handled, and carrier-free. Furthermore, topographical and mechanical analysis by atomic force microscopy showed that tissue equivalents formed on the alignment-inducing PA template had highly-ordered, compact collagen deposition, with a two-fold higher elastic modulus compared to the less compact tissues produced on the non-alignment template, the PA-coated glass. We suggest that this technology represents a new paradigm in tissue engineering and regenerative medicine, whereby all processes for the biofabrication and subsequent self-release of natural, bioprosthetic human tissues depend solely on simple templatetissue feedback interactions.

Self-assembled arginine-capped peptide bolaamphiphile nanosheets for cell culture and controlled wettability surfaces

The spontaneous assembly of a peptide bolaamphiphile in water, namely, RFL4FR (R, arginine; F, phenylalanine; L, leucine) is investigated, along with its novel properties in surface modification and usage as substrates for cell culture. RFL4FR self-assembles into nanosheets through lateral association of the peptide backbone. The L4 sequence is located within the core of the nanosheets, whereas the R moieties are exposed to the water at the surface of the nanosheets. Kinetic assays indicate that the self-assembly is driven by a remarkable two-step process, where a nucleation phase is followed by fast growth of nanosheets with an autocatalysis process. The internal structure of the nanosheets is formed from ultrathin bolaamphiphile monolayers with a crystalline orthorhombic symmetry with cross-β organization. We show that human corneal stromal fibroblast (hCSF) cells can grow on polystyrene films coated with films dried from RFL4FR solutions. For the first time, this type of amphiphilic peptide is used as a substrate to modulate the wettability of solid surfaces for cell culture applications.

Peptide growth factors signal differentially through protein kinase C to extracellular signal-regulated kinases in neonatal cardiomyocytes

The extracellular signal-regulated kinases 1/2 (ERK1/2) are activated in cardiomyocytes by Gq protein-coupled receptors and are associated with induction of hypertrophy. Here, we demonstrate that, in primary cardiomyocyte cultures, ERK1/2 were also significantly activated by platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or fibroblast growth factor (FGF), but insulin, insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF) had relatively minor effects. PDGF, EGF or FGF increased cardiomyocyte size via ERK1/2, whereas insulin, IGF-1 or NGF had no effect suggesting minimum thresholds/durations of ERK1/2 signaling are required for the morphological changes associated with hypertrophy. Peptide growth factors are widely accepted to activate phospholipase C gamma1 (PLCgamma1) and protein kinase C (PKC). In cardiomyocytes, only PDGF stimulated tyrosine phosphorylation of PLCgamma1 and nPKCdelta. Furthermore, activation of ERK1/2 by PDGF, but not EGF, required PKC activity. In contrast, EGF substantially increased Ras.GTP with rapid activation of c-Raf, whereas stimulation of Ras.GTP loading by PDGF was minimal and activation of c-Raf was delayed. Our data provide clear evidence for differential coupling of PDGF and EGF receptors to the ERK1/2 cascade, and indicate that a minimum threshold/duration of ERK1/2 signaling is required for the development of cardiomyocyte hypertrophy.

Supra-molecular assembly of a lumican-derived peptide amphiphile enhances its collagen-stimulating activity

C16-YEALRVANEVTLN, a peptide amphiphile (PA) incorporating a biologically active amino acid sequence found in lumican, has been examined for its influence upon collagen synthesis by human corneal fibroblasts in vitro, and the roles of supra-molecular assembly and activin receptor-like kinase ALK receptor signaling in this effect were assessed. Cell viability was monitored using the Alamar blue assay, and collagen synthesis was assessed using Sirius red. The role of ALK signaling was studied by receptor inhibition. Cultured human corneal fibroblasts synthesized significantly greater amounts of collagen in the presence of the PA over both 7-day and 21-day periods. The aggregation of the PA to form nanotapes resulted in a notable enhancement in this activity, with an approximately two-fold increase in collagen production per cell. This increase was reduced by the addition of an ALK inhibitor. The data presented reveal a stimulatory effect upon collagen synthesis by the primary cells of the corneal stroma, and demonstrate a direct influence of supra-molecular assembly of the PA upon the cellular response observed. The effects of PA upon fibroblasts were dependent upon ALK receptor function. These findings elucidate the role of self-assembled nanostructures in the biological activity of peptide amphiphiles, and support the potential use of a self-assembling lumican derived PA as a novel biomaterial, intended to promote collagen deposition for wound repair and tissue engineering purposes

A peptide hydrogel derived from a fragment of human cardiac troponin C

The human cardiac troponin C peptide fragment H-V9EQLTEEQKN EFKAAFDIFVLGA31-OH, which covers helix-A in the native protein, self-assembles into b-sheet fibrils in solution. These fibrils further entangle to give a hydrogel. This peptide may therefore serve as a template for development of novel biomaterials.

Peptide based hydrogels for cancer drug release: modulation of stiffness, drug release and proteolytic stability of hydrogels by incorporating D-amino acid residue(s)

Synthetic tripeptide based noncytotoxic hydrogelators have been discovered for releasing an anticancer drug at physiological pH and temparature. Interestingly, gel stiffness, drug release capacity and proteolytic stability of these hydrogels have been successfully modulated by incorporating D-amino acid residues, indicating their potential use for drug delivery in the future.