Amalia Aggeli

Self-assembling peptide nanotubes

Biological proteins and peptides have the intrinsic ability to self-assemble into elongated solid nanofibrils 1 , 2 , 3 , 4 , 5 , 6 , 7 , which may give rise to amyloid diseases 8 , 9 , 10 , 11 or inspire applications ranging from tissue engineering to nanoelectronics 12 , 13 , 14 , 15 , 16 . Proteinaceous fibrils are extensively studied and well understood, to the extent that detailed theoretical models have been proposed that explain and predict their behavior 17 , 18 . Another intriguing state of protein-like self-assembly is that of nanotubes (NTs), defined here as an elongated nano-object with a definite inner hole. In contrast to proteinaceous fibrils, nanotubes are much less frequently observed and far less well understood. However, they have attracted research interest internationally as key components for nanotechnology.

Production of self-assembling biomaterials for tissue engineering

Self-assembling peptide-based biomaterials are being developed for use as 3D tissue engineering scaffolds and for therapeutic drug-release applications. Chemical synthesis provides custom-made peptides in small quantities, but production approaches based upon transgenic organisms might be more cost-effective for large-scale peptide production. Long lead times for developing appropriate animal clones or plant lines and potential negative public opinion are obstacles to these routes. Microbes, particularly safe organisms used in the food industry, offer a more rapid route to the large-scale production of recombinant self-assembling biomaterials. In this review, recent advances and challenges in the recombinant production of collagen, elastin and de novo designed self-assembling peptides are discussed.

Engineering of peptide β-sheet nanotapes

A set of principles are outlined for the design of short oligopeptides which will self-assemble in appropriate solvents into long, semi-flexible, polymericβ-sheet nanotapes. Their validity is demonstrated by experimental studies of an 11-residue peptide (DN1) which forms nanotapes in water, and a 24-residue peptide (K24) which forms nanotapes in non-aqueous solvents such as methanol. Circular dichroism (CD) spectroscopy studies of the self-assembly behaviour in very dilute solutions (µm) reveal a simple transition from a random coil-to-β-sheet conformation in the case of DN1, but a more complex situation for K24. Association of DN1 is very weak up to a concentration of 40 µm at which there is a sudden increase in the fraction of peptide in the β-sheet structure, indicative of an apparent ‘critical tape concentration’. This is shown to arise from a two-step self-assembly process: the first step being a transition from a random coil to an extended β-strand conformation, and the second the addition of this β-strand to a growing β-sheet. Both peptides are shown to gel their solvents at concentrations above 2×10 -3 volume fraction: these gels are stable up to the boiling point of the solvents. Rheology measurements on gels of the 24-residue peptide in 2-chloroethanol reveal that the tapes form an entangled network with a mesh size of 10–100 nm for peptide volume fractions 0.03–0.003; the persistence length of the tape is 13 nm or greater, indicative of a moderately rigid polymer; the tapes are about a single molecule in thickness. The mechanical properties of the gels in many respects are comparable to those of natural biopolymers such as gelatin, actin, amylose and agarose.

Biomimetic self-assembling peptides as scaffolds for soft tissue engineering

Tissue engineered therapies are emerging as solutions to several of the medical challenges facing aging societies. To this end, a fundamental research goal is the development of novel biocompatible materials and scaffolds. Self-assembling peptides are materials that have undergone rapid development in the last two decades and they hold promise in meeting some of these challenges. Using amino acids as building blocks enables a great versatility to be incorporated into the structures that peptides form, their physical properties and their interactions with biological systems. This review discusses several classes of short self-assembling sequences, explaining the principles that drive their self-assembly into structures with nanoscale ordering, and highlighting in vitro and in vivo studies that demonstrate the potential of these materials as novel soft tissue engineering scaffolds.

Recombinant self-assembling peptides as biomaterials for tissue engineering.

Synthetic nanostructures based on self-assembling systems that aim to mimic natural extracellular matrix are now being used as substrates in tissue engineering applications. Peptides are excellent starting materials for the self-assembly process as they can be readily synthesised both chemically and biologically. P11-4 is an 11 amino acid peptide that undergoes triggered self-assembly to form a self-supporting hydrogel. It exists as unimers of random coil conformations in water above pH 7.5 but at low pH adopts an antiparallel β-sheet conformation. It also self-assembles under physiological conditions in a concentration-dependent manner. Here we describe an unimer P11-4 production system and the use of a simple site-directed mutagenesis approach to generate a series of other P11-family peptide expression vectors. We have developed an efficient purification strategy for these peptide biomaterials using a simple procedure involving chemical cleavage with cyanogen bromide then repeated filtration, lyophilisation and wash steps. We report peptide-fusion protein yields of ca. 4.64 g/L and we believe the highest reported recovery of a recombinant self-assembling peptide at 203 mg/L of pure recombinant P11-4. This peptide forms a self-supporting hydrogel under physiological conditions with essentially identical physico-chemical properties to the chemically synthesised peptide. Critically it also displays excellent cytocompatibility when tested with primary human dermal fibroblasts. This study demonstrates that high levels of a series of recombinant self-assembling peptides can be purified using a simple process for applications as scaffolds in tissue engineering.

Biomimetic self-assembling peptides as injectable scaffolds for hard tissue engineering

The production of bone-, dentine- and enamel-like biomaterials for the engineering of mineralized (hard) tissues is a high-priority in regenerative medicine and dentistry. An emerging treatment approach involves the use of short biomimetic peptides that self-assemble to form micrometer-long nanofibrils with well defined surface chemistry and periodicity that display specific arrays of functional groups capable of mineral nucleation. The fibrils also give rise to dynamically stable 3D scaffold gels for the potential control of crystal disposition and growth. Peptides can also be injected in their monomeric fluid state, with subsequent self-assembly and gelation in situ triggered by physiological conditions. In this way, they can infiltrate and self-assemble within irregular or microscopic cavities, for restorative treatment of bone defects, dentinal hypersensitivity or dental decay. Cell adhesion and proliferation is also supported by these scaffolds, offering further advantages for applications in hard tissue engineering. These self-assembling matrices also provide well defined model systems that can contribute greatly to the elucidation of the biological mechanisms of protein-mediated biomineralization.

Self-assembling β-Sheet Tape Forming Peptides

Biological proteins have intrinsically the ability to self-assemble, and this has been implicated in pathological situations called amyloid diseases. Conversely understanding protein self-assembly and how to control it can open up the route to new nanodevices and nanostructured materials for a wide range of applications in medicine, chemical industry and nanotechnology. Biological peptides and proteins have complex chemical structure and conformation. This makes it difficult to decipher the fundamental principles that drive their self-assembling behaviours. Here we review our work on the self-assembly of simple de novo peptides in solution. These peptides are designed so that: (i) the chemical complexity of the primary structure and (ii) the conformational complexity are both kept to a minimum. Each peptide adopts an extended β-strand conformation in solution and these β-strands self-assemble in one dimension to form elongated tapes as well as higher order aggregates with pure antiparallel β-sheet structure, without the presence of any other conformations such as turns, loops, α-helices or random coils. Experimental data of the self-assembling properties are fitted with an appropriate theoretical model to build a quantitative relationship between peptide primary structure and self-assembly. These simple systems provide us with the opportunity to reveal the generic properties of the pure β-sheet structures and expose the underlying physicochemical principles that drive the self-assembling behaviour of this biological motif.

Bioproduction and characterization of a pH responsive self-assembling peptide

Peptide P11‐4 (QQRFEWEFEQQ) was designed to self‐assemble to form β‐sheets and nematic gels in the pH range 5–7 at concentrations ≥12.6 mM in water. This self‐assembly is reversibly controlled by adjusting the pH of the solvent. It can also self‐assemble into gels in biological media. This together with its biocompatibility and biodegradability make P11‐4 an attractive building block for the fabrication of nanoscale materials with uses in, for example, tissue engineering. A limitation to large‐scale production of such peptides is the high cost of solid phase chemical synthesis. We describe expression of peptide P11‐4 in the bacterium Escherichia coli from constructs carrying tandem repeats of the peptide coding sequence. The vector pET31b+ was used to express P11‐4 repeats fused to the ketosteroid isomerase protein which accumulates in easily recoverable inclusion bodies. Importantly, the use of auto‐induction growth medium to enhance cell density and protein expression levels resulted in recovery of 2.5 g fusion protein/L culture in both shake flask and batch fermentation. Whole cell detergent lysis allowed recovery of inclusion bodies largely composed of the fusion protein. Cyanogen bromide cleavage followed by reverse phase HPLC allowed purification of the recombinant peptide with a C‐terminal homoserine lactone (rP11‐4(hsl)). This recombinant peptide formed pH dependent hydrogels, displayed β‐structure measured by circular dichroism and fibril formation observed by transmission electron microscopy. Biotechnol. Bioeng. 2009;103: 241–251.

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.

Self-assembly of amphiphilic β-sheet peptide tapes based on aliphatic side chains†

Amphiphilic β‐sheet nanotapes based on the self‐assembly of 9mer and 7mer de novo designed β‐strand peptides were studied in the dilute regime. The hydrophobic face of the tapes consisted predominantly of aliphatic (leucine) side chains, while the hydrophilic tape face contained polar side chains (glutamine, arginine and glutamic acid). Both peptides underwent a transition from a monomeric random coil to a self‐assembled β‐sheet tape upon increase of peptide concentration in aqueous solutions. P9‐6 exhibited lower critical concentration (c*) for self‐assembly and thus higher propensity for self‐assembly in water, compared to the shorter P7‐6. At neutral pH where there was little net charge per peptide, self‐assembly was favoured compared to low pH in which there was a net + 1 charge per peptide; the net charge decreased overall intermolecular attraction, manifested as an increase in c* for self‐assembly in low compared to neutral pH aqueous solutions. Propensity for self‐assembly and β‐sheet formation was found to be greatly enhanced in a polar organic solvent (methanol) compared to water. These studies combined with future more extensive comparative studies between amphiphilic tapes based on aliphatic amino acid residues and amphiphilic tapes based on aromatic residues will throw more light on the relative importance of hydrophobic versus aromatic interactions for the stabilisation of peptide assemblies. Systematic studies of this kind may also allow us to throw light on the fundamental principles that drive peptide self‐assembly and β‐sheet formation; they may also lead to a set of refined criteria for the effective design of peptides with prescribed combination of properties appropriate for specific applications. Copyright