Functionalised fibrils for bio-nanotechnology

Sally L. Gras*, Adam M. Squires †, Christopher M. Dobson ‡ and Cait E. MacPhee*Department of Chemical and Biomolecular Engineering,

University of Melbourne, Victoria 3010, Australia. Email: sgras@unimelb.edu.au

†Department of Chemistry, University of Reading, PO Box 224, Whiteknights, Reading RG6 6AD, United Kingdom.

‡ Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.

School of Physics, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JZ, United Kingdom.

Abstract—Amyloid fibrils offer great potential as nano-materials. Functionalised fibrils assembled from peptides coupled to bioactive ligands interact specifically with cells compared to non-functionalized fibrils, suggesting that these fibrils are also promising bio-scaffolds.

Keywords; bio-nanotechnology; amyloid; fibrils; self-assembly; peptide; bioactive; biomaterial; bio-scaffold.

Nanotechnology promises to extend the properties of current biomaterials. Scaffolds may be self-assembled using bottom-up manufacturing to produce diverse topographies and biological ligands at a nanometre scale. These features may promote cell adhesion, migration and differentiation and provide a new approach for understanding cell and tissue behaviour. Such scaffolds offer great potential for application in novel in vitro and in vivo technologies and devices.

Amyloid fibrils are self-assembling protein nano-fibrils that are attractive candidates as a basis for biocompatible materials. The fibril core is remarkably stable and consists of -sheetsstabilised by hydrogen bonding between the main chains of adjacent -strands [which are oriented in a cross-arrangement] [1] as shown in Figure 1. Subsequent -sheets are stacked within the core at a distance determined by the size of amino acid side chains. Large proteins can also form fibrils with additional structure present on the fibril surface [2] (Figure 1).

Fibrils adopt diverse nano-topographies including ribbon-like and rod-like fibrils ranging from 10-50 nm in diameter, as illustrated in Figure 2. Fibril nano-topography is determined by the size, number and arrangement of protofilament sub-units within the fibril. This structure can be controlled by the choice of solution conditions used for fibril assembly [3]. Fibrils may be formed from existing protein sequences with known properties [4-5]. Alternately, an understanding of the forces driving fibril formation, including sequence hydrophobicity, -sheet propensity and charge can be used to design de novo

fibrils with tailored physico-chemical and mechanical properties [6-7].

Figure 1. The ordered cross- structure of amyloid fibrils. A: a single -sheet from within the fibril core. Hydrogen bonding between the -strands stabilises this -sheet. B: several -sheets stacked to form the cross-structure of the fibril core. C: larger proteins may have some residues excluded from the fibril core and present as disordered structure on the fibril surface. -strands and -sheets are shown here in a parallel arrangement, other combinations of -strands or -sheets that are anti-parallel or out of register may also be possible.

Our interest concerns the use of amyloid fibrils as bio-nanoscaffolds. Fibrils have previously been used to construct nano-wires and create scaffolds displaying fluorophores, biotin, cytochrome, and active enzymes including barnase, carbonic anhydrase and glutathione S-transferase on the fibril surface [8-13]. These ligands are similar to the protein structure found on

Fibril axisFibril axisFibril axis

A B

C

Fibril axisFibril axisFibril axis

A B

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the surface of fibrils from large proteins (Figure 1). Functionalisation experiments [8-12] indicate that fibril self-assembly is largely insensitive to chemical modification and suggest that fibrils may be engineered to display a diverse range of ligands on the fibril surface.

Figure 2. The varied nano-topographies of amyloid fibrils imaged by Transmission Electron Microscopy. Thin straight fibrils ~10 wide (top left), thick straight fibrils ~ 30-50 nm wide (top, right), flat laterally associating fibrils ~30-50 nm wide (bottom left) and twisted ribbon-like fibrils ~ 30 nm wide (bottom right). Scale bars are 200 nm.

Our goal is to develop fibrils displaying biological ligands to promote and exploit cell behaviour in vitro. The nano-topography of amyloid fibrils recommends these structures as bio-materials (Figure 2). It has been shown that similar surface topographies including nano-grooves, columns and roughness can provoke and control cell behaviour [14-16]. Even small variations in surface height of approximately 10 nm can lead to significant responses in cell morphology, gene expression and extra-cellular matrix protein production [17], suggesting that fibrils are capable of inducing cellular responses. Functionalised fibrils may also be designed displaying bioactive ligands on a nano-length scale. These fibrils will have extended bio-interactive properties compared to non-functionalised fibrils. The nano-scale clustering, density and spatial distribution of bioactive ligands are known to influence cell behaviour [17-18]. The display of ligands on fibrils may therefore be tailored to further encourage cell-fibril interactions and promote desired cellular responses.

Several bioactive ligands may be used for surface display on functionalised fibrils. One candidate is the classic Arginine-Glycine-Aspartic Acid (RGD) sequence. This sequence, originally found in the extra-cellular matrix protein

fibronectin, is the minimal motif required for cell adhesion [20]. The RGD sequence binds to a range of cell surface integrin receptors which are responsible for cell responses to a wide variety of environmental cues [21]. Another candidate for ligand display is the sequence Tyrosine-Isoleucine-Glycine-Serine-Arginine (YIGSA) from the extra-cellular matrix protein laminin [22]. This motif interacts with cell laminin receptors, again allowing the cell to respond to the extra-cellular environment. The choice of flanking sequences adjacent to ligands can also be used to tune ligand affinity for different integrins and different cell types [21], allowing the development of functionalised fibrils with wide-ranging interactive properties.

Our strategy is to couple model self-assembling peptides with bioactive ligands to produce functionalised fibrils. We are exploring a number of fibrillar frameworks based on peptides. Well characterised systems include peptide fragments from transthyretin [23] which form long (> 1 m) regular nano-fibrils approximately 10 nm in width, some of which have been characterised by solid-state NMR [24-25]. Other short peptides include the SH3 domain, the cc peptide, and Urep2 [12-13, 26]. Several of these peptides have been successfully used as scaffolds to display cyotchrome, fluorophores or functional enzymes (as described above). These peptides are therefore ideal systems for biofunctionalisation and the peptide sequence may be extended to include bioactive or control ligands.

A wide range of experimental techniques may be used to examine functionalised peptides prior to and following fibril assembly. These techniques include methods traditionally used to examine amyloid fibrils [27] and assays used to assess biomaterials in vitro. Circular dichroism, Fourier transform infra red spectroscopy and X-ray diffraction can be used to probe the -sheet secondary structure and the dimensions of the fibril core [27], to confirm that fibril structure is not perturbed by functionalisation. Proteolysis, fluorescent labelling and immunogold labelling can be used to probe and quantify ligand display on the fibril surface. The bioactivity of the functionalised peptides and fibrils may also be examined using in vitro bioactivity assays including cell adhesion and dissociation assays.

Our experiments to date reveal that functionalised amyloid fibrils are promising bio-nanomaterials [28]. Our strategy coupling self-assembling peptides to bioactive ligands creates functionalised fibrils that exhibit specific interactions with cells. Such interactions are not observed for non-functionalised fibrils. Further examination of cell-fibril interactions should reveal whether functionalised fibrils may act as bio-scaffolds or tools for probing cell behaviour.

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ACKNOWLEDGMENTS

We thank Dr Anna Tickler for peptide synthesis and mass spectrometry, Darerca Owen for cell culture facilities and Dr Filip Meersman and Professor Mike Horton for discussions. S.L.G. is funded by a Gates Cambridge Scholarship, C.M.D is funded by the Wellcome and Leverhulme Trusts and C.E.M. is a Royal Society University Research Fellow.

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