STRUCTURES

BACKGROUND TO THE INVENTION

Field of the invention

The present invention relates to materials based on filamentous peptide- or protein- based structures. The invention has particular, but not exclusive, applicability to methods for forming nanostructured biofilms, and to such biofilms themselves.

Related art

WO 2004/052773 discloses the formation of a tubular or spherical nanostructure composed of peptides each having up to 4 amino acids. In tubular form, the nanostructures are said to be of interest in electron field emission devices and in sensor tips.

WO 2006/013552 discloses a method of forming a fibre made of peptide nanostructures. The nanostructures are provided in solution and the solution is subsequently fiberized. The fiberization process is typically a spinning process, such as an electrospinning process. This document also discloses the formation of a film of peptide nanostructures, in which peptide molecules are dissolved into an organic solvent, adding a hydrophilic solvent to form an interface between the organic solvent and the hydrophilic solvent, and allowing a film of peptide nanostructures to form at the interface. The peptides have up to 4 amino acids. WO 2007/043048 discloses the formation of hydrogels based on short aromatic peptides. The hydrogels typically contain more than 99% water. The peptides contain no more than 6 amino acids. Such hydrogels are of interest as tissue engineering scaffolds.

WO 2008/068752 discloses a nanostructure array in which elongated nanostructures are arranged perpendicularly to a plane. The nanostructures are formed from peptides. The peptide monomers are held in an organic solvent on a substrate and the solvent is slowly removed via evaporation. The result is an array of nanostructures, upstanding from the substrate.

The present inventors note that self-assembly is emerging as one of the few practical routes towards the reliable and convenient fabrication of nanostructured materials [References 1-3]. A majority of the materials currently used in technology lack, however, the ability to spontaneously generate controlled nanometre scale ordering, implying that fundamentally new types of materials are required to address this challenge. Self- assembly is the approach predominantly exploited by nature for the fabrication of functional components and materials from macromolecules in living systems affording an exquisite level of control, and consequently much attention has focussed on biomolecular recognition as the basis for generating novel materials from the bottom up in the laboratory.

Much progress has been made in the controlled assembly of biomolecules into one [References 2, 4, 5] and two dimensional artificial nanostructures [References 2, 6]. Processing nanostructures into useful macroscopic forms, however, remains a major challenge [Reference 7], requiring dimensions from the nano to micro and macro-scale to be bridged. SUMMARY OF THE INVENTION

The present inventors have devised the present invention, in order to address one or more of these challenges, and preferably to provide an improvement in relation to known macroscopic forms of nanostructures.

Accordingly, in a first aspect, the present invention provides a material comprising filamentous peptide- or protein-based nanostructures, in which said nanostructures are assembled to be connected together and aligned substantially parallel with respect to each other.

In a second aspect, the present invention provides a process for forming a material according to the first aspect, in which the filamentous peptide- or protein-based nanostructures self-assemble to be connected together and aligned substantially parallel with respect to each other.

Preferred and/or optional features will now be set out. These are applicable singly or in any combination with any aspect of the invention, unless the context demands otherwise.

Preferably, the material is a solid material. For example, the material may be provided as a film or as a sheet. Alternatively, the material may be provided as a substantially equi-axed body (but this is not preferred at present).

It is preferred that the mechanical properties of the material are dominated by (and most preferably wholly dependent on) the assembly and connection of the nanostructures. Preferably, the material contains at least 50% by weight of the nanostructures. More preferably, the material consists substantially only of the nanostructures. However, it is possible for the material to contain functional species to provide the material with additional functionality. Such species typically will not affect the mechanical properties of the material.

It is preferred thatjhe material is not supported on a substrate of a different material. Furthermore, it is preferred that the material does not include a matrix material within which the nanostructures are located. Still further, it is preferred that the material does not include a filler material. Thus, the material may be provided with adequate mechanical properties solely by the assembly and interconnection of the nanostructures.

The nanostructures are preferably bonded directly to each other. Preferably, the nanostructures are held together with non-covalent interactions. These bonds may be highly specific. Alternatively, the nanostructures may be held together by covalent bonds.

The filamentous nanostructures are preferably elongated, with a principal axis aligned along the direction of elongation of the nanostructure. Preferably, the principal axes of the nanostructures are substantially straight and aligned parallel with respect to each other, at least in individual alignment domains. Here, the term "domain" is used in a similar manner to the field of liquid crystals, to denote a region of material in which the alignment is substantially uniform. A body of the material may have a single domain only, or may be composed of a plurality of domains of different alignment, in the manner of a polycrystalline material. In the case where the body includes a plurality of domains, there may be no overall preferred alignment of the nanostructures in the body, in view of the averaging effect of the preferred alignment of the different domains.

Preferably, the alignment of the nanostructures (at least within one domain) is nematic alignment.

Preferably (at least within one domain), the degree of alignment of the nanostructures is such that at least 50% (more preferably at least 60%, at least 70%, at least 80% or at least 90%) of the nanostructures have principal axes aligned within plus or minus 40° (more preferably within plus or minus 30°, more preferably within plus or minus 20°, more preferably within plus or minus 10°, still more preferably within plus or minus 5°) of each other, when projected onto a measurement plane. Such measurements may be performed using known X-ray diffraction techniques, e.g. perpendicular to the measurement plane.

As discussed below, preferably the individual nanostructures themselves are formed by a self-assembly process. Furthermore, preferably the material is formed by self-assembly of the nanostructures. The self-assembly of the material may take place in a separate self-assembly step to the self-assembly of the nanostructures. Thus, the material typically has at least two scales of ordering: a first scale of ordering due to a regular structure of the nanostructures and a second scale of ordering due to a regular arrangement of the nanostructures (at least within a domain) in the material. At least the first scale of ordering may be investigated by X-ray diffraction. The second scale of ordering may be investigated by selected area electron diffraction, for example, over a range of areas of the material. The second scale of ordering is on the micron scale, in that the nanostructures themselves are not randomly distributed within the material but are aligned, this alignment extending over lengths of at least 1 μm, e.g. in a direction perpendicular to the nanostructure principal axis.

For each preferred embodiment, the length range of the first scale of ordering is preferably different from and non-overlapping with the length range of the second scale of ordering. This difference may be, for example, by a factor or at least two, more preferably a factor of at least ten or at least fifty. The first scale of ordering may itself have two nanometre scale orderings, corresponding to highly regular molecular repeats within the fibrils. The first nanometre scale ordering may be in the range 0.4-2 nm. The second nanometre scale ordering may be in the range 1-10 nm, with the proviso that, for a particular embodiment, the length of the second nanometre scale of ordering is not the same as for the first nanometre scale of ordering.

Preferably there is provided a body formed from (and more preferably consisting of) the material according to the first aspect. The body may have a thickness of at least 1 μm. More preferably, the body has a thickness of at least 5 μm, or at least 10 μm. The width and/or length of the body is typically significantly greater than the thickness, e.g. at least 1 mm. Preferably, the body is self-supporting under gravity.

Preferably, the material (or body) has a Young's modulus of at least 500 MPa, and more preferably at least 1 GPa, at least 2 GPa, at least 3 GPa or at least 4 GPa. Other elastic moduli of the material (or body) may be within similar ranges to the Young's modulus. For example, it is possible to measure the storage modulus using dynamic mechanical analysis (e.g. three-point bending carried out at 1 Hz) in a manner known to the person skilled in the art.

Preferably, the nanostructures are formed from peptides or proteins having more than 6 amino acids. The nanostructures may be formed from peptides or proteins having 250 or fewer amino acids. It is preferred that the nanostructures are amyloid fibrils.

Here, the number of amino acids may be the average number of amino acids in the peptides or proteins which make up the nanostructure. However, it may be preferable that the polydispersity of the peptides or proteins is limited such that substantially all peptides or proteins in the nanostructure have more than 6 amino acids. Similarly, the nanostructures may be formed from peptides or proteins substantially all of which have 250 or fewer amino acids.

The number of amino acids may be determined using MALDI TOF mass spectrometry (matrix-assisted laser desorption / ionization time of flight mass spectrometry). Suitable techniques are described in Reference 14: "MALDI MS, A Practical Guide to Implementation, Methods and Applications" edited by Franz Hillenkamp ad Jasna Peter- Katalinic, WILEY-VCH, 2007, in particular Chapter 3: "MALDI-MS in Protein Chemistry and Proteomics" by Karin Hjerno and Ole N Jensen, beginning on page 83, the content of which is hereby incorporated by reference in its entirety.

The nanostructures may have a rod-like geometry. Preferably, the diameter of the nanostructures is at least 2 nm. The diameter of the nanostructures may be 15 nm or less, preferably 10 nm or less.

The nanostructures may have functional species attached. When the nanostructures are assembled together to be connected and aligned, the functional species may be provided in a very regular array within the material. For example, the nanostructures may include electrically conductive or semiconducting species. The nanostructures may include species that are polarizable or have multiple charge states. The nanostructures may include a thermoelectric species. The nanostructures may include a magnetic (e.g. ferromagnetic) species, although such a species is not typically required in order to provide alignment of the nanostructures. The nanostructures may include a fluorophore. The nanostructures may include an inorganic nanoparticle species. For example, inorganic nanotubes such as single walled or multiwalled carbon nanotubes may be included. This is or interest in particular to achieve ordering of the inorganic nanoparticles (e.g. CNTs).

Preferably, the process for forming the material includes a first step of forming the nanostructures. The nanostructures may be formed in solution/suspension to form a substantially homogenous suspension of nanostructures in a liquid. The nanostructures typically self-assemble from peptide monomers. However, in a preferred embodiment, the nanostructures may be formed by growth from seed nanostructures in a solution/suspension of peptide monomers. In some embodiments, the formation of the nanostructures may lead to the formation of a gel with the liquid (i.e. an increase in viscosity of the liquid due to the formation and interaction of the nanostructures).

In the self-assembly of the nanostructures themselves, the conditions may be controlled so as to favour intermolecular interactions over intramolecular interactions.

Preferably, there is included a second step, distinct from the first step, in which the nanostructures arrange themselves to form the material, the nanostructures becoming connected together and aligned substantially parallel with respect to each other. Thus, this is typically a separate self-assembly process, distinct from the self-assembly of the nanostructures themselves.

In the second step, there may be added a self-assembly promotion agent. Typically, this agent is a plasticizing agent, e.g. a small molecule compound such as polyethylene glycol (typically small molecular weight polyethylene glycol). The self-assembly promotion agent is considered to allow the nanostructures to move and/or align more readily with respect to each other during the self-assembly process, thus promoting the eventual self-assembly of the nanostructures into an energetically-preferred assembled structure. It is to be noted that plasticizers do not necessarily affect the bulk viscosity of the suspension or hydrogel. The exact mechanism promoting alignment is not known, but it is considered by the inventors (without wishing to be bound by theory) that the small molecules compete with other nanostructures (e.g. fibrils) for bonding and may delay the formation of connections between the nanostructures until they have had time to align. It is noted that some workers consider that the presence of even inert molecules in solution enhances the alignment of nanostructures (e.g. rigid rods) due to depletion interactions.

The second step may be achieved by casting the suspension of the nanostructures into a film. The film may subsequently be dried. The first and/or second step may be carried out in mild conditions. For example, these step may be carried out at atmospheric pressure, e.g. in air. These steps may be carried out above 0 ⁰ C and below 100 ⁰ C and more preferably at or just above room temperature. In this way, the manufacturing conditions for the material may be compatible with the use of the material in biological or medical applications, or with delicate species included in the assembly of nanostructures.

Further optional features of the invention are set out in the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the present invention will now be described, by way of example, with reference to the drawings, in which:

Figs. 1 A-1 E show the strategy for the fabrication of nanostructured biofilms through multi- scale self-assembly. Protein molecules are assembled in a first stage into nanofibrils which are then stacked into films, as shown in the three stages of Fig. 1A, viewing from left to right. Fig. 1 B shows an atomic force micrograph of the component nanofibrils and Fig. 1C shows a scanning electron micrograph of the resulting free standing protein film. Optical images under crossed polarisers in Figs. 1 D and 1 E show no transmission through the protein film when the objective polariser is parallel to the fibril alignment in the film (Fig. 1D) and maximal transmission at a 45° angle (Fig. 1 E).

Figs. 2A-2C shows characterisation of the nanostructured films by X-ray diffraction studies. Fig. 2A, for example, includes an X-ray diffraction pattern (top) and a schematic illustration of the X-ray beam incidence with respect to the film. A film without (Fig. 2A) and with (Fig. 2B) plasticiser is illuminated from the top showing in Fig. 2A an isotropic orientation of the characteristic inter-sheet and inter-strand repeats and in Fig. 2B orientational order, with the intersheet and inter-strand orientations being perpendicular to each other as required by the fibril geometry. In Fig. 2C the same film as in Fig. 2A is illuminated from the side, demonstrating the alignment of the fibrils in the film plane.

Figs. 3A and 3B illustrate mechanical testing of protein films in three point bending geometry with an oscillating load applied to the suspended centre section (Fig. 3A) and a comparison of the storage modulus at 1 Hz with the Young's modulus of other materials (Fig. 3B).

Figs. 4A and 4B show the intensity of emission of light at 482 nm from nanostructured films containing aligned fluorophores excited at 450 nm. The emitted light was transmitted through a polarising filter with a fixed orientation and the orientation of the film was changed through 360° (filled squares in Fig. 4A). Fig. 4B shows a fluorescence microscopy image of a non-functionalised (i) and functionalised (ii) fibril biofilm.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS. FURTHER OPTIONAL FEATURES OF THE INVENTION

The present inventors have fabricated and characterized macroscopic nanostructured free-standing films assembled from protein molecules which exhibit robust material properties and a high level of ordering on the nano and micro scale.

Controlling biological self-assembly to produce novel materials has emerged as a central challenge in nanotechnology. Here the inventors describe the fabrication and characterisation of macroscopic free-standing nanostructured protein biofilms. It is shown that these films produced through self-assembly of protein molecules are highly rigid with Young's moduli of up to 6 GPa, comparable to the highest values for proteinacous materials found in nature, and exhibit hierarchical ordering from the nanometre to the micrometre scale. It is further demonstrated that the ordering generated in proteinaceous scaffolds can be exploited to organise other components which lack the propensity to self-assemble on their own. The results outline a new path toward artificial nanostructured biomaterials.

The present inventors designed a strategy (Fig. 1A) to exploit the natural propensity of protein molecules to create highly-specific non-covalent contacts in order to assemble macroscopic materials with controlled nanostructure. In a first step, protein molecules are assembled into elongated fibrils under conditions where the formation of inter- molecular interactions is favoured over intra-molecular ones. The resulting nanofibrils are highly stable, rigid and can be formed from a range of different proteins [References 5, 8]. In a second step, the fibrils are cast into thin films. During the casting process, the present inventors have found that the fibrils can be induced to stack with nematic order, a tendency which can be accentuated with the presence of plasticizing molecules (see below), thereby resulting in materials which have a hierarchy of length scales: nanometre ordering within the fibrils and micrometre scale ordering in the stacking of the fibrils. The nematic order on macroscopic scales in the nanostructured biofilms leads to interaction with visible light and can be observed clearly by polarisation microscopy: Fig. 1E shows a single domain film which transmits light uniformly when positioned at a 45° angle between crossed polarisers and is opaque when parallel to one of the polarisers.

Characterisation of the films by X-ray diffraction measurements (Fig. 2) reveals that they possess a high level of order on the nanometre scale. Insight into the structure of the material can be obtained from examining the characteristic distances and orientations of the two major length scales present. The individual jβ-strands composing the nano-fibrils are separated by 4.8 A along the fibril direction, and these sheets composed of arrays of strands associate laterally to form parallel assemblies giving rise to a characteristic distance of approximately 12 A in the perpendicular direction to the strand repeat [Reference 9]. When the nanostructures are cast from the hydrogel (see below) into the films, the long axis of the nanostructures are aligned in the film plane as observed by X- ray scattering. These results also show that the casting and drying process does not degrade the atomic level core structure of the fibrils. If plasticizing molecules such as small molecular weight polyethylene glycol are added to the suspension of nanostructures before the casting process, further ordering can be observed and the fibrils adopt nematic order in the solid phase. Liquid crystalline behaviour of elongated biomolecular assemblies in solution is known [Reference 10, 11], and the present inventors speculate (without wishing to be constrained by theory) that the presence of a plasticizer could enable this orientational order to be preserved in the solid phase through weakening of the inter-fibril bonding or by enhancing the tendency for orientational ordering as a result of the depletion interaction.

The original X-ray intensity patterns in Figs. 2A-2C used false colour to indicate variations of intensity. In general, in the versions of Figs. 2A-2C shown here, darker regions represent regions of low intensity and lighter regions represent regions of higher intensity. Exceptionally, all of the regions of high intensity which are shown in a dark shade are indicated with an "M", indicating a maximum.

The results of Fig. 2 were obtained by exposing a film of the material from the top with X- rays and collecting the resulting diffraction pattern. The alignment is visible in the anisotropy of the 4.8 A reflection, i.e. the highest intensity of diffracted x-rays in the Fig. 2B is in one direction - this gives the direction of the principal axis of the fibrils. The degree of alignment can be considered to be a standard deviation for the director angle of the principal axis of the fibrils, in a similar manner to the determination of the degree of alignment for nematic liquid crystals.

There are two nanometre scale repeats shown by the X-ray diffraction results. One is at 4.8 A, this is the distance between adjacent strands along the fibril, and one at 12 A, this is the distance in the perpendicular direction to the fibril axis between adjacent sheets. A fibril typically has 2-12 sheets (width) and anywhere from 50 to 100000 or more strands (length). The micron scale ordering is on long length scale that is too large to give rise to a direct X-ray reflection discernible in Fig. 2, but the nematic order can be seen in the anisotropy of the 4.8 A repeat. A characteristic length-scale for the inter-fibril ordering is the length of the fibril, i.e. 1 micron.

Biological structures hold great promise for fabricating self-organising structures as the molecular recognition capabilities inherent to many biomolecules can lead to complex assemblies. The material properties of many such structures, however, are limited; for instance the persistence length of DNA, even in the double helix form is less than 50 nm, thereby constraining the length scales on which organisation can be achieved as well as the robustness of the formed materials. In nature, many functional materials are instead assembled from proteins. The protein nanofibrils used as the basis of nanostructured films in this work are very robust, their mechanical properties being in the upper range of existing biomaterials [References 4, 5]. The present inventors probed whether these mechanical properties were maintained and transmitted to the macroscopic films composed of such fibrils. To this effect, the elastic modulus of the fibrils was probed in a three point bending geometry, where both ends of a film were fixed, and an oscillating load was applied to the suspended centre section. The results in Fig. 3B reveal that the elastic moduli of the films range from 4.5 to 6.2 GPa, and is therefore only lowered by a small fraction when compared with the mechanical properties of the individual nanostructures. This is in contrast to films generated from high-performance carbon based nanomaterials such as "bucky-paper" formed from carbon nanotubes, where the elastic modulus and tensile strength of the bulk material are typically several orders of magnitude lower than the corresponding characteristics of the individual nanostructures [Reference 12]. This lowering of the mechanical properties when the size of the material is scaled up is a fundamental limitation for the production of high-performance macroscopic forms of nanomaterials and has been ascribed to the difficulty in establishing robust contacts between the adjacent nanotubes. Protein nanofibrils on the other hand have an outer surface rich in functional groups which ensure efficient connectivity between the structures through hydrophobic packing and large surface area for van der Waals contacts.

The fabrication of spatially organised or oriented arrays of objects on the nanometre scale is a requirement for many practical applications of nanotechnology. Frequently the chemistry of functional materials themselves does not predispose them towards spontaneous organisation. The present inventors have illustrated the power of self- assembling protein scaffolds to control the organisation of objects by attaching fluorophores to the nanofibrils. When the fibrils adopt nematic order in the film state, they direct the alignment of the dipole moments of the fluorophores in a single direction, thereby producing a material which emits polarised light under optical excitation. The fluorophore used was the planar aromatic dye Thioflavin T (ThT) which can attach to β- sheet rich structures with the orientation of the emission dipole parallel to the sheet length which coincides with the long axis of the fibril [Reference 13]. The fluorescence of the molecule is maintained in the casting and drying process. The optical activity of the resulting material was tested by exciting the film with light polarised in a given direction; the emitted light was also transmitted through a polarisation filter with the same orientation as the excitation wave, and the intensity at the emission wavelength was recorded (see below). When the film is rotated through 360°, the intensity of the emission varies significantly (Fig. 4). Emission maxima, separated by 180° correspond to the dipole axis being parallel to the filter orientation, and the minima, separated from the maxima by 90° corresponding to perpendicular relative orientation, thereby demonstrating the that the molecular level alignment of the fluorophores could be achieved using the organised proteinaceous scaffold.

Thus, the high specificity of biomolecular interactions can be exploited to fabricate nanostructured biofilms through a multi-scale self-assembly process. Due to the unique combination of accurate self-assembly, robust material properties and chemical versatility with regard to possibilities for surface functionalisation, such protein based nanostructured films represent a attractive path towards realising novel multi-functional materials built from the bottom up.

Films were prepared as follows. Hen egg white lysozyme (Sigma Aldrich) was incubated as a 3% w/w solution in aqueous 10 mM hydrochloric acid at 65°C for 14 days to induce self-assembly into nanofibrils. The fibrillar content was improved in a second step by adding 5% v/v of this preformed fibril suspension as seed material at the beginning of the polymerisation reaction. At the end of the polymerisation reaction plasticisers (PEG 400 or glycerol) were added. Films were then cast by transferring 1 ml of the nanofibril- containing hydrogel onto a flat polytetrafluoroethylene film where the solvent and the volatile acid were left to evaporate for 24h and the resulting protein films could be removed with tweezers.

Films containing aligned fluorphores were prepared as follows. A filtered (220 nm pore size) solution of Thioflavin T (Sigma Aldrich) prepared at a concentration of 10 mg/ml and

1 μl was added to 1 ml of the nanofibril hydrogel, and mixed using a vortex mixer for 30s.

The fluorescence of the films was measured using a Cary Eclipse Fluorimeter equipped with polarisers. A 10 mm x 10 mm piece of the film was fixed between two round quartz plates. The active region where emission was measured was limited to a circle by using a mask attached to the top quartz plate. The sample was then rotated through 360° in increments of 45° and an emission spectrum was recorded from 460 to 600 nm. The excitation wavelength used was 440 nm.

X-ray diffraction studies were performed at the crystallographic diffraction data collection facility, Department of Biochemistry, University of Cambridge. X-rays with a wavelength of 1.54 A were produced by a rotating copper anode generator RU-H3R (Rigaku-MSC, Ltd), and collimated and focussed by Osmic Max-ux optics. Images were acquired on a Raxis-IV++ image plate (Rigaku-MSC, Ltd). Data acquisition times were between 5 and 20 min. Mechanical measurements were performed using three point bending geometry on a TA- lnstruments Q800 DMA setup, illustrated schematically in Fig. 3A. The storage modulus was computed from the measured stiffness k _s as E = /c _s L ³ /(6/), where / = Wh ³ IM is the cross-sectional moment of inertia and w, h, L denote the width, height and length of the suspended film.

The preferred embodiments of the invention have been described by way of example. On reading this disclosure, modifications of these embodiments, further embodiments and modifications thereof will be apparent to the skilled person and as such are within the scope of the invention.

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