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Title:
CELL CULTURE SUBSTRATES
Document Type and Number:
WIPO Patent Application WO/2019/033171
Kind Code:
A1
Abstract:
The present application describes a class of peptides comprising from (2) to (12) amino acid residues, wherein: the N-terminus of the peptide is linked to an optionally substituted, fused polycyclic group comprising an aromatic ring; at least one amino acid residue has a hydrophobic side chain; at least two amino acid residues have a cation-containing side chain, or at least one amino acid residue has two or more cation-containing side chains, or at least one amino acid residue has one or more side chains comprising two or more cations; and the net charge of the peptide is positive at a pH of from about (4) to about (10). The present application also describes hydrogels and cell culture substrates comprising the peptides, as well as the use of the cell culture substrates for culturing cells.

Inventors:
MARTIN, Adam (c/- Balaclava Road, North Ryde, New South Wales 2109, 2109, AU)
ITTNER, Lars Matthias (c/- Balaclava Road, North Ryde, New South Wales 2109, 2109, AU)
KE, Yazi Diana (c/- Balaclava Road, North Ryde, New South Wales 2109, 2109, AU)
Application Number:
AU2018/050874
Publication Date:
February 21, 2019
Filing Date:
August 16, 2018
Export Citation:
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Assignee:
MACQUARIE UNIVERSITY (Balaclava Road, North Ryde, New South Wales 2109, 2109, AU)
International Classes:
C07K5/10
Foreign References:
EP2518041A12012-10-31
Other References:
ARNUSCH, C. J. ET AL.: "Ultrashort Peptide Bioconjugates are Exclusively Antifungal Agents and Synergize with Cyclodextrin and Amphotericin B", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 56, no. 1, January 2012 (2012-01-01), pages 1 - 9, XP055572260
ELASHAL, H. E. ET AL.: "Site-selective chemical cleavage of peptide bonds", CHEMICAL COMMUNICATIONS, vol. 52, no. 37, 2016, pages 6304 - 6307, XP055572266, Retrieved from the Internet
BARLOS, K. ET AL.: "Fmoc/Trt-amino acids: comparison to Fmoc/tBu-ammo acids in peptide synthesis", JOURNAL OF PEPTIDE RESEARCH, vol. 51, no. 3, March 1998 (1998-03-01), pages 194 - 200, XP055572271
BAYAT, S. ET AL.: "Rational design of mimetic peptides based on aldo-ketoreductase enzyme as asymmetric organocatalysts in aldol reactions", RSC ADVANCES, vol. 4, 2014, pages 38859 - 38868, XP055572351, Retrieved from the Internet
MCCLOSKEY, A. P. ET AL.: "Ultrashort self-assembling Fmoc-peptide gelators for anti- infective biomaterial applications", JOURNAL OF PEPTIDE SCIENCE, vol. 23, no. 2, February 2017 (2017-02-01), pages 131 - 140, XP055572358, Retrieved from the Internet
WEI, Q. ET AL.: "Viscosity-controlled printing of supramolecular-polymeric hydrogels via dual-enzyme catalysis", JOURNAL OF MATERIALS CHEMISTRY B, vol. 4, 15 August 2016 (2016-08-15), pages 6302 - 6306, XP055459310, Retrieved from the Internet
XU, X. -D. ET AL.: "Biological Glucose Metabolism Regulated Peptide Self-Assembly as a Simple Visual Biosensor for Glucose Detection", MACROMOLECULAR RAPID COMMUNICATIONS, vol. 33, no. 5, 16 March 2012 (2012-03-16), pages 426 - 431, XP055572367, Retrieved from the Internet
FICHMAN, G. ET AL.: "Self-assembly of short peptides to form hydrogels: Design of building blocks, physical properties and technological applications", ACTA BIOMATERIALIA, vol. 10, no. 4, April 2014 (2014-04-01), pages 1671 - 1682, XP055572370, Retrieved from the Internet
ZHOU, M. ET AL.: "Self-assembled peptide-based hydrogels as scaffolds for anchoragedependent cells", BIOMATERIALS, vol. 30, no. 13, May 2009 (2009-05-01), pages 2523 - 2530, XP055572373, Retrieved from the Internet
Attorney, Agent or Firm:
GRIFFITH HACK (GPO Box 4164, Sydney, New South Wales 2001, 2001, AU)
Download PDF:
Claims:
CLAIMS :

1. A peptide comprising from 2 to 12 amino acid residues, wherein :

- the N-terminus of the peptide is linked to an optionally substituted, fused polycyclic group comprising an aromatic ring;

- at least one amino acid residue has a hydrophobic side chain ;

- at least two amino acid residues have a cation-containing side chain, or at least one amino acid residue has two or more cation-containing side chains, or at least one amino acid residue has one or more side chains comprising two or more cations; and

- the net charge of the peptide is positive at a pH of from about 4 to about 10.

2. The peptide according to claim 1, wherein the peptide comprises at least two amino acid residues having a cation- containing side chain.

3. The peptide according to claim 2, wherein at least one of the amino acid residues having a cation-containing side chain is selected from lysine, arginine and histidine residues.

4. The peptide according to claim 2 or 3, wherein at least one of the amino acid residues having a cation-containing side chain is a lysine residue.

5. The peptide according to any one of claims 2 to 4, wherein the at least two amino acid residues having a cation-containing side chain are each selected independently from lysine, arginine and histidine residues.

6. The peptide according to any one of claims 1 to 5, wherein the at least one amino acid residue having a hydrophobic side chain is selected from valine, isoleucine, leucine, phenylalanine, tyrosine and tryptophan residues.

7. The peptide according to any one of claims 1 to 6, wherein the at least one amino acid residue having a hydrophobic side chain is a phenylalanine residue.

8. The peptide according to any one of claims 1 to 7, wherein the optionally substituted fused polycyclic group comprising an aromatic ring is linked to the N-terminus of the peptide by a linker group that is from 1 to 3 atoms in length.

9. The peptide according to any one of claims 1 to 8, wherein the peptide comprises from 4 to 8 amino acid residues.

10. The peptide according to claim 9, wherein the amino acid residues are selected from valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, glutamine, asparagine, serine, threonine, cysteine, alanine, proline, glycine, and beta-alanine residues, and wherein

- the at least one amino acid residue having a hydrophobic side chain is selected from valine, isoleucine, leucine,

phenylalanine, tyrosine and tryptophan residues; and

- the at least two amino acid residues having a cation- containing side chain are independently selected from lysine, arginine and histidine residues.

11. A compound of Formula (I)

.L.

Ar'

n

salt thereof, wherein

Ar is an optionally substituted fused polycyclic group comprising an aromatic ring;

L is a bond or a linker that is 1, 2 or 3 atoms in length; n is an integer from 2 to 12;

each A is independently selected from the amino acid residues (A' ) to ( G' )

(Α') (Β') (C)

(D") (Ε') (F1) (G') . each R' group in the compound is an independently selected radical, wherein at least one R' group is a hydrophobic moiety, and wherein at least two R' groups are moieties containing a cation or at least one R' group is a moiety containing 2 or more cations; and

the net charge of the compound is positive at a pH of from about 4 to about 10.

12. The compound of Formula (I) according to claim 11, wherein the compound of Formula (I) has the Formula (la)

( l a ) .

13. The compound according to claim 11 or 12, wherein Ar is 9- fluorenyl.

14. The compound according to any one of claims 11 to 13, wherein L is -CH2OC(=0)-.

15. The compound according to any one of claims 11 to 14, wherein Ar-L- has the chemical structure:

16. The compound according to any one of claims 11 to 15, wherein n is 4.

17. The compound according to any one of claims 11 to 16, wherein the at least one R' group that is a hydrophobic moiety is -CH2Ph.

18. The compound according to any one of claims 11 to 17, wherein at least one of the R' groups that is a moiety containing a cation is a moiety containing a primary, secondary or tertiary amine.

19. The compound according to claim 18, wherein the R' group is -CHzCHzCHzCHzNi .

20. The compound according to claim 11 which is selected from Fmoc-FkFk and Fmoc-FFkk and stereoisomers thereof.

21. A hydrogel comprising a peptide or compound according to any one of claims 1 to 20.

22. A hydrogel comprising a peptide or compound according to any one of claims 1 to 20 and an aqueous solution having a pH of from about 4 to about 10.

23. A hydrogel comprising a peptide or compound according to any one of claims 1 to 20 and an aqueous solution having a pH of from about 6 to about 8.

24. A cell culture substrate comprising a peptide or compound according to any one of claims 1 to 20.

25. A cell culture substrate comprising a hydrogel as defined in any one of claims 21 to 23.

26. A 3-dimensional cell culture substrate comprising a cell culture substrate as defined in claim 24 or 25, wherein all three dimensions of the cell culture substrate are greater than about 50 μπι.

27. Use of a peptide or compound according to any one of claims 1 to 20, a hydrogel according to any one of claims 21 to 23, or a cell culture substrate according to any one of claims 24 to 26, for culturing a cell.

28. A method of making a cell culture substrate comprising mixing a peptide or compound according to any one of claims 1 to 20 and an aqueous solution.

29. The method according to claim 28, wherein the aqueous solution has a pH of from about 6 to about 8.

30. The method according to claim 28, wherein the aqueous solution has a pH of from about 7.2 to about 7.5.

31. The method according to any one of claims 28 to 30, further comprising mixing a peptide or compound according to any one of claims 1 to 20 and/or the aqueous solution with one or more cells.

32. A method of culturing a cell, comprising forming a cell culture substrate by the method of any one of claims 28 to 31, and incubating cells in or on the cell culture substrate under

conditions that promote viability, growth or proliferation of the cells .

33. A method of recovering one or more cells from a cell culture substrate, comprising contacting the cell culture substrate with a digestion enzyme to cause degelation, wherein the cell culture substrate comprises a peptide or compound according to any one of claims 1 to 20, and wherein the peptide or compound comprises one or more natural amino acid residues.

Description:
CELL CULTURE SUBSTRATES

The present application claims priority from Australian provisional application no. 2017903293, the entirety of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a class of peptides and their use in cell culture applications.

BACKGROUND

Cell culture is an important scientific technique primarily used to maintain or grow cells in an artificial environment. Cell culture substrates help provide the conditions necessary for the cells to adhere to surfaces, and survive and/or proliferate in such artificial environments.

Examples of cell culture substrates include both synthetic substrates (e.g. polylysine) and naturally derived substrates (e.g. Matrigel ® ) . Polylysine is commonly used in cell culture as it enhances cell adherence to cultureware. For example, poly-D-lysine is commonly used as a coating agent in the production of poly-D-lysine coated petri dishes for growing cells. Owing to manufacturing constraints, the length of the polymeric chains in polylysine can vary from batch-to-batch, which can lead to inconsistencies in culture conditions and thus inconsistencies in experimental results.

Matrigel ® is a solubilized basement membrane preparation extracted from the Engelbreth-Holm- Swarm (EHS) mouse sarcoma. The chief components of Matrigel ® are structural proteins such as laminin, entactin, collagen and heparan sulfate proteoglycans. Also present are growth factors like TGF-beta and EGF that prevent differentiation and promote proliferation of many cell types.

Matrigel ® contains other proteins in small amounts and, as a naturally derived product, its exact composition can vary from batch to batch. For this reason, Matrigel ® may not be appropriate for experiments that require precise knowledge of all proteins and their concentrations in the cell culture substrate.

Some cell types are more difficult to culture. For example, neurons require specific conditions to grow, limiting the cell culture substrates suitable for culturing such neuronal cells.

It would be advantageous if at least preferred embodiments of the present invention were to provide alternative cell culture

substrates. It would also be advantageous if at least preferred embodiments of the present invention were able to provide an alternative cell culture substrate suitable for culturing neuronal cells including neurons. It would also be advantageous if at least preferred embodiments of the present invention were to provide compounds that can be used for preparing cell culture substrates under conditions that are suitable for cell adhesion, survival and/or proliferation.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a peptide comprising from 2 to 12 amino acid residues, wherein:

- the N-terminus of the peptide is linked to an optionally substituted, fused polycyclic group comprising an aromatic ring;

- at least one amino acid residue has a hydrophobic side chain;

- at least two amino acid residues have a cation-containing side chain, or at least one amino acid residue has two or more cation-containing side chains, or at least one amino acid residue has one or more side chains comprising two or more cations; and

- the net charge of the peptide is positive at a pH of from about 4 to about 10. In an embodiment, the peptide comprises at least two amino acid residues having a cation-containing side chain.

In an embodiment, at least one of the amino acid residues having a cation-containing side chain is selected from lysine, arginine and histidine residues.

In an embodiment, at least one of the amino acid residues having a cation-containing side chain is a lysine residue.

In an embodiment, the at least two amino acid residues having a cation-containing side chain are each selected independently from lysine, arginine and histidine residues.

In an embodiment, the at least one amino acid residue having a hydrophobic side chain is selected from valine, isoleucine, leucine, phenylalanine, tyrosine and tryptophan residues.

In an embodiment, the at least one amino acid residue having a hydrophobic side chain is a phenylalanine residue.

In an embodiment, the optionally substituted fused polycyclic group comprising an aromatic ring is linked to the N-terminus of the peptide by a linker group that is from 1 to 3 atoms in length.

In an embodiment, the peptide comprises from 4 to 8 amino acid residues. In an embodiment, the 4 to 8 amino acid residues are selected from valine, isoleucine, leucine, methionine,

phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, glutamine, asparagine, serine, threonine, cysteine, alanine, proline, glycine, and beta-alanine residues, wherein

- the at least one amino acid residue having a hydrophobic side chain is selected from valine, isoleucine, leucine,

phenylalanine, tyrosine and tryptophan residues; and

- the at least two amino acid residues having a cation- containing side chain are independently selected from lysine, arginine and histidine residues. In a second aspect, the present invention provides a compound of Formula (I)

(I)

or a salt thereof, wherein:

Ar is an optionally substituted fused polycyclic group comprising an aromatic ring;

L is a bond or a linker that is 1, 2 or 3 atoms in length; n is an integer from 2 to 12; each A is independently selected from the amino acid residues (A' ) to ( G' )

(Α') (Β') (C)

(D') (Ε') (F-) (Θ') . each R' group in the compound is an independently selected radical, wherein at least one R' group is a hydrophobic moiety, and wherein at least two R' groups are moieties containing a cation or at least one R' group is a moiety containing 2 or more cations; and the net charge of the compound is positive at a pH of from about 4 to about 10.

In an embodiment, the compound of Formula (I) has the Formula (la)

(la) .

In an embodiment, Ar is 9-fluorenyl.

In an embodiment, L is -CH 2 OC(=0)-.

In an embodiment, Ar-L- has the chemical structure:

In an embodiment, n is 4.

In an embodiment, the at least one R' group that is a hydrophobic moiety is -CH 2 Ph.

In an embodiment, at least one of the R' groups that is a moiety containing a cation is a moiety containing a primary, secondary or tertiary amine. In an embodiment, the R' group is - CH 2 CH 2 CH 2 CH 2 NH 3 + .

In an embodiment, the compound is selected from Fmoc-FkFk and Fmoc- FFkk and stereoisomers thereof.

In a third aspect, the present invention provides a hydrogel comprising a peptide according to the first aspect of the present invention or a compound according to the second aspect of the present invention (a peptide according to the first aspect of the present invention or a compound according to the second aspect of the present invention is sometimes referred to below as a "compound of the present invention") . In an embodiment, the hydrogel comprises a compound of the present invention and an aqueous solution having a pH of from about 4 to about 10. In an embodiment, the aqueous solution has a pH of from about 6 to about 8. In a fourth aspect, the present invention provides a cell culture substrate comprising a compound of the present invention. In an embodiment, the cell culture substrate comprises a hydrogel according to the third aspect of the present invention.

In a fifth aspect, the present invention provides a 3-dimensional cell culture substrate comprising a cell culture substrate according to the fourth aspect of the present invention, wherein all three dimensions of the cell culture substrate are greater than about 50 (am.

In a sixth aspect, the present invention provides the use of a compound of the present invention, a hydrogel according to the third aspect of the present invention, or a cell culture substrate according to the fourth or fifth aspect of the present invention, for culturing a cell .

In a seventh aspect, the present invention provides a method of making a cell culture substrate comprising mixing a compound of the present invention and an aqueous solution.

In an embodiment, the aqueous solution has a pH of from about 6 to about 8. In an embodiment, the aqueous solution has a pH of from about 7.2 to about 7.5.

In an embodiment, the method further comprises mixing the compound of the present invention and/or the aqueous solution with one or more cells .

In an eighth aspect, the present invention provides a method of culturing a cell, comprising forming a cell culture substrate by the method of the seventh aspect of the present invention, and

incubating cells in or on the cell culture substrate under conditions that promote viability, growth or proliferation of the cells .

In a ninth aspect, the present invention provides a method of recovering one or more cells from a cell culture substrate, the method comprising contacting the cell culture substrate with a digestion enzyme to cause degelation, wherein the cell culture substrate comprises compound of the present invention, and wherein the compound comprises one or more natural amino acid residues.

BRIEF DESCRIPTION OF THE FIGURES

Preferred embodiments of the present invention are described below, by way of example only, with reference to the accompanying drawings in which:

Figure 1 shows the chemical structure of the two tetrapeptides described in Example 1: Fmoc-FFkk (1) and Fmoc-FkFk (2) .

Figure 2(a) and 2(b) are graphs of modulus (Pa) vs time (s) of time resolved rheology measurements, showing gelation of Fmoc-FFkk (a) and Fmoc-FkFk (b) over a time period of 5000 s, as described in Example 1.

Figure 2(c) and 2(d) are graphs of modulus (Pa) vs time (s) of thixotropy measurements of Fmoc-FFkk (a) and Fmoc-FkFk (b), showing the recovery of the network after application of a large external strain, as described in Example 1.

Figure 3(a) and 3(b) are graphs of modulus (Pa) versus frequency (Hz) for hydrogels of Fmoc-FFkk (a) and Fmoc-FkFk (b), showing frequency independent behaviour, typical of a hydrogel material.

Figure 3(c) and 3(d) are graphs of modulus (Pa) vs strain (%) for hydrogels of Fmoc-FFkk (a) and Fmoc-FkFk (b), showing deformation o the hydrogel network upon application of a large external strain.

Figure 4(a) and 4(b) are atomic force microscopy (AFM) images of a Fmoc-FFkk gel (a) and Fmoc-FkFk gel (b) (hydrogels were spread coated onto freshly cleaved mica substrates at 0.5% (w/v), as described in Example 1) . Scale bars = (a) 500 nm and (b) 1 μτη.

Figure 5(a) is a circular dichroism spectrum (CD spectrum; graph of ellipticity (mdeg) vs wavelength (nm)) of an Fmoc-FFkk gel (squares) and Fmoc-FkFk gel (triangles), as described in Example 1, indicative of their different secondary structures.

Figure 5(b) is an ATR-IR spectra (graph of transmission (%) vs wavenumber (crrr 1 )) of an Fmoc-FFkk gel (grey; upper spectra) and Fmoc-FkFk gel (black; lower spectra), as described in Example 1, indicative of their different secondary structures.

Figure 6(a) , 6(b) and 6(c) are a series of AFM images of glass coverslip coated with poly-d-lysine (a), Fmoc-FFkk (b) and Fmoc-FkFk (c), as described in Example 1, clearly showing the differences in surface coverage and morphology. Scale bars = 1 μπι. Figure 7 is a series of images displaying primary neurons observed on coverslips coated with poly-D-lysine (PDL; top), Fmoc-FFkk gel (middle) and Fmoc-FkFk gel (bottom) at times DIV1 to DIV5. DIV1 shows lamellipodia extending from cell body, DIV2 shows extension of lamellipodia, DIV3 shows generation of axons, DIV4 shows generation of dendrites and DIV5 shows the development of the network

structure. Neurons were stained for the structural marker proteins 3-tubulin and ΜΆΡ2, and with a DAPI nuclear stain, as described in Example 1. Labels indicate (a) lamellipodia, (b) axonal elongation and (c) dendrite formation. Scale bars = 10 μπι. Figure 8 (a) -(c) is a series of images of long term neuronal cultures (40 days in vitro) grown on coverslips coated with poly-D-lysine (a), Fmoc-FFkk (b) and Fmoc-FkFk (c) showing intact neuronal networks. Neurons were stained for the structural marker proteins 3-tubulin and ΜΆΡ2, and with a DAPI nuclear stain, as described in Example 1. Scale bars = 100 μπι.

Figure 8(d) is a graph showing the cell viability (%; y-axis) over time (10, 20, 30 and 40 days in vitro; x-axis) of primary neurons seeded on the peptide nanofibers Fmoc-FFkk (black) and Fmoc-FkFk (grey) . Cell viability determined using an Alamar Blue colorimetric assay with PDL used as the positive control (100% viability), as described in Example 1.

Figure 9 is a series of images taken after 10 days in vitro (DIV10; top) and 14 days in vitro (DIV14; bottom) showing the transfection of primary neurons (indicated by arrows in images) cultured on coverslips coated with PDL (left), Fmoc-FFkk (centre) and Fmoc-FkFk (right), as described in Example 1. Neurons were transfected at DIV7, and stained after fixing with MAP2 and DAPI . Scale bars = 50 Figure 10(a) is a series of images taken after 10 days in vitro (DIV10; top) and 30 days in vitro (DIV30; bottom) showing the synaptic development of neurons cultured on coverslips coated with PDL (left), Fmoc-FFkk (centre) and Fmoc-FkFk (right), as described in Example 1. Neurons were stained with the synaptic markers synaptophysin and PSD-95, and the nuclear stain DAPI. Scale bars = 50 μιη.

Figure 10(b) is a series of images taken after 20 days in vitro (DIV20; left) and 30 days in vitro (DIV30; right) showing the density of synapses along dendrites cultured on coverslips coated with PDL (top), Fmoc-FFkk (middle) and Fmoc-FkFk (bottom), as described in Example 1. Neurons were stained with the synaptic markers synaptophysin and PSD-95, and the nuclear stain DAPI. Arrows denote colocalisation of pre- and post-synaptic markers . Scale bars = 10 μπι.

Figure 10 (c) is a graph showing the quantification of synaptic density (synapses/pm; y-axis) over time (20, 25 and 30 days in vitro; x-axis) for neurons cultured on PDL (grey), Fmoc-FFkk (black) or Fmoc-FkFk (white), as described in Example 1.

Figure 11 (a) - (d) is a series of images showing primary neurons cultured within 3D hydrogels of (a, b) Fmoc-FFkk and (c, d) Fmoc- FkFk as described in Example 1. Primary neurons were fixed at DIV5 and stained for 3-tubulin, MAP2 and nucleus. Representative overlays for all channels are shown. Scale bars = 100 μπι. Figure 11(e) shows an image of neurons cultured for 5 days in vitro (DIV5) within a 3D hydrogel of Fmoc-FFkk. Neurons were stained for 3-tubulin, MAP2 and with a DAPI nuclear stain, as described in Example 1. Scale bar = 50 μτη. Figure 12 is a schematic diagram depicting the preparation of poly- D-lysine coverslips (top), a "2.5D" culture system (middle) and a 3D culture system for neuronal cultures (bottom) as described in

Example 3.

Figure 13 shows a photographic image of a 3D culture system before (left) and after (right) treatment with trypsin as described in

Example 3 for D-lysine containing peptide (top, no degradation) and L-lysine containing peptide (bottom, complete degradation) . Images show treatment with trypsin causing the degradation of the L-lysine containing peptide, causing the hydrogel to liquefy and sink to the bottom of the vial.

Figure 14(a) is a series of still images taken from videos which show the electrical activity of primary neurons cultured on PDL (top), Fmoc-FFkk (middle) and Fmoc-FkFk (bottom), demonstrating synchronized firing at DIV15 (0 sec (left), 2 sec (centre) and 4 sec (right)) . Primary neurons were cultured on PDL, Fmoc-FFkk or Fmoc- FkFk and transfected with GCaMP6s at DIV11 before imaging was undertaken four days later, as described in Example 1, section 1.4. Scale bar = 100 μπι.

Figure 14(b) and (c) are graphs depicting the quantification of firing events (y-axis, arbitrary units, scale bar = 10 counts in (b) and 20 counts in (c) ) over time (x-axis, scale bar = 20 ms in (b) and 10 ms in (c)) from Figure 14(a), as described in Example 1, section 1.4. These graphs show (b) random firing at DIV8 and (c) synchronous firing at DIV15.

Figure 15(a) and 15(b) are graphs of viscosity (cP) vs shear rate

(1/s) of (a) Fmoc-FFkk and (b) Fmoc-FkFk dissolved in water at 0.5% (w/v) as described in Example 1. Values above 1 cP indicate the presence of self-assembly, with Fmoc-FkFk showing strong shear rate dependent behaviour. Figure 16(a) and 16(b) are graphs of I(q) (a.u.) vs q(A _1 ) showing the Small angle neutron scattering (SANS) patterns and associated fits obtained from 1% (w/v) solutions of Fmoc-FFkk (a) and Fmoc-FkFk (b) dissolved in D 2 0 as described in Example 1.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In a first aspect, the present invention provides a peptide comprising from 2 to 12 amino acid residues, wherein:

- the N-terminus of the peptide is linked to an optionally substituted, fused polycyclic group comprising an aromatic ring;

- at least one amino acid residue has a hydrophobic side chain;

- at least two amino acid residues have a cation-containing side chain, or at least one amino acid residue has two or more cation-containing side chains, or at least one amino acid residue has one or more side chains comprising two or more cations; and

- the net charge of the peptide is positive at a pH of from about 4 to about 10.

Peptides in accordance with the first aspect may be used to make hydrogels when combined with aqueous solutions at biologically relevant pHs. Advantageously, these hydrogels are compatible with a wide variety of biological systems and are suitable for use as cell culture substrates. Furthermore, at least preferred peptides of the first aspect are capable of forming a hydrogel that is capable of supporting the growth of primary hippocampal neurons, which are difficult to culture on many other substrates.

Without wishing to be bound by theory, the inventors believe that it is the unique combination of the group linked to the N-terminus of the peptide, the hydrophobic side chain and the cation-containing side chains that is responsible for promoting the assembly of a hydrogel upon mixing with aqueous solutions. Advantageously, hydrogels formed using at least preferred peptides of the invention allow the long-term culturing of neurons in culture dishes. In contrast to existing substrates, these hydrogels are water soluble and do not require strongly basic or strongly acidic conditions and/or the use of additional agents such as traditional pH adjusters or buffers for solubilization. The use of pH adjusters and buffers has traditionally been problematic with prior art cell culture substrates, since these are generally toxic to cultured cells and lengthy washing procedures are required to remove them from the substrate prior to the addition of cells. In addition, the removal of pH adjusters and buffers from prior art media also lead to extra time and labour in preparing cell culture substrates. In the present invention, these additional steps (costing both time and labour) may be avoided or reduced by using the peptides of the present invention.

Some existing methods for culturing cells involve using poly-D- lysine coated plastic cultureware. It is technically challenging to control the length of D-lysine polymers which causes batch-to-batch variation. The variation of polymer length leads to differences in product stability, and can create inconsistencies in culture conditions and thus differences in experimental results. In addition, neuronal cell death is common when removing neuronal cells from the poly-D-lysine coated plastic cultureware using chemicals or enzymes .

The synthesis of the peptides of the invention can be controlled, which can give rise to more consistent properties compared to the commercial manufacture of poly-D-lysine or Matrigel ® .

The ability of the peptides of the invention to form hydrogels when added to aqueous solutions at biologically relevant pHs, allows cells to be introduced before or during gel formation, thereby allowing the gel to form while the cells are dispersed, giving the ability to form three dimensional gels with cells dispersed therein. This is particularly advantageous for sensitive cells (such as neuronal cells including neurons), which are incompatible with the pHs required for gel formation using conventional cell culture substrates . Number of amino acid residues

The peptide of the present invention comprises from 2 to 12 amino acid residues (i.e. the peptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues) . In some embodiments, the peptide comprises from 2 to 8 amino acid residues. In some embodiments, the peptide comprises from 4 to 8 amino acid residues. In some

embodiments, the peptide comprises from 4 to 6 amino acid residues. In some embodiments, the peptide comprises 4 amino acid residues (i.e. is a tetrapeptide ) .

Amino acid residues having a hydrophobic side chain

The peptide of the present invention comprises at least one amino acid residue that has a hydrophobic side chain. A hydrophobic side chain is a side chain that repels water. A hydrophobic side chain comprises one or more hydrophobic moieties (i.e. a moiety that repels water) . Hydrophobic moieties tend to be nonpolar (e.g.

contain functional groups that are either uncharged or have little charge) . Examples of hydrophobic moieties include straight or branched Ci-io-alkyl, C 3 - 6 -cycloalkyl , aryl and heteroaryl groups, wherein the Ci-io-alkyl, C 3 - 6 -cycloalkyl , aryl and heteroaryl groups may be optionally substituted with one, two or three Ci-6alkyl, C3-6- cycloalkyl, and/or halo (e.g. F, CI or Br) groups. Examples of hydrophobic moieties include -C 6 H 5 , -C 6 H 4 Me, -C 6 H 3 Me 2 , -C 6 H 2 Me 3 , - CeH4Et, —CeH 3 Et2, C6¾Et 3 , —C6H4F, C6¾F2, — CeH2F 3 , —C6H4CI, —CeH 3 Cl2,

C 6 H 2 C1 3 etc. In some embodiments the hydrophobic side chain comprises one or more of these hydrophobic moieties (for example a straight or branched Ci-10-alkyl covalently bonded to an aryl or heteroaryl group) . In some embodiments the hydrophobic side chain comprises an aryl or heteroaryl group. In some embodiments the hydrophobic side chain is or comprises a straight or branched Ci-i 0 alkyl group substituted with an optionally substituted aryl or optionally substituted heteroaryl. For example, in some embodiments, the hydrophobic side chain is or comprises Ci-6alkylphenyl,

especially -CH 2 Ph. As a person skilled in the art will appreciate, there are different methods to determine the hydrophobicity of a compound. An example of one such method is the partition coefficient as described in Hansch et al. [1975, Journal of Medicinal Chemistry, 865-868], where the distribution of a compound between two immiscible phases, with one phase being polar and the other phase non-polar, is used to approximate the compound's hydrophobicity. In some embodiments, the amino acid residue that has a hydrophobic side chain corresponds to an amino acid that has a LogP of at least 2.7 in its Fmoc-protected form, as measured by the method of Hansch et al . [1975, Journal of Medicinal Chemistry, 865-868] . In some embodiments, the amino acid residue has a side chain that has a calculated LogP of greater than 1.2 (for example, calculated using ChemDraw) .

In some embodiments, the hydrophobic side chain is selected from optionally substituted straight or branched Ci-ioalkyl (e.g. straight or branched Ci-ioalkyl substituted with an optionally substituted aryl (e.g. phenyl or haloaryl) or optionally substituted

heteroaryl), optionally substituted straight or branched Ci-i 0 alkoxy, optionally substituted straight or branched C 2 -iothioether (e.g.

optionally substituted -CH 2 CH 2 SCH 3 ) , optionally substituted straight or branched C 2 -ioalkenyl , optionally substituted straight or branched C2-ioalkynyl, optionally substituted aryl (e.g. haloaryl), optionally substituted heteroaryl, optionally substituted C 3 - 6 cycloalkyl and optionally substituted C 3 - 6 cycloalkenyl , wherein the optional substituents are selected from halo (e.g. F, CI or Br), Ci-6alkyl, haloCi- 6 alkyl , C 3 - 6 cycloalkyl , optionally substituted aryl (e.g.

phenyl or -C 6 H 4 OH) and optionally substituted heteroaryl (e.g.

indolyl ) .

Typically, the hydrophobic side chain has a molecular weight of less than about 500 g/mol (e.g. less than about 400 g/mol or less than about 300 g/mol) .

Amino acid residues that have a hydrophobic side chain may be "natural" amino acid residues or "non-natural" amino acid residues that have hydrophobic side chains. Examples of "natural" amino acids that have a hydrophobic side chain include L-valine, L-isoleucine , L-leucine, L-methionine, L-phenylalanine , L-tyrosine and L- tryptophan. L-Phenylalanine is preferred. Examples of "non-natural" amino acids that have a hydrophobic side chain include D-valine, D- isoleucine, D-leucine, D-methionine, D-phenylalanine , D-tyrosine and D-tryptophan . D-Phenylalanine is preferred. Other examples of "non- natural" amino acids that have a hydrophobic side chain include L- norleucine, D-norleucine, L-norvaline and D-norvaline.

Examples of amino acids having a hydrophobic side chain include amino acids (A-l) to (G-l)

wherein each R 1 is hydrophobic and independently selected from optionally substituted straight or branched Ci-i 0 alkyl (e.g. straight or branched Ci-i 0 alkyl substituted with an optionally substituted aryl (e.g. phenyl or haloaryl) or optionally substituted

heteroaryl), optionally substituted straight or branched Ci-i 0 alkoxy, optionally substituted straight or branched C2-iothioether,

optionally substituted straight or branched C2-ioalkenyl, optionally substituted straight or branched C 2 -ioalkynyl, optionally substituted aryl (e.g. haloaryl), optionally substituted heteroaryl, optionally substituted C 3 - 6 cycloalkyl and optionally substituted C 3 - 6 cycloalkenyl , wherein the optional substituents are selected from halo (e.g. F, CI or Br), Ci-6alkyl, haloCi-6alkyl , CVecycloalkyl , optionally substituted aryl and optionally substituted heteroaryl, and wherein, when there is more than one R 1 group, the R 1 groups may be the same or different. In some embodiments, the at least one amino acid residue that has a hydrophobic side chain is an amino acid residue of any of the amino acids (A-l) to (G-l) described above . Amino acid residues having a cationic side chain

The peptide of the present invention comprises at least two amino acid residues that have a cation-containing side chain, or at least one amino acid residue having two or more cation-containing side chains, or at least one amino acid residue having one or more side chains comprising two or more cations. When the peptide comprises two or more amino acid residues that have a cation-containing side chain, the amino acid residues that have a cation-containing side chain may be the same or different amino acid residues. Similarly, when the peptide comprises two or more amino acid residues that have two or more cation-containing side chains, or two or more amino acid residues that have one or more side chains comprising two or more cations, the amino acid residues may be the same or different amino acid residues .

As used herein, a "cation" is a species that is cationic (i.e.

positively charged) at a pH of less than about 10. For example, the cation exists as a cationic species when the peptide is in an aqueous solution having a pH of less than about 10 (e.g. less than about 9, less than about 8) . As a person skilled in the art will appreciate, the formation of a cation is typically an equilibrium process, with the ratio of cationic to neutral species being dependant on pH. As used herein, "cation" may refer to a species that is either wholly or partially cationic. For example, histidine in an aqueous solution near pH 7 exists as approximately 25% protonated (i.e. cationic) and 75% neutral. This would be considered "cationic" in the context of this specification.

The cation-containing side chain preferably contains a cation at biologically relevant pHs, e.g. a pH of 7.1 to 7.6 or 7.2 to 7.5, ideally 7.3 to 7.4. As a person skilled in the art will appreciate, moieties containing groups such as amines (which form ammonium ions at biological pHs), guanidine (e.g -NHC (=NH) NH 2 ) and imidazole (e.g. -C 3 H 3 N 2 ), may form a cation at pHs of less than about 10.

Accordingly, the cation-containing side chain may, in some

embodiments, be a side chain containing an amine. Examples of cation-containing side chains include Ci-i 0 -aminoalkyl (e.g. -(CH 2 )i- 10 NH 2 , - ( CH 2 ) m CH (NH 2 ) C n alkyl (where m may be 0 or an integer from 1 to 8, n is an integer from 1 to 9 and n + m = 1 to 9) , -

( CH 2 ) 0 NHC p alkyl (where o and p are each an integer from 1 to 9 and o + p = 2 to 10), - (CH 2 ) q N (C r alkyl) (C s alkyl) (where q, r and s are each an integer from 1 to 8 and q + r + s = 3 to 10) and imines (e.g. -

(CH 2 ) 0-9C (H) =N-H, - (CH 2 ) m C (H) =N- (C n alkyl) (where m may be 0 or an integer from 1 to 8, n is an integer from 1 to 9 and n + m = 1 to 9), - ( CH 2 ) 0 -C ( C p alkyl ) =N-H (where o may be 0 or an integer from 1 to 8, p is an integer from 1 to 9 and o + p = 1 to 9), -(CH 2 ) q -

C ( C r alkyl ) =N- ( C s alkyl ) (where q may be 0 or an integer from 1 to 7, r and s are each an integer from 1 to 7 and q + r + s = 2 to 9), -

(CH 2 ) t -N=C (H) (C u alkyl) (where t and u are each an integer from 1 to 8 and t + u = 2 to 9), - (CH 2 ) V -N=C (C„alkyl) (C x alkyl) (where v, w and x are each an integer from 1 to 7 and v + w + x = 3 to 9 ) ) . In some embodiments, the cation-containing side chain is a side chain selected from -(CH 2 ) 6 NH 2 , -(CH 2 ) 5 NH 2 , -(CH 2 ) 4 NH 2 , -(CH 2 ) 3 NH 2 , -(CH 2 ) 2 NH 2 and -CH 2 NH 2 , especially -(CH 2 ) 3 NH 2 , -(CH 2 ) 4 NH 2 and -(CH 2 ) 5 NH 2 , more especially -(CH 2 ) 4 NH 2 .

Quaternary ammonium groups (-N + R 4 , where each R is independently alkyl or aryl) are cationic independent of pH. Accordingly, a moiety comprising a quaternary ammonium group is a moiety comprising a cation. In some embodiments the cation-containing side chain is a straight or branched Ci-i 0 alkyl substituted with a quaternary ammonium group (e.g. Ci-ioalkylN + (Ci-6alkyl) (Ci-6alkyl) (Ci-6alkyl) ) .

Amino acid residues that have a cation-containing side chain may be "natural" amino acid residues or "non-natural" amino acid residues that have cation-containing side chains. Examples of "natural" amino acids that have a cation-containing side chain (at biologically relevant pHs) include L-lysine, L-arginine and L-histidine.

Preferred are L-lysine and L-arginine. L-Lysine is particularly preferred .

Examples of "non-natural" amino acids that have a cationic side chain include D-lysine, D-arginine and D-histidine. Preferred are D- lysine and D-arginine. D-Lysine is particularly preferred. Other examples of "non-natural" amino acids that have a cationic side chain include L-ornithine and D-ornithine.

Without wishing to be bound by theory, the inventors believe that one of the cationic groups acts to negate or cancel out the effect of the free carboxylic acid of the peptide chain. The second and subsequent cationic groups (when subsequent cationic groups are present) are beneficial for promoting the self-assembly of hydrogels and/or creating an environment in cell culture substrates that is beneficial for the adhesion, survival and/or proliferation of cells. In some embodiments, the cation-containing side chain is selected from optionally substituted straight or branched Ci-i 0 aminoalkyl (e.g. - (CH 2 ) 1 - 10 NH 2 , - (CH 2 )mCH (NH 2 ) Cnalkyl (where m may be 0 or an integer from 1 to 8, n is an integer from 1 to 9 and n + m = 1 to 9), - ( CH 2 ) 0 NHC p alkyl (where o and p are each an integer from 1 to 9 and o + p = 2 to 10), - ( CH 2 ) q N ( C r alkyl ) (C s alkyl) (where q, r and s are each an integer from 1 to 8 and q + r + s = 3 to l0) and imines (e.g. - (CH 2 ) 0-9C (H) =N-H, - (CH 2 ) m C (H) =N- (C n alkyl) (where m may be 0 or an integer from 1 to 8, n is an integer from 1 to 9 and n + m = 1 to 9), - ( CH 2 ) 0 -C ( C p alkyl ) =N-H (where o may be 0 or an integer from 1 to 8, p is an integer from 1 to 9 and o + p = 1 to 9), -(CH 2 ) q -

C ( C r alkyl ) =N- ( C s alkyl ) (where q may be 0 or an integer from 1 to 7, r and s are each an integer from 1 to 7 and q + r + s = 2 to 9), - (CH 2 ) t -N=C (H) (C u alkyl) (where t and u are each an integer from 1 to 8 and t + u = 2 to 9), - (CH 2 ) V -N=C (C„alkyl) (C x alkyl) (where v, w and x are each an integer from 1 to 7 and v + w + x = 3 to 9) .

In some embodiments, the cation-containing side chain is straight or branched Ci-ioalkyl substituted with one or more groups selected from -NH 2 , -NH ( optionally substituted Ci- 6 alkyl), -N ( optionally

substituted Ci- 6 alkyl) (optionally substituted Ci- 6 alkyl). In some embodiments, the Ci-i 0 alkyl group is substituted with 1, 2, 3, 4, 5 or 6 groups selected from -NH 2 , -NH ( optionally substituted Ci- 6 alkyl), -N ( optionally substituted Ci- 6 alkyl) (optionally substituted Ci-6alkyl) . The Ci-ioalkyl substituted with 1, 2, 3, 4, 5 or 6 groups selected from -NH 2 , -NH ( optionally substituted Ci- 6 alkyl), - N (optionally substituted Ci- 6 alkyl) (optionally substituted Ci- 6 alkyl), may optionally be further substituted with, for example, 1, 2, 3, 4, 5 or 6 substituents . The optional further substituents may, for example, be independently selected from halo, haloCi- 6 alkyl and Ci-

6 alkoxy .

In some embodiments the cation-containing side chain is a straight or branched Ci-ioalkyl substituted with a quaternary ammonium group (e.g. -N + (alkyl) 4 ) , guanidine group (e.g. -NHC (=NH) NH 2 ) or an imidazole group (e.g. -C 3 H 3 N 2 ), wherein the Ci-ioalkyl may optionally be further substituted.

Typically, the cation-containing side chain has a molecular weight of less than about 500 g/mol (e.g. less than about 400 g/mol or les than about 300 g/mol) .

Examples of amino acids having a cationic side chain include amino acids (A-2) to (G-2)

(D-2) (E-2) (F-2) (G-2) wherein R 2 is cationic and selected from optionally substituted straight or branched Ci-ioaminoalkyl (e.g. - (CH2) 1-10NH2, - ( CH2 ) mCH (NH2 ) C n alkyl (where m may be 0 or an integer from 1 to 8, n is an integer from 1 to 9 and n + m = 1 to 9), - ( CH 2 ) 0 NHC p alkyl (where o and p are each an integer from 1 to 9 and o + p = 2 to 10) , - (CH 2 ) q N (C r alkyl) (C s alkyl) (where q, r and s are each an integer from 1 to 8 and q + r + s = 3 to 10 ) ) , imines (e.g. - (CH 2 ) 0-9C (H) =N-H, - (CH 2 ) mC (H) =N- (C n alkyl) (where m may be 0 or an integer from 1 to 8, n is an integer from 1 to 9 and n + m = 1 to 9), - ( CH 2 ) 0 -C ( C p alkyl ) =N-H (where o may be 0 or an integer from 1 to 8, p is an integer from 1 to 9 and o + p = 1 to 9), - ( CH 2 ) q -C ( C r alkyl ) =N- ( C s alkyl ) (where q may be 0 or an integer from 1 to 7, r and s are each an integer from 1 to 7 and q + r + s = 2 to 9), - (CH 2 ) t -N=C (H) (C u alkyl) (where t and u are each an integer from 1 to 8 and t + u = 2 to 9), -(CH 2 ) V - N=C ( C„alkyl ) (C x alkyl) (where v, w and x are each an integer from 1 to 7 and v + w + x = 3 to 9) ) and straight or branched Ci-i 0 alkyl substituted with a quaternary ammonium group (e.g. -N + ( alkyl ) 4 ) , guanidine group (e.g. -NHC (=NH) NH 2 ) or an imidazole group

(e.g. -C 3 H 3 N 2 )) , wherein the straight or branched Ci-i 0 alkyl may optionally be further substituted. In Formulas (D-2)-(G-2), the R 2 groups may be the same or different. In some embodiments, the amino acid residue that has a cation-containing side chain is an amino acid residue of any of the amino acids (A-2) to (G-2) described above. In some embodiments, R 2 is selected from -(CH 2 ) 6 NH 2 , -

(CH 2 )5NH 2 , -(CH 2 ) 4 NH 2 , -(CH 2 ) 3 NH 2 , -(CH 2 ) 2 NH 2 and -CH 2 NH 2 , especially - (CH 2 ) 3 NH 2 , -(CH 2 ) 4 NH 2 and -(CH 2 ) 5 NH 2 , more especially -(CH 2 ) 4 NH 2 .

"Additional" amino acid residues The peptide of the present invention may comprise amino acids in addition to the at least one amino acid residue that has a

hydrophobic side chain, and the amino acid residue or residues that have a cation-containing side chain.

Additional amino acids that may be included in the peptide include "natural" and "non-natural" amino acids.

Examples of other amino acids that may be included in the peptide include L-glutamine, L-asparagine, L-serine, L-threonine, L-cysteine, L-alanine, L-valine, L-proline, L-aspartic acid, L-glutamic acid, glycine, beta-alanine, D-glutamine, D-asparagine, D-serine, D- threonine, D-cysteine, D-alanine, D-valine, D-proline, D-aspartic acid and D-glutamic acid.

In some embodiments, less than 70% of the amino acid residues are other than amino acid residues having a hydrophobic side chain and amino acid residues having a cation-containing side chain (e.g. less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10%) . In some embodiments, there are no amino acid residues other than amino acid residues having a hydrophobic side chain and amino acid residues having a cation-containing side chain (i.e. the peptide contains only amino acid residues having a hydrophobic side chain and amino acid residues having a cation- containing side chain) .

In some embodiments, the ratio of charged amino acid residues to hydrophobic amino acid residues is from 1:3 ( charged : hydrophobic ) 5:1, for example, from about 1:2 to about 5:1, about 1:1 to about 5:1, about 1:1 to about 4:1, about 1:1 to about 3:1, about 1:1 to about 2:1 or about 1:1.

Optionally substituted, fused polycyclic group, comprising an aromatic ring

The N-terminus of the peptide is linked to an optionally

substituted, fused polycyclic group comprising an aromatic ring.

The fused polycyclic group comprises 2 or more fused rings, wherein at least one of the rings is aromatic. For example, the fused polycyclic group may contain 2, 3 or 4 rings that are fused together, at least one of the rings being aromatic. In some embodiments, the polycyclic group comprises 2, 3 or 4 fused rings, wherein 2 of the rings are aromatic. In some embodiments, the polycyclic group comprises 3 or 4 fused rings, wherein 3 of the rings are aromatic. In some embodiments, the polycyclic group comprises 4 fused rings, wherein 4 of the rings are aromatic. One or more of the rings may contain a heteroatom (e.g. N, S, 0) and may therefore be heterocyclic or heteroaromatic . Typically each of the rings comprises from 5 to 10 ring atoms (e.g. is a 5-membered ring or a 6-membered ring) .

In some embodiments, the fused polycyclic group comprising an aromatic ring is substituted. In such embodiments, the substituents are typically selected from Ci- 3 alkyl, Ci- 3 haloalkyl , halo (e.g. F, CI, Br), Ci- 3 alkoxy, C 2 -3alkenyl, C 2 -3alkynyl, N0 2 , NMe 2 or SMe . Examples of the optionally substituted, fused polycyclic group comprising an aromatic ring include radicals of fluorene (e.g. 9- fluorenyl), phenothiazine (e.g. 10-phenolthiazinyl ) , indole (e.g. 2- indolyl and 3-indolyl), carbazole (e.g. 9-carbozolyl ) , benzimidazole (e.g. 2-benzimidazolyl and 3-benzimidazolyl ) , benzimidazolone (e.g. 1-benzimidazolonyl ) , benzoindole, phenanthroline, bipyridine, anthracene, pyrene and perylene. One specific example that the inventors have found to be useful is 9-fluorenyl.

The optionally substituted, fused polycyclic group comprising an aromatic ring may be directly bound to the N-terminus of the peptide or may be bound by a "linker".

In some embodiments, the optionally substituted, fused polycyclic group comprising an aromatic ring is linked to the N-terminus of the peptide by a linker that is from 1 to 3 atoms in length. The linker is typically alkyl, but may comprise heteroatoms such as 0, N, S.

Examples of linkers include -CH 2 CH 2 CH 2 -, -C (=0) CH 2 CH 2 - , -CH 2 C ( =0 ) CH 2 - , -CH2OC (=0) -, -C(=0)0CH 2 -, -CH 2 C (=0) 0-, -CH 2 CH 2 0-, -CH 2 OCH 2 -, - C(=0)NHCH 2 -, -CH 2 C ( =0 ) NH- , -CH 2 CH 2 NH- , -CH 2 NHCH 2 - , -CH 2 CH ( CH 3 ) CH 2 - , - CH 2 CH 2 -, -C(=0)CH 2 -, -C(=0)0-, -CH 2 C(=0)0-, wherein one end is bound to the fused polycyclic group and the other end to the N-terminus of the peptide, and wherein the linker is bound to the N-terminus via a carbon atom of the linker. One specific example that the inventors have found to be useful is -CH 2 0C(=0)-.

Charge

The peptides of the first aspect of the present invention have a positive net charge at a pH of from about 4 to about 10. The net positive charge may be determined by a number of different ways. For example, the net charge at a given pH can be calculated by a person skilled in the art by taking into account the theoretical or measured pKas of the constituent amino acids. Alternatively, the net charge can be predicted using computer software. Alternatively, the net charge at a given pH may be derived from the isoelectric point of the peptide. A person skilled in the art will be able to readily assess or predict the net charge of a peptide.

In some embodiments, the peptide comprises from 4 to 8 amino acid residues selected from valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, glutamine, asparagine, serine, threonine, cysteine, alanine, proline, glycine, and beta-alanine residues, wherein

- the at least one amino acid residue having a hydrophobic side chain is selected from valine, isoleucine, leucine,

phenylalanine, tyrosine and tryptophan residues; and

- the at least two amino acid residues having a cation- containing side chain are independently selected from lysine, arginine and histidine residues.

In another aspect, the present invention provides a peptide comprising from 4 to 8 amino acid residues selected from valine, isoleucine, leucine, methionine, phenylalanine, tyrosine,

tryptophan, lysine, arginine, histidine, glutamine, asparagine, serine, threonine, cysteine, alanine, proline, glycine, and beta- alanine residues, wherein the peptide comprises at least one amino acid residue selected from valine, isoleucine, leucine,

phenylalanine, tyrosine and tryptophan residues and at least two amino acid residues independently selected from lysine, arginine and histidine residues, and wherein the N-terminus of the peptide is linked to an optionally substituted, fused polycyclic group comprising an aromatic ring.

For the avoidance of doubt, the amino acid residues referred to above may be, each independently, D- or L-amino acids.

The peptides of the present invention may be prepared by methods that are known to those skilled in the art. Examples include traditional solution phase synthesis, solid phase synthesis, native chemical ligation (an option often used in constructing larger peptides (e.g. more than 10 amino acid residues)) and automated synthesis .

In a second aspect, the present invention provides a compound of Formula (I) -OH

Ar'

(I)

or a salt thereof, wherein:

Ar is an optionally substituted fused polycyclic group comprising an aromatic ring;

L is a bond or a linker that is 1, 2 or 3 atoms in length; n is an integer from 2 to 12;

each A is independently selected from the amino acid residues (A' ) to ( G' )

(Α') (Β') (C)

each R' group in the compound is an independently selected radical, wherein at least one R' group is a hydrophobic moiety, and wherein at least two R' groups are moieties containing a cation or at least one R' group is a moiety containing 2 or more cations; and

the net charge of the compound is positive at a pH of from about 4 to about 10.

In some embodiments, the compound of Formula (I) has the Formula (la)

(la) In some embodiments, Ar comprises 2, 3 or 4 fused rings, wherein at least one of the rings is aromatic.

In some embodiments, Ar is selected from 9-fluorenyl,

phenothiazinyl , indolyl and carbazolyl. In an embodiment, Ar is 9- fluorenyl .

In some embodiments, L is a linker that is three atoms in length.

In some embodiments, L is selected from -CH2CH2CH2-, -C (=0) CH2CH2-, - CH 2 C (=0) CH2-, -CH2OC (=0) -, -C (=0) OCH2-, -CH2C (=0) 0-, -CH2CH2O-, -CH2OCH2-, -C (=0) NHCH2-, -CH 2 C (=0)NH-,-CH2CH 2 NH-,-CH 2 NHCH2-, -CH 2 CH ( CH 3 ) CH 2 - , - CH2CH2-, -C(=0)CH 2 -, -C(=0)0- and -CH 2 C(=0)0-. In some embodiments, L is -CH 2 0C(=0)-.

In some embodiments, Ar-L- has the chemical structure:

In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some embodiments, n is from 4 to 8. In some embodiments, n is from 4 to 6. In some embodiments, n is 4.

In some embodiments, each A in Formula (I) is an amino acid residue selected from the 20 "natural" amino acids or a stereoisomer thereof .

Typically R' is a radical having a molecular weight of less than about 500 g/mol (e.g. less than about 400 g/mol or less than about 300 g/mol) . In some embodiments, each R' is selected from -H, optionally substituted straight or branched Ci-ioalkyl (e.g. straight or branched Ci-ioalkyl substituted with an optionally substituted aryl (e.g. phenyl or haloaryl) or optionally substituted

heteroaryl), optionally substituted straight or branched Ci-i 0 alkoxy, optionally substituted straight or branched C 2 -iothioether (e.g.

optionally substituted -CH2CH2SCH3 ) , optionally substituted straight or branched C 2 -ioalkenyl , optionally substituted straight or branched C 2 -ioalkynyl, optionally substituted aryl (e.g. haloaryl), optionally substituted heteroaryl, optionally substituted C 3 - 6 cycloalkyl , optionally substituted C 3 - 6 cycloalkenyl , optionally substituted straight or branched Ci-i 0 aminoalkyl , optionally substituted imines (e.g. - (CH 2 ) 0-9C (H) =N-H, - (CH 2 ) ra C (H) =N- (C n alkyl) (where m may be 0 or an integer from 1 to 8, n is an integer from 1 to 9 and n + m = 1 to 9), - ( CH2 ) o -C ( C p alkyl ) =N-H (where o may be 0 or an integer from 1 to 8, p is an integer from 1 to 9 and o + p = 1 to 9), -(CH 2 ) q - C ( C r alkyl ) =N- ( C s alkyl ) (where q may be 0 or an integer from 1 to 7, r and s are each an integer from 1 to 7 and q + r + s = 2 to 9), - (CH 2 ) (H) (C u alkyl) (where t and u are each an integer from 1 to 8 and t + u = 2 to 9), - (CH 2 ) V -N=C (C„alkyl) (C x alkyl) (where v, w and x are each an integer from 1 to 7 and v + w + x = 3 to 9)) and straight or branched Ci-i 0 alkyl substituted with a quaternary ammonium, guanidine or imidazole group, wherein the Ci-ioalkyl may optionally be further substituted.

In some embodiments, the at least one R' group that is a hydrophobic moiety is selected from optionally substituted straight or branched Ci-ioalkyl (e.g. straight or branched Ci-i 0 alkyl substituted with an optionally substituted aryl (e.g. phenyl or haloaryl) or optionally substituted heteroaryl), optionally substituted straight or branched Ci-ioalkoxy, optionally substituted straight or branched C 2 -i 0 thioether (e.g. optionally substituted -CH 2 CH 2 SCH3 ) , optionally substituted straight or branched C 2 -i 0 alkenyl , optionally substituted straight or branched C 2 -i 0 alkynyl , optionally substituted aryl (e.g. haloaryl), optionally substituted heteroaryl, optionally substituted C3-

6 cycloalkyl and optionally substituted C 3 - 6 cycloalkenyl , wherein the optional substituents are selected from halo (e.g. F, CI or Br), Ci- 6alkyl, haloCi-6alkyl , C 3 -6cycloalkyl , optionally substituted aryl (e.g. phenyl or -C 6 H 4 OH) and optionally substituted heteroaryl (e.g. indolyl) .

In some embodiments, the at least one R' group that is a hydrophobic moiety is selected from optionally substituted straight or branched Ci-ioalkyl (e.g. straight or branched Ci-i 0 alkyl substituted with an optionally substituted aryl (e.g. phenyl or haloaryl) or optionally substituted heteroaryl), optionally substituted straight or branched Ci-iohaloalkyl or optionally substituted -CH 2 Ph. In some embodiments, at least one of the moieties containing a cation is an optionally substituted straight or branched Ci- l oaminoalkyl . In some embodiments, at least two of the moieties containing a cation are optionally substituted straight or branched Ci-ioaminoalkyl, especially C 2 - 6 aminoalkyl .

In some embodiments, the at least two R' groups that are moieties containing a cation are selected from optionally substituted straight or branched Ci-i 0 aminoalkyl , optionally substituted imines (e.g. - (CH 2 ) o-gC (H) =N-H, - ( CH 2 ) ra C ( H ) =N- ( C n alkyl ) (where m may be 0 or an integer from 1 to 8, n is an integer from 1 to 9 and n + m = 1 to 9), - ( CH 2 ) o-C ( C p alkyl ) =N-H (where o may be 0 or an integer from 1 to 8, p is an integer from 1 to 9 and o + p = 1 to 9), -(CH 2 ) q - C ( C r alkyl ) =N- ( C s alkyl ) (where q may be 0 or an integer from 1 to 7, r and s are each an integer from 1 to 7 and q + r + s = 2 to 9), - (CH 2 ) t _ N=C (H) (C u alkyl) (where t and u are each an integer from 1 to 8 and t + u = 2 to 9), - (CH 2 ) V -N=C (C„alkyl) (C x alkyl) (where v, w and x are each an integer from 1 to 7 and v + w + x = 3 to 9)) and straight or branched Ci-ioalkyl substituted with a quaternary ammonium, guanidine or imidazole group, wherein the Ci-ioalkyl may optionally be further substituted.

In some embodiments, Ar is 9-fluorenyl.

In some embodiments, L is-CH 2 OC (=0) - .

In some embodiments, Ar-L- has the chemical structure:

In some embodiments, n is 4.

In some embodiments, the at least one hydrophobic moiety is -CH 2 Ph.

In some embodiments, at least one of the moieties containing a cation is -CH 2 CH 2 CH 2 CH 2 NH 3 + . In some embodiments, at least two of the moieties containing a cation are -CHzCHzCHzCHzNi .

In some embodiments, the compound of Formula (I) is selected from

Ar- CHzOC ( =0) -KKFF (SEQ ID NO: 1) , Ar-CHzOC (=0) -KKW (SEQ ID NO: 2) ,

Ar- CHzOC ( =0) -YKYK (SEQ ID NO: 3) , Ar-CHzOC (=0) -YYKK (SEQ ID NO: 4) ,

Ar- CHzOC ( =0) -KKYY (SEQ ID NO: 5) , Ar-CHzOC (=0) -WKWK (SEQ ID NO: 6) ,

Ar- CHzOC ( =0) -WWKK (SEQ ID NO: 7) , Ar-CHzOC (=0) -KKWW (SEQ ID NO: 8) ,

Ar- CHzOC ( =0) -IKIK (SEQ ID NO: 9) , Ar-CHzOC (=0) -IIKK (SEQ ID NO: 10) ,

Ar- CHzOC ( =0) -KKII (SEQ ID NO: 11) , Ar-CHzOC (=0) -LKLK (SEQ ID NO: 12)

Ar- CHzOC ( =0) -LLKK (SEQ ID NO: 13) , Ar-CHzOC (=0) -KKLL (SEQ ID NO: 14)

Ar- CHzOC ( =0) -FKFKF (SEQ ID NO 15) , Ar-CHzOC (=0) -FFFKK (SEQ ID NO:

16) , Ar-CHzOC (=0) -KKFFF ( SEQ ID NO: 17), Ar-CHzOC (=0) -KFKFK (SEQ ID NO: 18), Ar-CHzOC (=0) -FFKKK (SEQ ID NO: 19 ) , Ar-CHzOC (=0) -KKKFF (SEQ ID NO: 20) , Ar-CHzOC (=0) -VKVKV (SEQ ID NO: 21 ) , Ar-CH 2 OC ( =0 ) -VWKK (SEQ ID NO: 22) and Ar-CH 2 OC (=0) -KKKW (SEQ ID NO: 23), wherein Ar is selected from 9-fluorenyl, 9-carbozolyl , 10-phenolthiazinyl, indolyl, benzimidazolyl, benzimidazolonyl, naphthyl and anthracenyl, or a stereoisomer thereof (i.e. where one or more of the L-amino acids is replaced by its D-stereoisomer) .

In some embodiments, the compound of Formula (I) is selected from Fmoc-FkFk and Fmoc-FFkk or a stereoisomer thereof.

Fmoc-FkFk (sometimes referred to as Fmoc- L Phe- D Lys- L Phe- D Lys ) has the structure

Fmoc-FFkk (sometimes referred to as Fmoc- L Phe- L Phe- D Lys- D Lys ) has the structure

In some embodiments, the compound of Formula (I) is selected from Fmoc-FFKK ( SEQ ID NO: 24), Fmoc-FKFK (SEQ ID NO: 25) or a

stereoisomer thereof. In some embodiments, the compound of Formula (I) is selected from Cbz-FFKK (SEQ ID NO: 26) and Cbz-FKFK (SEQ ID NO: 27) or a stereoisomer thereof (where Cbz is

(9-carbazolyl) -CH 2 OC (=0) - (i.e. Ar-L- =

( 9-carbazolyl ) - CH 2 OC (=0) - ) ) . In some embodiments, the compound of Formula (I) is selected from Cbz-FFkk and Cbz-FkFk or a stereoisomer thereof.

In some embodiments, the compound of Formula (I) is selected from Fmoc-WKK (SEQ ID NO: 28), Fmoc-VKVK (SEQ ID NO: 29), Fmoc-Wkk, Fmoc-VkVk, Fmoc-KFKFK (SEQ ID NO: 30), Fmoc-FFKKK (SEQ ID NO: 31), Cbz-WKK (SEQ ID NO: 32), Cbz-VKVK (SEQ ID NO: 33), Cbz-Wkk and Cbz-VkVk or a stereoisomer thereof (where Cbz is

(9-carbazolyl) - CH 2 OC (=0) - (i.e. Ar-L- = ( 9-carbazolyl ) - CH 2 OC (=0) -)) .

The compounds of the present invention may be prepared by methods that are known to those skilled in the art. Examples include peptide synthesis from amino acids using traditional solution phase synthesis, solid phase synthesis, native chemical ligation (an option often used in constructing larger peptides (e.g. more than 10 amino acid residues) ) and automated synthesis. Methods are known for preparing various amino acids that are used in the compounds of the present invention. A person skilled in the art will be able to prepare various amino acids from common starting materials and use those to prepare the compounds of Formula (I) and Formula (la) .

Features of the first aspect of the invention described herein may also apply to the second aspect of the invention. Similarly, features of the second aspect of the invention described herein may also apply to the first aspect of the invention.

The peptides and compounds of the present invention typically have good solubility at biologically relevant pHs . For example, at least preferred peptides /compounds are readily soluble in aqueous solutions having a pH of about pH 7.4. In some embodiments, the solubility is in excess of 5 mg/mL (e.g. >10 mg/mL or >15 mg/mL) and may be in excess of about 20 mg/mL. Peptides previously used in cell cultures were typically only solubilised at high pH (e.g. pH>9) .

This represents a distinct advantage of the peptides/compounds of the present invention over these previously used peptides in the context of cell culturing. For example, peptides/compounds of the present invention may have improved compatibility with biological systems, particularly those that are sensitive to high or low pH . This allows the use of the peptides/compounds of the present invention in biological systems that are sensitive to high or low pH.

In a third aspect, the present invention provides a hydrogel comprising a peptide or compound of the present invention. Hydrogels comprising a peptide or compound of the present invention are sometimes referred to herein as "networks" or similar (e.g.

"nanofiber networks", "nanofiber substrates", "peptide nanofibers") . In some embodiments, the hydrogel comprises a peptide or compound of the present invention and an aqueous solution having a pH of from about 1 to about 10 (e.g. from about 2 to about 9, from about 3 to about 9, from about 4 to about 8, from about 5 to about 8, from about 6 to about 8, from about 7 to about 8 or from about 7.1 to about 7.6) . In some embodiments, the aqueous solution has a pH of from about 6 to about 8.

Advantageously, since the peptides according to the first aspect and compounds according to the second aspect can form hydrogels at biologically relevant pHs (e.g. from about 6 to about 8, about 7.1 to about 7.6, about 7.2 to about 7.5, or about 7.3 to about 7.4.), cells may be added before or during gelation and survive owing to the relatively benign conditions of gel formation. Prior art methods to form a gel typically require much lower or much higher pHs in order to form the gel. These pHs are generally not compatible with living cells or tissues. The present invention provides a way of being able to include cells in a media whilst gelation occurs to thereby form a gel with cells distributed within the gel.

In a fourth aspect, the present invention provides a cell culture substrate comprising a peptide or compound of the present invention or a hydrogel according to the third aspect.

In a fifth aspect, the present invention provides a 3-dimensional cell culture substrate comprising a cell culture substrate according to the fourth aspect, wherein all three dimensions of the cell culture substrate are greater than about 50 μπι (e.g. more than about 100 μπι, 200 μπι, 500 μπι, 1 mm, 2 mm, 3mm, 5 mm, 10 mm, 15 mm or 20 mm) . Such a 3-dimensional cell culture substrate may also sometimes be referred to as a medium, matrix or scaffold (or 3D medium, 3D matrix or 3D scaffold) .

Many conditions have been developed that allow for the creation of a controlled, artificial and in vitro environment in which cells may adhere, survive and/or proliferate. Conditions (e.g. nutrient formulations, pH and osmolality of cell culture media) can be varied depending on parameters such as cell type, cell density and culture system employed.

The inventors have found (and demonstrated) that hydrogels

comprising at least preferred peptides or compounds in accordance with the invention can support the proliferation of neuronal cells including neurons for up to 40 days in vitro. In a sixth aspect, the present invention provides the use of a peptide or compound of the present invention for culturing a cell.

In a seventh aspect, the present invention provides a method of making a cell culture substrate comprising mixing a peptide or compound of the present invention and an aqueous solution. The aqueous solution may be, or comprise, a conventional cell culture medium used in prior art cell culturing systems. The aqueous solution may be added to the peptide/compound or the

peptide/compound may be added to the aqueous solution. The pH of the aqueous solution may be between about 1 and about 10. Preferably, the pH is from about 6 to about 8. For more sensitive cells, a pH of from about 7.1 to about 7.6 (e.g. from about 7.2 to about 7.5, about 7.3 to about 7.4) may be desired.

In some embodiments, the peptide/compound forms a hydrogel. In some embodiments, a salt or other gelation agent (e.g. divalent metal salt, or pH switching agent (such as a weak acid or base, depending on the peptide to be used) ) may be included in the aqueous solution to initiate or promote the formation of a hydrogel.

In some embodiments, the cells that are to adhere, survive and/or proliferate in or on the substrate are mixed with the aqueous solution before the gelation occurs. The cells may be added to the aqueous solution before the aqueous solution is mixed with the peptide or compound of the present invention. In some embodiments, the cells that are to adhere, survive and/or proliferate in or on the substrate are mixed with the aqueous solution during the gelation. In some embodiments, the cells that are to adhere, survive and/or proliferate in or on the substrate are added to the hydrogel after the gelation occurs .

In an eighth aspect, the present invention provides a method of culturing a cell, comprising forming a cell culture substrate by the method of the seventh aspect of the present invention, and

incubating cells in or on the cell culture substrate under

conditions that promote viability, growth or proliferation of the cells .

In a ninth aspect, the present invention provides a method of recovering one or more cells from a cell culture substrate, comprising contacting the cell culture substrate with a digestion enzyme to cause degelation, wherein the cell culture substrate comprises a peptide or compound according to the present invention, and wherein the peptide or compound comprises one or more natural amino acid residues. As a person skilled in the art will appreciate, the digestion enzyme should be a digestion enzyme capable of digesting the one or more natural amino acids.

Definitions

As used herein, "peptide" refers to compound comprising a chain of two or more amino acid residues (sometimes referred to as amino acid monomers or simply amino acids) linked by peptide bonds (amide bonds ) .

Amino acids are organic compounds containing both an amine group and a carboxylic acid group. Amino acids also typically have a side chain (i.e. a moiety bonded to a carbon atom between the amine and the carboxylic acid) . As used herein, "amino acid" may refer to any amino acid (i.e. an organic compound containing both an amine group and a carboxylic acid group) and typically refers to an organic compound containing both an amine and a carboxylic acid and having a molecular weight of from about 70 to about 700 g/mol (e.g. from about 70 to about 400 g/mol or from about 70 to about 200 g/mol) .

The peptides of the present invention typically comprise -amino acid residues. In a-amino acids, the amine group is bonded directly to the carbon atom alpha to the carboxyl group (this carbon atom is sometimes referred to as the a-carbon) . In a-amino acids, the side chain, if present, is also bonded directly to the a-carbon.

The peptides of the present invention may also comprise β-amino acid residues. β-Amino acids are amino acids in which the amine group is covalently bonded to a carbon atom which is beta to the carboxyl group (β-carbon) . The side chain, if present, may be covalently bonded to either the α-carbon or the β-carbon.

Other extended amino acids (e.g. gamma- (γ-) or delta- (δ-) amino acids) are also known and the peptide of the present invention may also comprise amino acid residues of such amino acids. Amino acids typically contain a chiral centre (asymmetric centre) and may therefore give rise to stereoisomers. The peptides of the present invention may contain any combination of stereoisomers.

In -amino acids, the two enantiomers are commonly known as D- or L- amino acids . "Natural" amino acids are typically L-amino acids (glycine is a notable exception as it does not contain a

stereocentre) . The peptides of the present invention may comprise D- amino acid residues, L-amino acid residues, amino acid residues that are neither D- nor L-, or a mixture thereof.

There is a group of amino acids that are commonly referred to as the "natural" amino acids (also sometimes referred to as the "standard" or "canonical" amino acids) . These "natural" amino acids are:

Arginine - Arg - R

Lysine - Lys - K

Aspartic acid - Asp - D

Glutamic acid - Glu - E

Glutamine - Gin - Q

Asparagine - Asn - N

Histidine - His - H

Serine - Ser - S

Threonine - Thr - T

Tyrosine - Tyr - Y

Cysteine - Cys - C

Tryptophan - Trp - W

Alanine - Ala - A

Isoleucine - lie - I

Leucine - Leu - L

Methionine - Met - M • Phenylalanine - Phe - F Valine - Val - V

• Proline - Pro - P

Glycine - Gly - G

These 20 amino acids are encoded directly by the codons of the universal genetic code. Amino acids other than these 20 amino acids are commonly referred to as "non-standard" or "non-canonical" amino acids. These amino acids are typically non-proteinogenic (i.e. they cannot be incorporated into proteins during translation) . Two known non-standard proteinogenic amino acids are selenocysteine and pyrrolysine .

Amino acids other than the 20 "natural" amino acids listed above are commonly referred to as "non-natural" amino acids, regardless of whether or not they are present in nature. "Non-natural" amino acids are typically not naturally encoded or found in the genetic code of any organisms. "Non-natural" amino acids include β-, γ- and δ- amino acids; the D-enantiomers of the 20 "natural" amino acids referred to above; and a-amino acids having side chains that are not found in nature. Although examples exist of "non-natural" amino acids that occur in nature (e.g. γ-aminobutyric acid (GABA) and β-alanine), "non-natural" amino acids are usually synthetic.

When using the 1 letter abbreviation for the "natural" amino acids, it is common to use the upper case to denote the L-enantiomers and lower case to denote the D-enantiomers (e.g. "F" denotes L- phenylalanine and "f" denotes D-phenylalanine ) . In this document, if the stereochemistry is not specified, it is intended that this be a reference to either or both enantiomers (i.e. the L-enantiomer and/or the D-enantiomer ) unless otherwise specified, or where the context requires otherwise due to express language or necessary implication.

When two or more amino acids combine to form a peptide, H2O is removed (i.e. condensation/dehydration reaction), and what remains of each amino acid is an amino-acid residue. -Amino-acid residues are therefore structures that lack a hydrogen atom of the amino group (e.g. -NH-CHR-COOH) , or the hydroxyl moiety of the carboxyl group (e.g. NH 2 -CHR-CO- ) , or both (e.g. -NH-CHR-CO- ) . All units of a peptide chain are therefore amino-acid residues. In practice, the term "amino acid" is sometimes used to describe an "amino acid residue".

The residue in a peptide that has an amino group that is not acylated by another amino-acid residue is called the N-terminal (or N-terminus) . The residue that has a carboxyl group that does not acylate another amino-acid residue is called the C-terminal (or C- terminus ) .

Unless otherwise herein defined, the following terms will be understood to have the general meanings which follow. The terms referred to below have the general meanings which follow when the term is used alone and when the term is used in combination with other terms, unless otherwise indicated. Hence, for example, the definition of "alkyl" applies to "alkyl" as well as the "alkyl" portions of "arylCi-6alkyl", "heteroarylCi-6alkyl" etc.

The term "alkyl" refers to a straight chain or branched chain saturated hydrocarbyl group. Preferred are Ci-6alkyl and Ci-4alkyl groups. The term "C x - y alkyl" refers to an alkyl group having x to y carbon atoms, For example, Ci- 6 alkyl refers to an alkyl group having 1 to 6 carbon atoms. Examples of Ci-6alkyl include methyl (Me), ethyl (Et), propyl (Pr), isopropyl (i-Pr), butyl (Bu), isobutyl (i-Bu), sec-butyl (s-Bu), tert-butyl (t-Bu), pentyl, neopentyl, hexyl and the like. Unless the context requires otherwise, the term "alkyl" also encompasses alkyl groups containing one less hydrogen atom such that the group is attached via two positions, i.e. divalent.

The term "alkenyl" refers to a straight chain or branched chain hydrocarbyl group having at least one double bond of either E- or Z- stereochemistry where applicable. Preferred are C 2 - 6 alkenyl and C 2 - 4 alkenyl groups. The term "C x - y alkenyl" refers to an alkenyl group having x to y carbon atoms. Examples of C 2 - 6 alkenyl include vinyl, 1- propenyl, 1- and 2-butenyl and 2-methyl-2-propenyl . Unless the context requires otherwise, the term "alkenyl" also encompasses alkenyl groups containing one less hydrogen atom such that the group is attached via two positions, i.e. divalent.

The term "alkynyl" refers to a straight chain or branched chain hydrocarbyl group having at least one triple bond. Preferred are C 2 - 6 alkynyl and C 2 - 4 alkynyl groups. The term "C x - y alkynyl" refers to an alkynyl group having x to y carbon atoms. Examples of C2-6alkynyl include ethynyl, 1-propynyl, 1- and 2-butynyl, 2-pentynyl, 3- pentynyl, 4-pentynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl and the like. Unless the context indicates otherwise, the term "alkynyl" also encompasses alkynyl groups containing one less hydrogen atom such that the group is attached via two positions, i.e. divalent.

The term "halo" refers to a halogen, such as F, CI or Br. The term "haloalkyl" refers to an alkyl substituted with one or more halo groups .

The term "alkoxy" refers to an alkyl group as defined above covalently bound via an 0 linkage, such as methoxy, ethoxy, propoxy, isoproxy, butoxy, tert-butoxy and pentoxy. Preferred are Ci- 6 alkoxy, Ci- 4 alkoxy and Ci- 3 alkoxy groups.

The term "carboxylate" or "carboxyl" refers to the group -COO ~ or -COOH.

The term "Ci-i 0 aminoalkyl" refers to a Ci-i 0 alkyl group (e.g. a Ci- 6alkyl group) substituted with one or more groups selected from -NH2, -NH ( optionally substituted Ci- 6 alkyl) and -N ( optionally substituted Ci- 6 alkyl) (optionally substituted Ci- 6 alkyl) . In some embodiments, the Ci-ioalkyl is substituted with 1, 2, 3, 4, 5 or 6 groups selected from -NH 2 , -NH ( optionally substituted Ci- 6 alkyl) and -N ( optionally substituted Ci-6alkyl) (optionally substituted Ci-6alkyl) .

The term "amino" refers to the primary group -NH 2 , as well as substituted amino groups (both monosubstituted and disubstituted) .

The term "substituted amino" or "secondary amino" refers to an amino group having a hydrogen of the primary group -NH 2 replaced with, for example, an alkyl group ( "alkylamino" ) , an aryl or aralkyl group ( "arylamino", "aralkylamino" ) and so on. Ci- 6 alkylamino and Ci- 3 alkylamino groups are preferred, such as for example, methylamino ( -NHMe ) , ethylamino (-NHEt) and propylamino (-NHPr) .

The term "disubstituted amino" or "tertiary amino" refers to an amino group having the two hydrogens of the primary group -NH 2 replaced with, for example, an alkyl group, which may be the same or different ( "di ( alkyl ) amino" ) , an aryl and alkyl group

("aryl (alkyl) amino") and so on. Di ( Ci- 6 alkyl ) amino and

di ( Ci- 3 alkyl ) amino groups are preferred, such as, for example, dimethylamino (-NMe2), diethylamino (-NEt 2 ), dipropylamino (-NPr 2 ) and variations thereof (e.g. -N(Me) (Et) and so on) .

The term "amido" or "amide" refers to the group -C(0)NH 2 .

The term "substituted amido" or "substituted amide" refers to an amido group having a hydrogen replaced with, for example, an alkyl group ( "alkylamido" or "alkylamide" ) , an aryl ( "arylamido" ) , aralkyl group ( "aralkylamido" ) and so on. Ci-3alkylamide groups are

preferred, such as, for example, methylamide (-C(O)NHMe), ethylamide (-C(O)NHEt) and propylamide (-C(O)NHPr) and reverse amides thereof (e.g. -NHC(0)Me, -NHC(0)Et and -NHC(O)Pr) .

The term "disubstituted amido" or "disubstituted amide" refers to an amido group having the two hydrogens replaced with, for example, an alkyl group ( "di ( alkyl ) amido" or "di ( alkyl ) amide" ) , an aralkyl and alkyl group ( "alkyl ( aralkyl ) amido" ) and so on. Di ( Ci- 3 alkyl ) amide groups are preferred, such as, for example, dimethylamide

(-C(0)NMe 2 ) , diethylamide (-C(0)NEt 2 ) and dipropylamide (-C(0)NPr 2 ) and variations thereof (e.g. -C(0)N(Me)Et and so on) and reverse amides thereof.

The term "aryl" refers to a carbocyclic (non-heterocyclic) aromatic ring or mono-, bi- or tri-cyclic ring system. The aromatic ring or ring system is generally composed of 6 to 10 carbon atoms. Examples of aryl groups include but are not limited to phenyl, biphenyl, naphthyl and tetrahydronaphthyl . 6-membered aryls such as phenyl are preferred. The term "arylalkyl" or "aralkyl" refers to an arylCi- 6 alkyl- such as benzyl.

The term "heterocyclyl" refers to a moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound which moiety has from 3 to 10 ring atoms (unless otherwise specified) , of which 1, 2, 3 or 4 are ring heteroatoms, each heteroatom being independently selected from 0, S and N, and the remainder of the ring atoms are carbon atoms. The term "heterocycloalkyl" refers to a heterocyclyl moiety comprising a saturated cyclic group comprising one or more ring carbons and one or more ring heteroatoms .

"Heterocycloalkenyl" refers to a heterocyclyl moiety comprising a cyclic group comprising at least one carbon-carbon double bond and one or more ring heteroatoms. "Heterocycloalkynyl" refers to a heterocyclyl moiety comprising a cyclic group comprising at least one carbon-carbon triple bond and one or more ring heteroatoms.

In this context, the prefixes 3-, 4-, 5-, 6-, 7-, 8-, 9- and 10- membered denote the number of ring atoms, or range of ring atoms, whether carbon atoms or heteroatoms. For example, the term "3-10- membered heterocylyl", as used herein, refers to a heterocyclyl group having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms. Examples of heterocylyl groups include 5-6-membered monocyclic heterocyclyls and 9-10 membered fused bicyclic heterocyclyls.

Examples of monocyclic heterocyclyl groups include, but are not limited to, those containing one nitrogen atom such as aziridine (3- membered ring), azetidine (4-membered ring), pyrrolidine

(tetrahydropyrrole), pyrroline (e.g., 3-pyrroline, 2,5- dihydropyrrole ) , 2if-pyrrole or 3if-pyrrole (isopyrrole, isoazole) or pyrrolidinone (5-membered rings), piperidine, dihydropyridine, tetrahydropyridine (6-membered rings), and azepine (7-membered ring) ; those containing two nitrogen atoms such as imidazoline, pyrazolidine (diazolidine ) , pyrazoline (dihydropyrazole ) (5-membered rings), piperazine (6-membered ring); those containing one oxygen atom such as oxirane (3-membered ring), oxetane (4-membered ring), oxolane (tetrahydrofuran) , oxole (dihydrofuran) (5-membered rings), oxane (tetrahydropyran) , dihydropyran, pyran (6-membered rings), oxepin (7-membered ring) ; those containing two oxygen atoms such as dioxolane (5-membered ring), dioxane (6-membered ring), and dioxepane (7-membered ring) ; those containing three oxygen atoms such as trioxane (6-membered ring) ; those containing one sulfur atom such as thiirane (3-membered ring), thietane (4-membered ring), thiolane ( tetrahydrothiophene ) (5-membered ring), thiane ( tetrahydrothiopyran ) (6-membered ring), thiepane (7-membered ring); those containing one nitrogen and one oxygen atom such as

tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole,

dihydroisoxazole (5-membered rings), morpholine, tetrahydrooxazine, dihydrooxazine, oxazine (6-membered rings); those containing one nitrogen and one sulfur atom such as thiazoline, thiazolidine (5- membered rings), thiomorpholine (6-membered ring); those containing two nitrogen and one oxygen atom such as oxadiazine (6-membered ring) ; those containing one oxygen and one sulfur such as: oxathiole (5-membered ring) and oxathiane (thioxane) (6-membered ring); and those containing one nitrogen, one oxygen and one sulfur atom such as oxathiazine (6-membered ring) .

The term "heterocyclyl" encompasses aromatic heterocyclyls and non- aromatic heterocyclyls .

The term "aromatic heterocyclyl" may be used interchangeably with the term "heteroaromatic" or the term "heteroaryl" or "hetaryl". The heteroatoms in the aromatic heterocyclyl group may be independently selected from N, S and 0.

"Heteroaryl" is used herein to denote a heterocyclic group having aromatic character and embraces aromatic monocyclic ring systems and polycyclic (e.g. bicyclic) ring systems containing one or more aromatic rings. The term aromatic heterocyclyl also encompasses pseudoaromatic heterocyclyls. The term "pseudoaromatic" refers to a ring system which is not strictly aromatic, but which is stabilized by means of delocali zation of electrons and behaves in a similar manner to aromatic rings. The term aromatic heterocyclyl therefore covers polycyclic ring systems in which all of the fused rings are aromatic as well as ring systems where one or more rings are non- aromatic, provided that at least one ring is aromatic. In

polycyclic systems containing both aromatic and non-aromatic rings fused together, the group may be attached to another moiety by the aromatic ring or by a non-aromatic ring.

Examples of heteroaryl groups are monocyclic and bicyclic groups containing from five to ten ring members . The heteroaryl group can be, for example, a five membered or six membered monocyclic ring or a bicyclic structure formed from fused five and six membered rings or two fused six membered rings or two fused five membered rings . Each ring may contain up to four heteroatoms selected from nitrogen, sulphur and oxygen. The heteroaryl group will contain up to 4 heteroatoms, more typically up to 3 heteroatoms, more usually up to 2 heteroatoms. In one embodiment, the heteroaryl group contains at least one ring nitrogen atom. The nitrogen atoms in the heteroaryl group can be basic, as in the case of an imidazole or pyridine, or essentially non-basic as in the case of an indole or pyrrole nitrogen. In general the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.

Aromatic heterocyclyl groups may be 5-membered or 6-membered mono ¬ cyclic aromatic ring systems.

Examples of 5-membered monocyclic heteroaryl groups include but are not limited to furanyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl

(including 1,2,3- and 1,2,4- oxadiazolyls and furazanyl, i.e. 1,2,5- oxadiazolyl ) , thiazolyl, isoxazolyl, isothiazolyl, pyrazolyl, imidazolyl, triazolyl (including 1,2,3-, 1,2,4- and 1,3,4- triazolyls), oxatriazolyl , tetrazolyl, thiadiazolyl (including 1,2,3- and 1,3,4- thiadiazolyls ) and the like.

Examples of 6-membered monocyclic heteroaryl groups include but are not limited to pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, pyranyl, oxazinyl, dioxinyl, thiazinyl, thiadiazinyl and the like. Examples of 6-membered aromatic heterocyclyls containing nitrogen include pyridyl (1 nitrogen), pyrazinyl, pyrimidinyl and pyridazinyl (2 nitrogens) .

Aromatic heterocyclyl groups may also be bicyclic or polycyclic heteroaromatic ring systems such as fused ring systems (including purine, pteridinyl, naphthyridinyl , lif-thieno [ 2 , 3- c] pyrazolyl , thieno [ 2 , 3-b] furyl and the like) or linked ring systems (such as oligothiophene, polypyrrole and the like) . Fused ring systems may also include aromatic 5-membered or 6-membered heterocyclyls fused to carbocyclic aromatic rings such as phenyl, naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl and the like, such as 5-membered aromatic heterocyclyls containing nitrogen fused to phenyl rings, 5- membered aromatic heterocyclyls containing 1 or 2 nitrogens fused to phenyl ring.

The term "C 2 -iothioether" refers to a group of the formula -C n alkyl-S- C m alkyl, where each of n and m is an integer from 1 to 9 and n + m = 2 to 10. Such groups include, for example, -CH 2 -S-Ci- 6 alkyl , -CH 2 CH 2 - S-Ci- 6 alkyl, -CH (CH 3 ) CH 2 -S-Ci- 6 alkyl, -CH 2 CH 2 -S-CH 3 etc.

Unless otherwise defined, the term "optionally substituted" as used herein indicates a group may or may not be substituted with 1, 2, 3, 4 or more groups, preferably 1, 2 or 3 groups, more preferably 1 or 2 groups, independently selected from the group consisting of alkyl (e.g. Ci- 6 alkyl), alkenyl (e.g. C 2 - 6 alkenyl ) , alkynyl (e.g. C 2 - 6 alkynyl), cycloalkyl (e.g. C 3 - 8 cycloalkyl ) , hydroxyl, oxo, alkoxy (e.g. Ci- 6 alkoxy) , aryloxy, arylCi- 6 alkoxy, thioalkyl (e.g. -S-Ci- 6alkyl), halo, haloCi-6alkyl (such as -CF 3 and -CHF 2 ), haloCi-6alkoxy (such as -OCF 3 and -OCHF 2 ), carboxyl, esters, cyano, nitro, amino, substituted amino, disubstituted amino, acyl, ketones, amides, aminoacyl, substituted amides, disubstituted amides, aryl,

arylCi- 6 alkyl , heterocyclylCi- 6 alkyl , arylC 2 - 6 alkenyl ,

heterocyclylC 2 - 6 alkenyl , arylC 2 - 6 alkynyl, heterocyclylC 2 - 6 alkynyl, heteroarylCi- 6 alkyl , heteroarylC 2 - 6 alkenyl , heteroarylC 2 - 6 alkynyl, heterocyclyl and heteroaryl, wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl and groups containing them may be further optionally substituted. Optional substituents in the case of heterocycles containing N may also include but are not limited to Ci- 6 alkyl i.e. N-Ci_ 6 alkyl.

For optionally substituted "alkyl", "alkenyl" and "alkynyl", the optional substituent or substituents are, unless otherwise defined, preferably selected from C 3 - 8 cycloalkyl, amino, substituted amino, disubstituted amino, aryl, heteroaryl, halo (e.g. F, CI, Br, I), haloCi- 6 alkyl , heterocyclyl, Ci- 6 alkoxy, oxo, aryloxy, carboxyl and esters. Each of these optional substituents may also be optionally substituted with any of the optional substituents referred to above.

As a person skilled in the art will appreciate, peptides are typically capable of forming salts (e.g. addition salts with suitable acids and/or bases) . Salts of peptides are well known in the art and a person skilled in the art will be able to choose suitable salts of the peptides/compounds of the present invention, person skilled in the art will also be able to prepare suitable salts of the compounds of the present invention. Common salts of peptides include Li + , Na + , K + , Mg 2+ , Ca 2+ , CI " , Br, I " , " OAc, + NH 4 . Contemplated herein are all suitable salts of the peptides and compounds of the present invention.

EXAMPLES

The present invention is further described below by reference to the following non-limiting Examples.

Example 1

1 Tetrapeptides Fmoc-FFkk and Fmoc-FkFk

Fmoc-FFkk, 1 Fmoc-FkFk, 2

The two tetrapeptides Fmoc-FFkk (1) and Fmoc-FkFk (2), bearing phenylalanine and lysine residues, were prepared as described below in section 1.1. The inventors found that these peptides are water soluble (0.5% w/v solutions of 1 and 2 having a pH of about 5 and about 4, respectively) and do not require a commonly employed pH switch in order to form self-supporting hydrogels. The initial pH of 4 or 5 may be neutralized (e.g. to biologically relevant pHs) upon the addition of Neurobasal media.

As described below, the inventors characterise the sol and gel state of the tetrapeptides (sometimes referred to herein as "nanofiber networks", "nanofiber substrates", "peptide nanofibers" and the like), before using them to support the growth of notoriously sensitive primary hippocampal neurons. It is shown that primary neurons cultured on thin hydrogel layers exhibit spreading, neurite extension and synapse formation, comparable with the currently used gold standard, poly-D-lysine . Long term survival of neurons is also demonstrated, with electrical activity of mature neurons confirmed using a GCaMP reporter assay (section 1.4) . 1.1 Peptide synthesis

The peptides in Example 1 may be prepared using solid phase peptide synthesis using well-known techniques (e.g. Merrifield et al . , Journal of the American Chemical Society, 1963, pp2149-2154) .

Functionalised resin beads may be used as a solid support to build up the peptide sequences through iterative coupling and deprotection reactions. Once the peptides Fmoc-FFkk and Fmoc-FkFk have been synthesised on the resin, cleavage of the peptide from the resin may be effected through the use of tri fluoroacetic acid, before removal of the solvent and lyophilisation . Further to this, the peptides may then be purified using semi-preparatvie HPLC before lyophilisation.

The peptides in Example 1 were typically prepared using the process described below. Solid phase peptide synthesis of tetrapeptides

Initial amino acid loading

2-chlorotrityl chloride resin (100-200 mesh; 1% DVB; 1.1 mmol/g) (500 mg, 0.55 mmol) was weighed into a 10 mL polypropylene syringe equipped with a porous polypropylene frit (Torviq SF-1000), which was used as the reaction vessel. The resin was washed with

dichloromethane (3 x 5 mL) before being allowed to swell in dichloromethane (5 mL) for at least 0.5 h prior to the loading of the first amino acid.

A solution of Fmoc-Lys ( Boc) -OH (3 equiv. , 773 mg) was dissolved in a mixture of dry dichloromethane (2 mL) , N, N-dimethyl formamide (2 mL) and N, N-diisopropylethylamine (DIPEA) (8 equiv., 0.8 mL) and taken up into the syringe with resin and stirred overnight using an orbital shaker. The resin was then washed with dichloromethane (3 x 4mL) and N, N-dimethylformamide (DMF) (3 x 4 mL) . N-terminal Fmoc deprotection

A solution of 20% (v/v) piperidine in DMF (2 x 4 mL) was added to the resin once for 1 min, then a fresh aliquot was taken up again and stirred for 10 mins . The solution was subsequently expelled and the resin washed with DMF (5 x 4 mL) . The resulting resin-bound amine was used immediately in the next peptide coupling step.

Amino acid coupling

Fmoc-protected amino acids (3 equiv. ) were dissolved in a 0.45 M DMF solution of 1-hydroxybenzotriazole hydrate (HOBt'H20) /Ν,Ν,Ν " ,N " - tetramethyl-O- (IH-benzotriazol-l-yl) uronium hexafluorophosphate (HBTU) (3 equiv.) and DIPEA (6 equiv., 0.6 mL) and this coupling solution added to the resin and stirred for 45 mins using an orbital shaker. The solution was expelled and the resin washed with DMF (5 x 4 mL) .

After another N-terminal Fmoc deprotection, iterative couplings were performed in order to build up the required peptide sequence.

Cleavage of the peptide

After the final coupling step, the resin was washed with DMF (3 x 4 mL) and dichloromethane (3 x 4 mL) . A solution of 3:7

dichloromethane : tri fluoroacetic acid with one drop of water was then added to the resin, and the resin stirred for 2 hours using an orbital shaker. The cleavage solution was then expelled, the resin washed with dichloromethane (2 x 4 mL) and the solvents evaporated under a stream of nitrogen. The resulting residue was lyophilised and purified by semi-preparative HPLC using an acetonitrile/water gradient, giving a white fluffy solid.

Fmoc-FFkk: IR: 3281 (m) , 3063 (w) , 3029 (w) , 2939 (m) , 1690 (m) , 1631 (s), 1534 (s), 1451 (m) , 1397 (m) , 1320 (w) , 1261 (m) , 1202 (m) , 1134 (m) , 1086 (w) , 1039 (m) , 990 (w) , 836 (m) , 799 (m) , 756

(m) , 739 (s), 721 (m) , 699 (s); ¾ NMR (400 MHz, DMSO-d 6 ) δ 8.36 (s, 1H) , 8.28 - 8.21 (m, 1H) , 7.86 (qt, J = 8.2 Hz, 3H), 7.61 (t, J = 8.1 Hz, 1H) , 7.41 (td, J = 7.5 Hz, 2H) , 7.34 (td, J = 7.5 Hz, 1H), 7.26 - 7.14 (m, 8H) , 6.27 (s, 1H) , 4.62 (br. s, 1H) , 4.30 - 4.15 (m, 2H) , 4.10 (t, J = 8.2 Hz, 1H) , 4.01 - 3.93 (m, 1H) , 3.43 (br, s, 1H) , 2.99 - 2.81, m, 3H) , 2.69 (br. q, 3H) , 1.68 (br. s, 1H) , 1.49 (br. s, 5H) , 1.34 - 1.20 (m, 4H) . 13 C NMR (101 MHz, DMSO-d 6 ) δ 174.53, 170,61, 170.37, 165.31, 142,56, 139.41, 137.43, 129.32, 129.19, 128.93, 128.15, 127.96, 127.62, 127.29, 127.06, 126.18, 125.23, 121.39 ,120.03, 109.78, 55,93, 53.80, 53.27, 52.20, 46.52, 31.50, 31.38, 26.82, 26.37, 22.38, 21.51; HR-MS (ESI) : calcd for C45H 5 4N 6 0 7 + H + : 791.4107, found 791.4128.

Fmoc-FkFk: IR: 3289 (m) , 3061 (w) , 3029 (w) , 2935 (m) , 1638 (s), 1528 (s), 1451 (m) , 1394 (m) , 1339 (w) , 1256 (m) , 1202 (s), 1177 (m) , 1133 (m) , 1086 (w) , 1034 (m) , 836 (m) , 799 (m) , 756 (m) , 739 (s), 721 (m) , 699 (s); ¾ NMR (400 MHz, DMSO-d 6 ) δ 8.70 (br. s, 1H) , 8.34 (s, 1H), 7.86 (qt, J = 8.2 Hz, 2H) , 7.80 - 7.74 (m, 1H) , 7.62 (d, J = 7.2 Hz, 1H), 7.41 (td, J = 7.3 Hz, 2H) , 7.34 (td, J = 7.5 Hz, 1H) , 7.29 - 7.05 (m, 8H), 6.28 (s, 1H) , 4.49 - 4.28 (m, 1H) ,

4.21 - 4.08 (m, 2H), 3.99 - 3.88 (m, 1H) , 3.66 (br, s, 1H), 3.11 - 3.02 (m, 1H), 2.95 - 2.55 (m, 5H) , 1.35 (br. q, 8H) . 13 C NMR (101 MHz, DMSO-dg) δ 174.07, 173.01, 170,88, 169.64, 142,57, 139.42, 138.31, 138.02, 137.43, 129.38, 128.93, 128.13, 128.09, 127.30, 126.13, 121.39, 120.04, 109.78, 55,46, 55.11, 51.18, 46.52, 38.41, 37.41, 32.58, 32.19, 27.42, 27.06, 22.01,;; HR-MS (ESI) : calcd for C45H54N6O7 + H + : 791.4107, found 791.4120.

1.2 Peptide characterisation

Fmoc-diphenylalanine (Fmoc-FF) and related derivatives self-assemble into hydrogels, however the majority of these gelators must be dissolved in basic conditions using dilute sodium hydroxide

(typically pH > 9) . Furthermore, gelation typically requires a pH switch using either a mineral acid or lactone which slowly

hydrolyses into an acid. These hydrogel formation methods are often not compatible with cell culture, due to the extremes of pH that are utilised, and once the hydrogel is formed the pH must be adjusted for the hydrogel to be used for cell culturing.

The incorporation of positively charged lysine residues onto the Fmoc-FF scaffold results in a shift in the hydrophobicity of the peptide, rendering it water soluble at neutral pH. The position of the lysine residues was altered in order to investigate the effect of amino acid sequence on the properties of the subsequent

hydrogels, giving peptides 1 ( Fmoc-FFkk) and 2 (Fmoc-FkFk), Figure 1. Analogues bearing only one lysine residue were synthesised, however were too insoluble to be investigated further. It should be noted that tetrapeptides 1 and 2 feature D-lysine, in order to make the hydrogels resistant towards proteolysis.

The tetrapeptides 1 and 2 were observed to self-assemble into fibres or wormlike micelles at neutral pH, as evidenced through viscosity and small angle neutron scattering (SANS) measurements (described below) . This allows gelation to be triggered through the addition of salts, either using a buffer or cell culture media. This completely circumvents the need for a pH switch, or heat-cooling to induce gelation. Thus, tetrapeptides 1 and 2 can form hydrogels through the more biocompatible route method of charge screening at room temperature. For the following measurements, gels were formed through addition of NeuroBasal medium, with a view towards their applications for culturing primary neurons. However, other cell culture media such as DMEM or OptiMEM can also induce gelation The kinetics of gelation for peptides 1 and 2 was investigated through time resolved rheology (Figure 2a, b) . Fmoc-FFkk rapidly reaches an initial plateau after one minute, after which time slow ageing of the gel is observed over the following four hours before another plateau is reached. For Fmoc-FkFk, no rapid initial plateau is reached, instead the gel slowly ages and stiffens over the course of the measurements. It should be noted that for both peptides, initially G' >G' ' , suggesting that gelation is rapid. From the frequency sweeps (Figure 3a, b), both gels display moderate stiffness (1 kPa for 1 , 3 kPa for 2 ) and LVE values of approximately 2% strain (Figure 3c, d) . Thixotropy measurements show that whilst recovery from large external shearing forces is rapid, both gels do not recover to their initial stiffnesses, potentially due to an irreversible rearrangement of the gel network (Figure 2c, d) .

The fibrous network of the gels was then investigated through atomic force microscopy measurements. In both cases, a dense network of fibres is formed, with the compact arrangement of fibres precluding measurement of their diameter. The amorphous, globular deposits in the images are likely due to the presence of additives in the NeuroBasal medium used to induce gelation. The arrangement of the tetrapeptide molecules within the fibres was investigated through circular dichroism and ATR-IR spectroscopy. Gels of Fmoc-FFkk show a negative peak in the CD centred around 220 nm (Figure 5a), typical of the β-sheet structure previously observed for short peptides based upon the Fmoc-diphenylalanine motif. The reordering of the amino acid sequence to give Fmoc-FkFk results in a change in secondary structure - a shoulder peak at 220 nm is still visible; however the main negative peak is centred at 205 nm, suggesting a random coil or disordered arrangement. This is not surprising, given that the diphenylalanine motif has been

interrupted through insertion of a lysine. This is confirmed by the ATR-IR, with the absorption in the Amide I region for Fmoc-FkFk slightly shifted towards higher wavenumbers relative to Fmoc-FFkk (1637 crrr 1 compared to 1627 crrr 1 ) (Figure 5b) . Circular dichroism measurements

CD measurements were performed using a ChirascanPlus CD

spectrometer, with data collected between wavelengths of 180 - 500 nm with a bandwidth of 1 nm, sample ratio of 0.1 s/point and step of 1 nm. In a typical experiment, 0.5% (w/v) peptide sols were prepared as above and diluted 1:8 (v/v) in water. Temperature was kept constant at 20 °C and all experiments were repeated three times and averaged into a single plot.

Viscosity measurements

Viscosity measurements were performed on an Anton Paar MCR 302 rheometer using a 25 mm stainless steel parallel plate geometry configuration and analysed using RheoPlus v3.61 software. Typical viscosity measurements involved casting 550 ]iL of a 0.5% (w/v) peptide solution onto one of the stainless steel plates, lowering the other plate to the measurement position, and monitoring viscosity over a range of shear rates (0.1 - 100 s _1 ) . A Peltier temperature control hood and solvent trap was used to reduce evaporation and maintain a temperature of 25 °C. Graphs showing the viscosity measurements of peptides Fmoc-FkFk (a) and Fmoc-FFkk (b) dissolved in water at 0.5% (w/v) are shown in Figure 15(a) and Figure 15 (b) . AFM measurements

Glass coverslips were coated with 100 ]iL of a 0.5% (w/v) solution of peptide dissolved in water and left to incubate overnight. Excess media was then aspirated and the coverslip attached to a stainless steel disc. Imaging was undertaken on a Bruker Multimode 8 atomic force microscope in Scanasyst mode in air, whereby the imaging parameters are constantly optimised through the force curves that are collected, preventing damage of soft samples. Bruker Scanasyst- Air probes were used, with a spring constant of 0.4 - 0.8 N/m and a tip radius of 2 nm.

Small angle neutron scattering measurements and modelling

Peptide solutions were prepared at a concentration of 1% (w/v) transferred to a demountable titanium cell of 2 mm path length before being measured at detector distances of 2 and 14 m. Isotropic scattering patterns were radially averaged and combined for a q range of 0.005 - 0.40 A, where q = 4nsin(9)/A, and λ is neutron wavelength (5 A) with a wavelength spread, Δ (λ / λ 12%) and 2Θ is the scattering angle. Data was corrected for the background, empty cell scattering and the sensitivity of the individual detector pixels. The data was reduced using IgorPro software employing NIST macros specific to QUOKKA to an absolute intensity scale.

Data was modelled using SasView, with a flexible cylinder model chosen for each scattering pattern, based upon AFM characterisation data. The scattering length density (SLD) of the peptide was calculated to be 1.77 χ 10 ~6 , with this value and the SLD of the solvent (D 2 O) fixed. Other parameters were allowed to vary freely and following a few optimisation cycles, the background was fixed. After this, multiple different starting points were used for the Kuhn length, radius and length of the cylinder, to ensure that a global, physically realistic minimum was found.

Small angle neutron scattering (SANS) patterns and associated fits obtained from 1% (w/v) solutions of Fmoc-FFkk (a) and Fmoc-FkFk (b) dissolved in D 2 0 are shown in Figure 16 (a) -(b) . Fmoc-FFkk can be fit to a flexible cylinder model with a diameter of 6.5 nm, a Kuhn length of 5.9 nm and fibre length of 45 nm. Fmoc-FkFk can be fit to a fractal model with diameter 4 nm and fractal dimension of 2.7, consistent with a self-assembled fibre network.

1.3 Primary neuronal cultures

With an understanding of the self-assembly of the tetrapeptides in hand, the inventors focused on applying these hydrogels as

substrates for culturing primary neurons. As the peptides form fibres or wormlike micelles when dissolved in water, the inventors used this peptide solution (no culture media added) to coat glass coverslips before seeding neurons atop them. As the peptides can be dissolved in water, this eliminates any washing of the coverslip required before seeding of neurons. This represents a significant time saving as the current gold standard for neuronal culture, poly- D-lysine (PDL) is often dissolved in a borate buffer and requires the coverslip to be washed several times before it can be used. For the following experiments, peptide solutions were prepared at 5 mg/mL and 100 or 150 μL cast onto glass coverslips, which were then incubated overnight before aspiration of any excess liquid.

For poly-D-lysine (PDL) coated coverslips, a solution of PDL in borate buffer (0.2 mg/mL) was prepared and 100 ]iL added to freshly flamed glass coverslips in 24 well plates and incubated overnight.

Following this, excess media was aspirated and the coverslip washed three times with sterile water before neurons were plated onto the treated coverslip.

The level of coverslip coverage for PDL, Fmoc-FFkk and Fmoc-FkFk was assessed through atomic force microscopy (Figure 4(a) and 4(b) . It can be seen that for PDL, a uniform film is obtained, whereas a coating of fibres on the glass coverslip is observed for Fmoc-FFkk and Fmoc-FkFk. Notably, changing the order of amino acids in the tetrapeptide alters the fibre morphology observed. For Fmoc-FFkk, thick fibres of approximately 8 nm which exhibit bundling behaviour are observed. Altering the amino acid sequence to Fmoc-FkFk gives smaller fibres of 4 nm in diameter, randomly oriented on the substrate. This results in better coverage on the glass coverslip, which is advantageous for cell culture. Primary neurons were obtained from pregnant C57BL6 mice of embryonic day 16.5. Briefly, the abdominal cavity of time-mated females was opened to remove the uterus. Embryos were placed on ice, decapitated and brains removed. After meninges were carefully removed, cortices and hippocampi were dissected and incubated with trypsin (Sigma) at 37 °C for 15-20 min, followed by trituration with fire-polished glass Pasteur pipettes (Livingstone) to obtain single cell

solutions. Cells were counted using a hemocytometer and plated onto glass coverslips in Dulbecco' s Modified Eagle Medium (Life

technologies) medium containing 10% heat-inactivated fetal bovine serum (Hyclone) . Medium was changed to Neurobasal containing B27 supplement and Glutamax (all Life technologies) for continued culturing .

Cells were fixed at the appropriate timepoint in vitro (DIV) using 4% paraformaldehyde and after permeabilisation, stained with primary antibodies for either: β3 tubulin (Millipore) , MAP2 (Millipore) , GFAP (AbCam) , IBA-1 (AbCam) , synaptophysin (AbCam) or PSD-95

(Millipore) using established protocols. Secondary antibody conjugation was performed with Alexa dyes (488, 555 or 647) and DAPI was used for nuclear visualisation. Coverslips were mounted using Fluoromount (Southern Biotech) and imaged with either an Olympus BX51 epifluorescence microscope (10, 20 or 40x air objectives) or a Zeiss 880 confocal microscope (60x/1.25 oil objective) .

Primary hippocampal neurons from mouse embryos that were 16.5 days old were seeded atop coverslips which had been coated with either

PDL, Fmoc-FFkk or Fmoc-FkFk solutions, followed by fixing at days 1- 5 and staining with antibodies for MAP2 (red, dendrites) and β3- tubulin (green, axons), and DAPI for the cell nucleus (blue) (Figure 7) . Primary neurons are known to undergo a very well defined development for the first several days after seeding. Peptide coated coverslips supported development of neurons, on par with the current standard of poly-D-lysine . After one day in culture, lamellipodia are present, with minor processes extending after two days. On day three, axonal extension occurs, followed by the development of dendrites and a highly connected neural network (Figure 7) . Here, it is clear that coverslips coated with peptides 1 and 2 support the initial development of primary neurons.

Following this, long term culturing of primary neurons on peptide coated coverslips was examined. Neurons were seeded on top of coated coverslips (PDL, Fmoc-FFkk or Fmoc-FkFk as depicted) and cultured for 40 days, before fixing and staining for ΜΆΡ2 , β3- tubulin and DAPI . It is known that the viability of primary neurons decreases over longer intervals, as levels of secreted products in the culture media can build to toxic levels . The results are shown in Figure 8 (a) -(c) , with neuronal processes intact for all

coverslips examined. Therefore, peptides 1 and 2 are viable candidates for the long term culturing of neurons. Especially notable is the lack of degradation of the substrate, suggesting that the D-lysine is not susceptible to proteolytic degradation. Cell viability was also further investigated. Primary hippocampal neurons were cultured on coverslips coated with Fmoc-FFkk, Fmoc- FkFk, or PDL for 10, 20, 30, or 40 days. Cell viability was determined using an Alamar Blue assay. Coverslips coated with PDL were used as a positive (100% viability) control. At DIV10 and DIV20, the viability of neurons cultured on peptide nanofibers showed a trend toward reduction. However, at longer time points, no significant differences in viability relative to PDL were observed (Figure 8(d)) . These results demonstrate the viability of long term cultures . Each experiment was repeated three times . At the

appropriate time point, 50 ]iL of warmed Alamar Blue was added to wells containing primary neurons cultured on either PDL or peptide nanofiber substrates, followed by incubation for Ah. Absorption was then recorded at 570 nm and subtracted from reference absorption at 596 nm. Control wells included no cells and a negative control of 10% (v/v) DMSO.

The ability to transfect primary neurons on coverslips coated with Fmoc-FFkk and Fmoc-FkFk was then investigated. This was done to ensure that there was no interference of the peptide with the function of the widely used transfection agent Lipofectamine LTX, or any residual DNA. For this experiment a green fluorescence protein ( GFP ) -encoding DNA construct was used and complexed with Lipofectamine LTX reagent before being added to neurons which had been cultured for 7 days on peptide or PDL coated coverslips. After fixing at 10 and 14 days in vitro, neurons were also stained with ΜΆΡ2 (red) to visualise neuronal processes (Figure 9) . Expression of GFP was observed for primary neurons cultured on all surfaces, with expression levels similar (approximately 5%) across all coverslips. Thus it can be concluded that the peptide coating on the coverslips does not interfere with the transfection of primary neurons.

Next, the synaptic development of primary neurons was assessed.

Neurons were cultured on peptide or PDL coated coverslips for up to thirty days, fixed at various intervals ranging from 10 to 30 days and then stained for the synaptic markers synaptophysin (a marker for pre-synaptic vesicles; green) and PSD-95 (post-synapse; red) alongside the nuclear stain DAPI (blue) as shown in Figure 10(a) . A clear colocalisation of the stains was observed, with the punctate nature of the fluorescence expected for synaptic compartments. Clear development of synapses along neuronal processes was observed from day 10 up to day 30. This indicates that peptide coated coverslips are able to recapitulate synaptic development. The density of synapses along dendrites was observed over days 10 to 30 (Figure 10(a)) with evident marker colocalisation (Figure 10(b)).

Quantification of synaptic density (Figure 10(c)) revealed similar results for neurons cultured on PDL, Fmoc-FFkk or Fmoc-FkFk, consistent with the nanofiber network (Fmoc-FFkk or Fmoc-FkFk) supporting the synaptic development of neurons. Quantification of synaptic density was achieved using ImageJ software to measure neurite length and instances of colocalisation in pre- and postsynaptic markers.

In addition to supporting the full range of neuronal development, including long term cultures and synaptic development, the ability to transition from a thin, 2D fibre coating to a fully- fledged 3D network can be achieved simply through the addition of NeuroBasal medium to the peptides dissolved in water, which induces gelation. This is compared to PDL, which cannot form 3D networks. Initial results of neurons seeded within hydrogels showed that neuronal growth occurs (Figure 11 (a) -(d) and Figure 11(e)) . 1.4 Electrical Activity of Primary Neurons on Peptide Nanofiber Subs trates

To further assess the maturation of primary neurons cultured on peptide nanofibers, the inventors performed adeno-associated virus (AAV) -mediated transductions with GFP at DIV7 before fixing cells at DIV11 and 14 to visualize the development of dendritic spines. In all cases, the development of spines was evident at DIV14 and comparable between PDL and peptide nanofiber substrates .

Importantly, these experiments also showed that primary neurons cultured on the peptide nanofiber substrates are suitable for AAV- mediated gene expression. To test this further, a transfection was performed with Lipofectamine, resulting in typically low

transfection efficiencies (~2%), comparable to results previously reported for cultures using PDL coating. Notably, across primary neurons cultured on PDL or the peptide nanofibers, similar

transduction efficiencies were observed for AAV-mediated

transduction or liposome-mediated trans fections , implying that the presence of the cationic peptide does not interfere with the transduction process. This suggests the lysine residues do not interfere with the cationic liposomes of the Lipofectamine reagent or the AAV.

In further studies, primary neurons were transduced with a GCaMP construct (AAV-hSynl-GCaMP6s-nls-dTomato ) to visualize electrical activity. Adeno-associated virus vectors for neuronal expression (hSynl) of the GCaMP6s calcium sensor were cloned by conventional restriction enzyme cloning. All plasmids were amplified in E. coli DH5 . AAV vectors were propagated in E. coli Stbl3 to avoid recombination events. All constructs were verified by sequencing. Upon binding of calcium, GCaMP undergoes a conformational change which results in fluorescence emission. Primary neurons were transduced at both DIV4 and DIV11, while imaging was undertaken at DIV8 and DIV15, respectively. In all cases, spontaneous synaptic activity for neurons cultured on peptide nanofibers was observed. Primary neurons imaged at DIV8 undergo random firing events, whereas primary neurons recorded at DIV15 fired in tandem (Figure 14a) .

Firing events for multiple neurons over time were quantified and averaged, showing random firing events occurring on all substrates at DIV8 (Figure 14(b)) and highly synchronous firing at DIV15 (Figure 14(c) ) . Similar network activity was observed for both PDL and both peptide substrates, confirming the development of

electrically active neuronal networks. These results demonstrate the maturation, viability and functionality aspects of the cultures.

Experimental details for these studies are as follows:

AAV generation/packaging

Adeno-associated virus vectors for neuronal expression (hSynl) of the GCaMP6s calcium sensor were cloned by conventional restriction enzyme cloning. All plasmids were amplified in E. coli DH5 . AAV vectors were propagated in E. coli Stbl3 to avoid recombination events. All constructs were verified by sequencing.

Transduction of primary neurons

Lipofectamine: 300 ]iL of conditioned media was removed from each well, and 100 ]iL of a transfection mix containing 0.6 ]iL

Lipofectamine LTX reagent and 0.2 ]ig GFP DNA in Neurobasal was carefully added to each well. After lh incubation, media was aspirated from wells and the wells supplemented with the initially removed conditioned media.

AAV: A 10 solution containing 0.3 pL either AAV-hSynl-GFP or AAV- hSynl-GCaMP6s-P2A-nls-dTomato (corresponding to approximately 10 11 viral particles) was added to wells containing primary neurons at appropriate timepoints . AAV-hSynl-GCaMP6s-P2A-nls-dTomato was a gift from Jonathan Ting (Addgene plasmid # 51084) . Transduction was confirmed and imaging undertaken on an Olympus SZ61 with a 20x air obj ective .

Analysis of GCaMP firing events

Changes in GCamp6s fluorescence of single cells was analysed using ImageJ software. Between nine and eleven visually responsive cells were chosen for each timepoint and substrate. Individual cells were contoured using ring-shaped ROIs covering the whole cell soma and changes in fluorescence intensity over a time course of approximately 20s was measured by averaging all pixels within the ROI . Mean pixel intensity of each cell was corrected for baseline fluorescence .

Example 2

2 Comparative examples

Phe-Phe-Asp Phe-Phe-Asp-Asp Gly-Phe-Phe

C1 C2 C3 C4

Gly-Phe-Phe-Arg-Gly-Asp The comparative examples were prepared by a similar process to that described in Example 1. The peptides were prepared with the capping groups C1-C4 , wherein CI is an Fmoc group, C2 is a phenothiazine group (i.e. ( 9-phenothiazyl ) -CH 2 OC (=0) - ) , C3 is an indole group (i.e. ( 3-indolyl) - CH 2 OC (=0) -) and C4 is a Cbz group (i.e.

(9-carbazolyl) -CH 2 OC (=0) -) .

Initially the diphenylalanine peptides (Phe-Phe) were tested and were unsuccessful, as in order to obtain hydrogels, or self- assembly, a low pH was required (pH approx 4 ) , which is not compatible with cell culture. The Gly-Phe-Phe sequence was also tested as it self-assembles at ~pH 7, however it was deemed too hydrophobic to support primary neurons. Following on from this, efforts were made to make the peptides more hydrophilic, resulting in the generation of Phe-Phe-Asp and Phe-Phe-Asp-Asp (SEQ ID NO: 34 ) peptides. However, these peptides proved to be too acidic (due to the extra carboxylate groups) to support primary neurons. Finally, the Gly-Phe-Phe-Arg-Gly-Asp (SEQ ID NO: 35) sequence was evaluated, as it is hydrophilic and contains an RGD motif derived from fibronectin, however it too could not support primary neurons.

Example 3

One of main challenges associated with 3D cultures is disassembly of gelators, releasing cells.

Current materials require harsh chemicals or mechanical force. When D-lysine is replaced by L-lysine, resulting hydrogels of Fmoc- FFKK and Fmoc-FKFK can be disassembled through addition of trypsin.

To prepare a 2D culture system using poly-lysine, a flamed coverslip is coated with not more than 150 ]iL of poly-lysine dissolved in borate buffer (pH 8) at a concentration of 0.5 mg/mL. The coated coverslip is incubated overnight, followed by aspiration of any remaining liquid and washing at least three times with sterile water .

The preparation of a 2.5D culture system (thin mat of fibres) involves dissolving the peptide in water at a concentration of 5 mg/ml before coating the flamed coverslip with not more than 100 ]iL of this solution and incubating overnight. Following this, any excess solution is aspirated.

For a 3D culture system, the peptide of choice is dissolved in water at a concentration of 10 mg/mL, followed by the addition of an equal volume of cell culture media (here Neurobasal) . To obtain cells uniformly distributed throughout the hydrogel, a cell suspension in culture media should be used. At least 50 ]iL of the peptide-cell suspension mixture is then added to a flamed coverslip, before incubation overnight, resulting in formation of a hydrogel. To disassemble hydrogels, subsequently releasing cells, first hydrogels are prepared as above through mixing a peptide solution and a cell suspension in cell culture media. After the required time (not less than 24 hours), an equal volume of trypsin-EDTA (5x solution) is added to the hydrogel, then incubated for 5 minutes. The hydrogel is then gently agitated to assist with disassembly, thus releasing the suspended cells and converting the hydrogel into a solution.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.