A Self-Assembling Short Amphiphilic Peptide And Related Methods And Uses

Abstract

There is provided a self-assembly amphiphilic peptide having the formula (I): XYZ (I), wherein X is a polar moiety at the N-terminus; X and Z each independently has between 1 to 4 residues of aliphatic amino acids or analogs or derivatives thereof, and wherein the average degree of hydrophobicity of the residues in block Z is more than the average degree of hydrophobicity of the residues in block Y. Disclosed are compositions and hydrogel comprising the peptide thereof. Also disclosed are methods of treatment for tissue regeneration, wound healing and methods of culture of stem cells, tissues and organoids.

Claims

1. An amphiphilic peptide having the formula (I):
XYZ  (I) wherein X is a polar moiety at the N-terminus; Y and Z each independently has between 1 to 4 residues of aliphatic amino acids or analogs or derivatives thereof, and wherein the average degree of hydrophobicity of the residues in block Z is more than the average degree of hydrophobicity of the residues in block Y.

2. The amphiphilic peptide of claim 1, wherein the polar moiety is selected from the group consisting of a polar functional group, a polar amino acid, and a small polar biomolecule.

3. The amphiphilic peptide of claim 1, wherein the polar moiety is selected from the group consisting of a polar functional group, a polar amino acid, and a small polar biomolecule, wherein the polar functional group is selected from the group consisting of amine, acetyl, hydroxyl, thiol, maleimide, and acid; or the polar amino acid is selected from the group consisting of lysine, histidine, glycine, serine and aspartic acid; or the small polar biomolecule is selected from the group consisting of biotin, alcohol, and saccharide.

4. The amphiphilic peptide of claim 1, wherein the amphiphilic peptide comprises a depsipeptide analog.

5. The amphiphilic peptide of claim 1, wherein the amphiphilic peptide comprises a depsipeptide analog, wherein depsipeptide analog comprises an α-hydroxy acid analog.

6. The amphiphilic peptide of claim 1, wherein the N-terminus is acetylated and/or C-terminus is amidated.

7. The amphiphilic peptide of claim 1, wherein the aliphatic amino acids comprise D-amino acids.

8. The amphiphilic peptide of claim 1, wherein the chirality of each of the residues in Z is the same.

9. The amphiphilic peptide of claim 1, wherein Z comprises a residue of an aliphatic amino acid with hydrophobic side chain, or analogs or derivatives thereof.

10. The amphiphilic peptide of claim 1, wherein the amphiphilic peptide is no more than 7 residues in length.

11. The amphiphilic peptide of claim 1, wherein the amphiphilic peptide is capable of self-assembling into a hydrogel.

12. The amphiphilic peptide of claim 1, wherein the amphiphilic peptide is selected from the group consisting of: KVI, KGAVLI, KGAVIL, KGAVIA, RVI, RGAVLI, RGAVIL, RGAVIA, HVI, HGAVLI, HGAVIL, HGAVIA, OrnVI, OrnGAVLI, OrnGAVIL, OrnGAVIA, DapVI, DapGAVLI, DapGAVIL, DapGAVIA, Dab VI, DabGAVLI, DabGAVIL, DabGAVIA, KgAVLI, KGaVLI, KgAVIL, KGaVIL; KGAVLI-NH.sub.2, KGAVIL-NH.sub.2, KgAVLI-NH.sub.2, KGaVLI-NH.sub.2, KgAVIL-NH.sub.2, and KGaVIL-NH.sub.2; Ac-KVI-NH.sub.2, Ac-KGAVLI-NH.sub.2, Ac-KGAVIL-NH.sub.2, Ac-KGAVIA-NH.sub.2, Ac-RVI-NH.sub.2, Ac-RGAVLI-NH.sub.2, Ac-RGAVIL-NH.sub.2, Ac-RGAVIA-NH.sub.2, Ac-HVI-NH.sub.2, Ac-HGAVLI-NH.sub.2, Ac-HGAVIL-NH.sub.2, Ac-HGAVIA-NH.sub.2, Ac-OrnVI-NH.sub.2, Ac-OrnGAVLI-NH.sub.2, Ac-OrnGAVIL-NH.sub.2, Ac-OrnGAVIA-NH.sub.2, Ac-DapVI-NH.sub.2, Ac-DapGAVLI-NH.sub.2, Ac-DapGAVIL-NH.sub.2, Ac-DapGAVIA-NH.sub.2, Ac-DabVI-NH.sub.2, Ac-DabGAVLI-NH.sub.2, Ac-DabGAVIL-NH.sub.2, Ac-DabGAVIA-NH.sub.2, Ac-KgAVLI-NH.sub.2, Ac-KGaVLI-NH.sub.2, Ac-KgAVIL-NH.sub.2, Ac-KGaVIL-NH.sub.2; GAVLI, SGAVLI, SGAVIL, SGAVIA, TGAVLI, TGAVIL, TGAVIA, SGAVLI, SGAVIA, SgAVLI, SGaVLI, SgAVIA, and SGaVIA; SGAVLI-NH.sub.2, SGAVIA-NH.sub.2, SgAVLI-NH.sub.2, SGaVLI-NH.sub.2, SgAVIA-NH.sub.2, and SGaVIA-NH.sub.2; Ac-GAVLI-NH.sub.2, Ac-SGAVLI-NH.sub.2, Ac-SGAVIL-NH.sub.2, Ac-SGAVIA-NH.sub.2, Ac-TGAVLI-NH.sub.2, Ac-TGAVIL-NH.sub.2, Ac-TGAVIA-NH.sub.2, Ac-SgAVLI-NH.sub.2, Ac-SGaVLI-NH.sub.2, Ac-SgAVIA-NH.sub.2, Ac-SGaVIA-NH.sub.2; DVI, DGAVLI, DGAVIL, EVI, EGAVLI, EGAVIL, and EGAVIA; and Ac-DVI-NH.sub.2, Ac-DGAVLI-NH.sub.2, Ac-DGAVIL-NH.sub.2, Ac-EVI-NH.sub.2, Ac-EGAVLI-NH.sub.2, Ac-EGAVIL-NH.sub.2, and Ac-EGAVIA-NH.sub.2, wherein Orn=ornithine, Dap=2,3-diaminopropionic acid, Dab=2,4-diaminobutyric acid, g=glycolic acid and a=L-lactic acid.

13. The amphiphilic peptide of claim 1 comprised in a composition or a hydrogel.

14. The amphiphilic peptide of claim 1, wherein amphiphilic peptide has one or more properties selected from the group consisting of: stable, biocompatible, biodegradable, biomimetic, xenofree, injectable, thixotrophic, substantially non-mutagenic, substantially resistant to enzymatic degradation, responsive to stimulus, responsive to change in pH, responsive to change in salt concentration, responsive to change in temperature, compatible with bioprinting and has a storage modulus of at least 1 kPa.

15. (canceled)

16. A method of treating a subject in need of tissue regeneration, the method comprising administering the amphiphilic peptide of claim 1 or a composition or a hydrogel comprising the amphiphilic peptide into the subject in need thereof.

17. (canceled)

18. A method of cell, tissue or organoid culture, the method comprising: culturing the cell, the tissue or the organoid in contact with the amphiphilic peptide of claim 1, or a composition or a hydrogel of the amphiphilic peptide.

19. The amphiphilic peptide of claim 1, wherein the amphiphilic peptide is comprised in a hydrogel and when single cells are seeded on or in the hydrogel, the hydrogel is more capable of promoting cell migration and/or generating single colony as compared to a hydrogel composed of peptides having sequences that are inverted from the sequences of said amphiphilic peptide.

20. The amphiphilic peptide of claim 1 when used in culturing stem cell, tissue or organoid.

Description

BRIEF DESCRIPTION OF FIGURES

[0133] FIG. 1. Second generation self-assembling ultrashort peptide motif. (a) Sequence and structure of the first (exemplified by Ac-ILVAGK-NH.sub.2) and second (exemplified by Ac-KGAVLI-NH.sub.2) generation amphiphilic motifs. While the directionality of the peptide backbone is reversed, both peptide motifs consist of a chain of aliphatic amino acids with increasing hydrophilicity, terminating with a polar residue such as lysine. (b) During self-assembly, the inverted sequence transitions from random coil (dotted line) to alpha-helical (dashed line) to beta secondary structures (solid line) with increasing peptide concentration. This suggests that the peptides form intermolecular helical pairs which subsequently stack into beta-turn fibrils that aggregate into nanofibers and sheets visible under (c) field emission scanning electron microscopy. The resulting macromolecular scaffolds entrap water to form clear hydrogels. (d) The peptides demonstrate salt-enhanced gelation, (e) forming stiff hydrogels with storage moduli of up to 20 kPa in buffered saline solutions.

[0134] FIG. 2. Self-assembly of the inverted tripeptide sequence. Peptide conformational changes from random coil (dotted line) to α-helical intermediates (dashed line) to β-fibrils (solid line) as concentration increases. The peptide dimers subsequently stack in fibrils that aggregate into nanofibers and sheets, which entrap water to form hydrogels. The nanofibrous architecture, as observed using field emission scanning microscopy, resembles extracellular matrix. The fibers extend into the millimeter range and readily condense into sheets. The fibers form interconnected three-dimensional scaffolds which are porous.

[0135] FIG. 3. Second generation ultrashort peptides with (a) free N-terminus and (b) glycine and histidine as the polar moieties are capable of macromolecular assembly into gels.

[0136] FIG. 4. Self-assembly of Depsipeptides. (a) Like their parent peptide sequences, depsipeptides also self-assemble into hydrogels in aqueous conditions. Gelation can be enhanced by increasing pH, salt and depsipeptide concentration. (b) Depsipeptides undergo the same secondary structure transitions from random coil to α-helical and subsequently to β-type structures with increasing concentration.

[0137] FIG. 5. Ultrashort tripeptides with amidated aspartic acid at the C-terminus exhibit self-assembly into nanofibrous hydrogels following dissolution in water and PBS. (a), (b) Nanofibrous microarchitecture of Ac-DVI-NH.sub.2 as revealed by field emission scanning microscopy. (c) Thixotropic behavior of Ac-DVI-NH.sub.2 hydrogels is reflected by recovery of the intact gel following disturbance by vortexing. Left: Ac-DVI-NH.sub.2 in a gel state before vortexing. Middle: Ac-DVI-NH.sub.2 changed to a fluid state immediately after vortexing. Right: Ac-DVI-NH.sub.2 returned to gel state after standing for 10 minutes after vortexing.

[0138] FIG. 6. 3D culture of human pluripotent stem cells. (a) H1 human embryonic stem cells were cultured in Ac-KGAVLI-NH.sub.2 hydrogel droplets under the following conditions: (i) in a proliferation media at a cell concentration of 2×10.sup.6 cells/mL; (ii) in a proliferation media at a cell concentration of 5×10.sup.6 cells/mL; and (iii) in an endoderm differentiation media at a cell concentration of 5×10.sup.6 cells/mL. The cells migrated and proliferated within the gel to cluster around a central nucleus at seeding densities exceeding 5×10.sup.6 cells/m L, while several colonies were observed to be formed at the lower seeding density of 2×10.sup.6 cells/m L. This behavior is independent of media formulation, and in over 95% of the colonies for H1 and H9 cells, only one central stem cell colony was obtained. (b) The H1 human embryonic stem cells retained their pluripotency, as evident from the staining of Oct4 and Tral-60 biomarkers, when cultured in mTESR media. (c) The Ac-KGAVLI-NH.sub.2 peptide was non-mutagenic, as evident from the lack of chromosomal aberrations after 5 passages of 3D culture. The hydrogel can thus be applied towards bioprinting of consistent stem cell colonies for high-throughput screening applications.

[0139] FIG. 7. 3D “one-pot” endoderm organoid derivation. (a) Encapsulated H1 embryonic stem cells were directly differentiated into definitive endoderm and subsequently hindgut spheroids without going through intermediate 2D culture steps. (b) Definitive endoderm differentiation was verified by confocal staining of Sox17 and FoxA2 biomarkers. 90% of the cells express Sox17, as determined by flow cytometry. (c) Similarly, expression of hindgut biomarker Cdx2 was observed on day 9, following further differentiation.

[0140] FIG. 8. 3D differentiation of H9 embryonic stem cells encapsulated in 8 mg/mL Ac-KGAVLI-NH2 hydrogel droplets. (a) Definitive endoderm differentiation was verified by confocal staining of Sox17 and FoxA2 biomarkers. 90% of the cells express Sox17, as determined by flow cytometry. (b) Similarly, expression of hindgut biomarker Cdx2 was observed on day 9, following further differentiation.

[0141] FIG. 9. Porcine wound healing study of peptide hydrogel wound dressings; Three peptide hydrogels applied using a polydimethylsiloxane mould to 8 cm×8 cm excisional wound. Wound dressing changes carried out weekly for 6 weeks with application of fresh hydrogel. P1: Ac-HGAVLI-NH2; P2: SGAVLI-NH2; P3: Ac-KGAVLI-NH2; C: control

EXAMPLES

[0142] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural, electrical and optical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments.

Materials and Methods

[0143] Materials. All peptides used in this study were either synthesized by solid-phase peptide synthesis, purified using high-performance liquid chromatography mass spectrometry and lyophilized, or purchased from Bachem AG (Bubendorf, Switzerland). Human H1 and H9 embryonic stem cells were purchased from WiCell Research Institute (Madison, Wis.). Reagents for culture of human embryonic stem cells were purchased from Stem Cell Technologies (British Columbia, Canada). All other cell culture reagents were purchased from Life Technologies (Carlsbad, Calif.). For immunohistochemistry, the primary antibodies used were Ab19857 rabbit polyclonal IgG against Oct 4 (Abcam, Cambridge, Mass.), SC-21705 mouse monoclonal IgM against Tra-I-60 (Santa Cruz Biotechnology Inc, Dallas, Tex.), AF1924-SP goat polyclonal IgG against human Sox17 (R&D Systems, USA), MAB2400-SP rabbit monoclonal IgG against human FoxA2 (R&D Systems, USA) and, MAB3665-SP mouse monoclonal IgG against human Cdx2 (R&D Systems, USA). 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Carlsbad, Calif.) was used to stain the actin cell nuclei.

[0144] Preparation of Hydrogels.

[0145] Lyophilized peptide powder was dissolved in milliQ water and mixed for 30 seconds by vortexing to obtain a homogenous solution. 10% volume of 10-times phosphate-buffered saline was subsequently added and mixed by pipetting. Gelation occurred between seconds (PBS) to overnight (water), depending on the peptide concentration and solution used.

[0146] Circular Dichroism Spectroscopy.

[0147] CD spectra were collected with a Jasco CD spectrophotometer fitted with a temperature controller, using quartz suprasil cuvettes with an optical path length of 1 mm. All samples were prepared in milIQ water and equilibrated for an hour at room temperature before measurement. Data acquisition was performed for wavelength range from 190-260 nm with a spectral bandwidth of 1.0 nm. All spectra were baseline-corrected using milliQ water as baseline. The mean residue ellipticity (MRE) was calculated as follows:

[0148] [θ]=θ/(10.Math.N.Math.c.Math.l) where 8 represents the ellipticity in millidegrees, N the number of amino acid residues, c the molar concentration in mol.Math.L-1, and I the cell path length in cm.

[0149] Field Emission Scanning Electron Microscopy.

[0150] Hydrogel samples were flash frozen in liquid nitrogen and subsequently freeze-dried. Lyophilized samples were sputtered with platinum in a JEOL JFC-1600 High Resolution Sputter Coater. The coated sample was then examined with a JEOL JSM-7400F FESEM system using an accelerating voltage of 2-5 kV.

[0151] Rheology.

[0152] Hydrogel samples were prepared in polydimethysiloxane moulds to obtain approximately 1 mm thick, 8 mm diameter discs. Dynamic strain and oscillatory frequency sweep experiments were carried out using the ARES-G2 Rheometer (TA Instruments, Piscataway, N.J.) with 8 mm titanium parallel plate geometry. The readings of 3 samples were averaged for each condition.

[0153] 3D Encapsulation of Stem Cells.

[0154] H1 embryonic stem cells cultured on Matrigel were dissociated into single cells using TrypLE Express, and re-suspended in 50% mTESR in PBS at an approximate concentration of 4×10.sup.6, 10.sup.7 or 1.6×10.sup.7 cells/mL. 0.5 μL of cells was injected into a droplet of 2 μL 10 mg/mL peptide solution. Warmed culture media (mTESR) containing ROCK inhibitor Y-27632 was added for the first day and replaced by either mTESR or endoderm differentiation media subsequently. The endoderm differentiation protocol was adapted from Spence et al (2011). Briefly, the encapsulated cells were exposed to RPMI media containing 100 ng/mL Activin A, 2 mM glutamax, 1% penicillin-streptomycin and increasing concentrations of defined fetal bovine serum. After 3 days, the hydrogel droplets were washed with RPMI and incubated in hindgut differentiation media (RPMI media containing 1% defined fetal bovine serum, 1% penicillin-streptomycin, 2 mM glutamax, 500 ng/mL FGF4 and 500 ng/mL Wnt3A.

[0155] Immunohistochemistry and Confocal Microscopy Imaging.

[0156] Cell samples were fixed in 4% paraformaldehyde for 15 minutes and permeabilized in 0.01% Triton-X for 10 minutes. The encapsulated human embryonic stem cells were incubated at 4° C. overnight in 5% Bovine-Serum Albumin containing primary antibodies. The corresponding secondary antibodies and DAPI were applied for 90 minutes before the samples were imaged. Confocal microscopy was performed using a Zeiss LSM 510 microscope at the Institute of Medical Biology Microscopy Unit (A*STAR, Singapore).

EXAMPLES

[0157] Before the present disclosure, the effect of sequence inversion has never been reported or systematically studied. Because of the strict rules governing the design of self-assembling helical peptides, the directionality change in peptide backbone was expected to disrupt intra-helical hydrogen bonding and thus macromolecular organization. Beta-sheet peptide self-assembly was expected to be affected by a smaller extent due to their characteristic motif of alternating hydrophilic-hydrophobic residues, as well as the planar nature of intermolecular interactions. Almost palindromic beta-sheet sequences have been described.

[0158] Surprisingly, the self-assembly of ultrashort peptides into helical fibers was found to be unaffected by sequence inversion and the consequent reversal in peptide backbone direction. Trimeric and hexameric inverted sequences, exemplified by Ac-KVI-NH.sub.2 and Ac-KGAVLI-NH.sub.2 (FIG. 1a, FIG. 2), are observed to undergo the same secondary structure transitions as their parent sequences. Circular dichroism spectra at increasing concentrations of peptide are surrogate snapshots of the structural transitions that occur during macromolecular assembly. At low concentrations, the monomers adopt a random coil confirmation with a slight positive n-π* transition near 217 nm and large negative transition around 190 nm (FIG. 1b). At higher concentrations, alpha-helices with their characteristic signature of a negative n-π* transition near 222 nm and split π-π* transition with a negative peak near 208 nm were observed. Further increases in concentration saw the development of beta-turn structures with negative bands at 218 nm. As the structural transition profiles are virtually identical to that of the original parent sequences of Ac-IVK-NH.sub.2 and Ac-ILVAGK-NH.sub.2, it is surmised that they follow the same self-assembly mechanism wherein the peptide monomers form anti-parallel pairs and subsequently stack to form beta-turn fibrils. It is postulated that since the turns within each helical fibril are not covalently linked, each succeeding pair of peptides can rotate laterally during fibril assembly to maximize intermolecular hydrogen bonding and hydrophobic interactions. In contrast, freedom of movement is restricted in the longer 28-mer coiled-coil and 30-mer collagen mimetic motifs. As such, there is no mention in published literature that the reverse sequences of heptad coiled-coils and collagen-mimetic peptides can self-assemble. The present experiment indicates that in the case of ultrashort peptides, the building block that dictates the macromolecular assembly is the amphiphilic motif, regardless of peptide backbone orientation.

[0159] Interestingly, the inverted sequence with a free N-terminus KGAVLI-NH.sub.2 also undergoes the same conformational transitions (FIG. 3a). In the original Gen-1 motif, acetylation is integral to self-assembly. Without it, peptides do not self-assemble, possibly due to the ionization of the free amine group at the N-terminus which leads to unfavourable charge interactions with the other peptides that discourage self-assembly. In eliminating the need for N-terminal acetylation, the inverted motif significantly widens the field of candidates accessible for biomedical applications. It would offer better solubility profiles and stimuli-enhanced gelation for peptide subclasses with neutral residues as the polar moiety. More importantly, it implicates that polar, bioactive moieties which are not amino acids can be substituted into the assembling motif to generate functionalized assemblies. Solid phase peptide synthesis occurs from C- to N-terminus. As the last moiety to be coupled, the functional groups on the N-terminal moiety may not require as extensive protection, making the overall synthesis easier. The self-assembling motif can thus be harnessed for the display of bioactive epitopes. In contrast, the chemistry of the moiety-of-interest may not lend itself to facile coupling onto the resin, which is needed for preparing Gen-1 peptides. The hypothesis is supported by observations that Ac-GAVLI-NH.sub.2, representatives from the serine subclass (Ac-SGAVIA-NH.sub.2 and SGAVLI-NH.sub.2) and histidine subclass (Ac-HGAVLI-NH.sub.2 and Ac-HGAVIA-NH.sub.2) all formed thixotrophic gels in dimethylsulfoxide (FIG. 3b). In particular, the gelation behavior of Ac-GAVLI-NH.sub.2 suggests that the polar moiety need not be an amino acid and can be fulfilled by N-acetylation or other polar functional groups. By extension, depsipeptide analogs of the inverted motif also self-assemble into hydrogels in a stimuli-responsive fashion (FIG. 4a). These candidates were prepared by substituting the second (glycine) or third (L-alanine) amino acid with their alpha-hydroxy acid analogs (glycolic acid and L-lactic acid), giving rise to Ac-KgAVLI-NH.sub.2 and Ac-KGaVLI-NH.sub.2. In doing so, the N-terminus portions of the amide backbone are replaced with ester linkages. This results in reduced hydrogen bonding, as ester bonds are hydrogen acceptors but not donors. Despite so, circular dichroism spectra of these depsipeptide analogs show the same secondary structure transitions from random coil to α-helical and subsequently to β-type structures with increasing concentration. (FIG. 4b). By expanding the library of self-assembling sequences to encompass both non-acetylated peptides and depsipeptides, subclasses of motifs with better biodegradability can be defined, as N-terminal acetylation limits enzymatic degradation while ester bonds are more labile compared to amide bonds.

[0160] The inverted hexapeptide analogs self-assembled into nanofibrous hydrogels (FIG. 1c,d), similar to their Gen-1 counterparts. The gelation behavior is likewise enhanced by the addition of buffered salts and by increasing pH. Faster gelation kinetics at lower peptide concentrations were observed following mixing with phosphate-buffered saline (see Table 1 below).

TABLE-US-00001 TABLE 1 Secondary Minimum gelation Solubility structure concentration in Peptide in water transitions buffered saline Ac-ILVAGK-NH.sub.2 ++ Yes  3 mg/mL Ac-KGAVLI-NH.sub.2 +++ Yes  3 mg/mL KGAVLI-NH.sub.2 +++ Yes 20 mg/mL Ac-IVK-NH.sub.2 ++++ Yes >30 mg/mL  Ac-KVI-NH.sub.2 ++++ Yes >30 mg/mL 

[0161] The inverted sequence motif demonstrated better solubility in water and similar gelation behavior compared to the original motif. Both motifs demonstrate salt- and pH-enhanced gelation, having lower gelation concentrations in buffered saline. Unlike the original motif, N-terminal acetylation is not a pre-requisite for self-assembly as KGAVLI-NH.sub.2 with its free amine terminus also undergoes the same secondary structure transitions and forms hydrogels in buffered saline. In general, hexamer peptides demonstrate better gelation with lower minimum gelation concentrations.

[0162] The storage moduli of hydrogels prepared in buffered saline are also comparable to Ac-ILVAGK-NH.sub.2 at 10 kPa magnitude (FIG. 1e). Hexameric peptides are by far superior gelators, as the tripeptides remained solutions in all the conditions evaluated. It is postulated that self-assembly and by extension gelation, is a delicate balance between solubility and aggregation. The observations thus underscore the importance of the hydrophobic contribution towards self-assembly.

[0163] Expanding the observations beyond the lysine subclass, representative candidates with acidic and neutral polar moieties were explored. In particular, unlike its Gen-1 counterparts, Ac-DVI-NH.sub.2 bears C-terminus amidation to avoid zwitterion formation (Table 2 and FIG. 5). This inhibited solvation in physiologically buffered solutions, allowing the peptide to self-assemble into nanofibrous scaffolds (Table 2 and FIG. 5b).

TABLE-US-00002 TABLE 2 TGA Degradation Peptide [Peptide] .sub.min, water [Peptide] .sub.min, PBS Temperature (° C.) Ac-IVD 40.8 mg/mL 19.2 mg/mL 323.6 Ac-IVD-NH.sub.2 44.9 mg/mL 33.9 mg/mL 328.5 Ac-DVI-NH.sub.2 11.6 mg/mL  9.9 mg/mL 321.5

[0164] The resulting hydrogel is thixotrophic (FIG. 5c). This observation is contrary to computational simulations previously developed by Smadbeck et al. In their study, a two-stage computation design framework which incorporates metrics for potential energy, fold specificity and approximate association affinity was applied to the design of ultrashort peptides. Ac-IVD was designated as the structural template for generating novel tripeptide hydrogel candidates. The inverted sequences were not amongst the shortlisted promising candidates. Further validations that included shuffled sequence variations suggested that Ac-EVI, the only inverted sequence experimentally evaluated, may not form hydrogels. While it is plausible that C-terminus amidation may have an unexpectedly larger contribution towards assembly, the marked differences with the present experimental observations nonetheless reflect the disconnection between in silico and in vitro models. This highlights the need for deeper understanding of the parameters involved in peptide self-assembly, in order to build more predictive computational algorithms.

[0165] An unexpected advantage of inverting the printable Ac-ILVAGK-NH.sub.2 Gen-1 sequence was improved biological properties for pluripotent stem cell culture. In a time-course study, cell behaviour was tracked in representative hydrogel droplets, and the same hydrogel droplets were imaged for six days. Using Ac-KGAVLI-NH.sub.2 as the matrix for bioprinting hydrogel droplets encapsulating cells, higher retention of the expanding colonies was observed at day 7. Almost all of the hydrogel droplets were intact after 7 days of culture. More interestingly, at threshold cell encapsulation densities exceeding 5×10.sup.6 cells/m L, single cell suspensions of embryonic stem cells H1 and H9 were more likely to aggregate into a single colony when encapsulated in Ac-KGAVLI-NH.sub.2 (FIG. 6a) whereas several colonies developed within Ac-ILVAGK-NH.sub.2. The proportion of hydrogel droplets with just a single colony exceeded 95%. Lower cell seeding densities (2×10.sup.6 cells/mL) resulted in the formation of several colonies within a single droplet. The same observations were made when differentiation media was applied. When cultured in defined mTESR media, the cells retained their pluripotency, as evident from the staining of Oct4 and Tral-60 biomarkers (FIG. 6b). The peptide was non-mutagenic, as evident from the lack of chromosomal aberrations after 5 passages of 3D culture (FIG. 6b). The enhanced stability, and more importantly, consistent and reproducible conditions for single colony development makes Ac-KGAVLI-NH.sub.2 hydrogels ideal matrices for stem cell differentiation into organoids. Stem cell can thus be encapsulated within Ac-KGAVLI-NH.sub.2 hydrogel droplets for long term culture. Coupled with its printability, this peptide can be exploited for automation of stem cell culture and “one-pot” organoid derivation for high-throughput screening of therapeutics. Lower cell encapsulation densities tended to give rise to several colonies within a single drop and would thus be more useful for stem cell expansion.

[0166] The example self-assembling sequences are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Basic Neutral Acidic Amino Amino Amino Acid Acid Acid Constituents Constituents Constituents N-terminal Ac-KVI-NH.sub.2 Ac-GAVLI-NH.sub.2 Ac-DVI-NH.sub.2 acetylated Ac-KGAVLI-NH.sub.2 Ac-SGAVLI-NH.sub.2 Ac-DGAVLI-NH.sub.2 Ac-KGAVIL-NH.sub.2 Ac-SGAVIL-NH.sub.2 Ac-DGAVIL-NH.sub.2 Ac-KGAVIA-NH.sub.2 Ac-SGAVIA-NH.sub.2 Ac-EVI-NH.sub.2 Ac-RVI-NH.sub.2 Ac-TGAVLI-NH.sub.2 Ac-EGAVLI-NH.sub.2 Ac-RGAVLI-NH.sub.2 Ac-TGAVIL-NH.sub.2 Ac-EGAVIL-NH.sub.2 Ac-RGAVIL-NH.sub.2 Ac-TGAVIA-NH.sub.2 Ac-EGAVIA-NH.sub.2 Ac-RGAVIA-NH.sub.2 Ac-HVI-NH.sub.2 Ac-HGAVLI-NH.sub.2 Ac-HGAVIL-NH.sub.2 Ac-HGAVIA-NH.sub.2 Ac-OrnVI-NH.sub.2 Ac-OrnGAVLI-NH.sub.2 Ac-OrnGAVIL-NH.sub.2 Ac-OrnGAVIA-NH.sub.2 Ac-DapVI-NH.sub.2 Ac-DapGAVLI-NH.sub.2 Ac-DapGAVIL-NH.sub.2 Ac-DapGAVIA-NH.sub.2 Ac-DabVI-NH.sub.2 Ac-DabGAVLI-NH.sub.2 Ac-DabGAVIL-NH.sub.2 Ac-DabGAVIA-NH.sub.2 Free N- KGAVLI-NH.sub.2 SGAVLI-NH.sub.2 terminus KGAVIL-NH.sub.2 SGAVIA-NH.sub.2 Depsipeptides Ac-KgAVLI-NH.sub.2 Ac-SgAVLI-NH.sub.2 Ac-KGaVLI-NH.sub.2 Ac-SGaVLI-NH.sub.2 KgAVLI-NH.sub.2 SgAVLI-NH.sub.2 KGaVLI-NH.sub.2 SGaVLI-NH.sub.2 Ac-KgAVIL-NH.sub.2 Ac-SgAVIA-NH.sub.2 Ac-KGaVIL-NH.sub.2 Ac-SGaVIA-NH.sub.2 KgAVIL-NH.sub.2 SgAVIA-NH.sub.2 KGaVIL-NH.sub.2 SGaVIA-NH.sub.2

EXAMPLES

Amphiphilic Short Peptide Motif for Wound Healing

[0167] A porcine wound healing study was performed to evaluate the peptide hydrogels for wound healing applications. An 8 cm×8 cm excisional full thickness wound was created on the back of the animal, and three of the peptide hydrogels were applied using a polydimethylsiloxane mould. Wound dressing changes were carried out weekly for 6 weeks, with application of fresh hydrogel. The results point towards an effective reduction of wound size for all three peptides tested (i.e. P1: Ac-HGAVLI-NH.sub.2; P2: SGAVLI-NH.sub.2; P3: Ac-KGAVLI-NH.sub.2). Representative images of the original wound sites and appearance after 1, 4 and 6 weeks are shown in FIG. 9.

[0168] In recent years, stem cell-derived organoid cultures are increasingly being used as models to study tissue and disease development, and evaluate therapeutic candidates. In particular, the use of patient-derived organoids has revolutionized personalized medicine as they are superior predictive in vitro models. Intestinal organoids prepared from colon biopsies have successfully been used to identify patients who will respond to an experimental cystic fibrosis therapy. The development of such organoid models was boosted by advances in defined growth factor cocktails, which mimic the various stem cell niches. The biochemical cues stimulate the self-organization of cells into structures that partially recapitulate key tissue traits such as the spatial arrangement of heterogeneous cells, cellular interactions and some biological processes. A crucial step for organoid development is the encapsulation of stem cell-derived progenitors or adult stem cells into Matrigel (solubilized basement membrane preparation extracted from murine sarcoma). This suggests that cell-substrate interactions in a 3D microenvironment are integral for cell migration during organoid differentiation. Due to its nanofibrous macromolecular architecture, ultrashort peptide hydrogels would be an ideal substitute for Matrigel. When paired with defined media, peptide hydrogels constitute a completely defined culture environment, free of xenogenic components. Although such a synthetic matrix would be devoid of natural ligands, bioactive motifs can be easily incorporated using various conjugation strategies. This will confer better control over the biochemical makeup of the microenvironment, and predispose differentiation into preferred lineages. As a proof-of-concept, the protocol described by Spence et al. was adapted for the direct 3D differentiation of embryonic stem cells into hindgut spheroids (FIG. 7a). The colonies were first differentiated into definitive endoderm by applying Activin A with increasing concentrations of defined serum. Definitive endoderm induction was verified by confocal imaging of Sox17 and FoxA2 biomarkers (FIG. 7b, FIG. 8a). 90% of the cells express Sox17, comparable to 92% of cells differentiated under 2D (Matrigel culture) conditions. Further differentiation into mid- and hindgut was achieved through application of high concentrations of FGF4 and Wnt3A, as seen from the expression of Cdx2 biomarker (FIG. 7c, FIG. 8b).

[0169] The motif is more important than sequence in dictating the self-assembly of ultrashort peptides. While the physicochemical properties are largely unchanged, sequence inversion of ultrashort self-assembling peptides can have significant effect on its biological properties. Most notably, when used as synthetic 3D stem cell culture substrates, it was observed that Ac-KGAVLI-NH.sub.2 prompted enhanced cell migration and better consistency in generating single colonies for reproducible organoid derivation, compared to its Gen1 analog. Its stimuli-responsive gelation properties can be harnessed for bioprinting, to reproducibly encapsulate cells for organoid differentiation. This scalable and customizable manufacturing technique can be automated for large-scale culture or for generating defined multi-domain tissue constructs for screening therapeutics, studying disease development and elucidating cellular interactions.

[0170] It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

REFERENCE

[0171] 1. Mendes, A. C.; Baran, E. T.; Reis, R. L.; Azevedo, H. S., Self-assembly in nature: using the principles of nature to create complex nanobiomaterials. Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology 2013, 5 (6), 582-612. [0172] 2. Loo, Y.; Zhang, S.; Hauser, C. A., From short peptides to nanofibers to macromolecular assemblies in biomedicine. Biotechnol Adv 2012, 30 (3), 593-603. [0173] 3. (a) Fallas, J. A.; O'Leary, L. E.; Hartgerink, J. D., Synthetic collagen mimics: self-assembly of homotrimers, heterotrimers and higher order structures. Chem Soc Rev 2010, 39 (9), 3510-27; (b) Gauba, V.; Hartgerink, J. D., Self-assembled heterotrimeric collagen triple helices directed through electrostatic interactions. J Am Chem Soc 2007, 129 (9), 2683-90. [0174] 4. (a) Bromley, E. H.; Sessions, R. B.; Thomson, A. R.; Woolfson, D. N., Designed alpha-helical tectons for constructing multicomponent synthetic biological systems. J Am Chem Soc 2009, 131 (3), 928-30; (b) Papapostolou, D.; Smith, A. M.; Atkins, E. D.; Oliver, S. J.; Ryadnov, M. G.; Serpell, L. C.; Woolfson, D. N., Engineering nanoscale order into a designed protein fiber. Proc. Natl. Acad. Sci. USA 2007, 104 (26), 10853-8. [0175] 5. (a) Frederix, P.; Patmanidis, I.; Marrink, S. J., Molecular simulations of self-assembling bio-inspired supramolecular systems and their connection to experiments. Chemical Society reviews 2018, 47 (10), 3470-3489; (b) Makam, P.; Gazit, E., Minimalistic peptide supramolecular co-assembly: expanding the conformational space for nanotechnology. Chemical Society reviews 2018, 47 (10), 3406-3420. [0176] 6. (a) Zhang, S., Emerging biological materials through molecular self-assembly. Biotechnol Adv 2002, 20 (5-6), 321-39; (b) Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A., Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane. Proc. Natl. Acad. Sci. USA 1993, 90 (8), 3334-8. [0177] 7. Loo, Y.; Lakshmanan, A.; Ni, M.; Toh, L. L.; Wang, S.; Hauser, C. A., Peptide Bioink: Self-Assembling Nanofibrous Scaffolds for Three-Dimensional Organotypic Cultures. Nano letters 2015, 15 (10), 6919-25. [0178] 8. Hauser, C. A.; Deng, R.; Mishra, A.; Loo, Y.; Khoe, U.; Zhuang, F.; Cheong, D. W.; Accardo, A.; Sullivan, M. B.; Riekel, C.; Ying, J. Y.; Hauser, U. A., Natural tri- to hexapeptides self-assemble in water to amyloid beta-type fiber aggregates by unexpected alpha-helical intermediate structures. Proc Natl Acad Sci USA 2011, 108 (4), 1361-6. [0179] 9. Smadbeck, J.; Chan, K. H.; Khoury, G. A.; Xue, B.; Robinson, R. C.; Hauser, C. A.; Floudas, C. A., De novo design and experimental characterization of ultrashort self-associating peptides. Accounts of chemical research 2014, 10 (7), e1003718. [0180] 10. Clevers, H., Modeling Development and Disease with Organoids. Cell 2016, 165 (7), 1586-1597. [0181] 11. (a) Saini, A., Cystic Fibrosis Patients Benefit from Mini Guts. Cell Stem Cell 19 (4), 425-427; (b) Dekkers, J. F.; Wiegerinck, C. L.; de Jonge, H. R.; Bronsveld, I.; Janssens, H. M.; de Winter-de Groot, K. M.; Brandsma, A. M.; de Jong, N. W.; Bijvelds, M. J.; Scholte, B. J.; Nieuwenhuis, E. E.; van den Brink, S.; Clevers, H.; van der Ent, C. K.; Middendorp, S.; Beekman, J. M., A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nature medicine 2013, 19 (7), 939-45. [0182] 12. Yin, X.; Mead, B. E.; Safaee, H.; Langer, R.; Karp, J. M.; Levy, O., Engineering Stem Cell Organoids. Cell Stem Cell 2016, 18 (1), 25-38. [0183] 13. Spence, J. R.; Mayhew, C. N.; Rankin, S. A.; Kuhar, M. F.; Vallance, J. E.; Tolle, K.; Hoskins, E. E.; Kalinichenko, V. V.; Wells, S. I.; Zorn, A. M.; Shroyer, N. F.; Wells, J. M., Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 2011, 470 (7332), 105-9.

APPLICATIONS

[0184] Peptide self-assembly is driven by secondary structure and intermolecular interactions, which are in turn dictated by peptide sequence. In view of the strict rules governing the design of helical self-assembling motifs, it is surprising that the self-assembly of ultrashort peptides into helical fibers is found to be unaffected by sequence inversion and the consequent reversal in peptide backbone direction.

[0185] During self-assembly, trimeric and hexameric inverted sequences are observed to undergo the same secondary structure transitions as their parent sequences, forming rigid, nanofibrous hydrogels in physiologically buffered saline. The results suggest that motif is more important than sequence in dictating the self-assembly of ultrashort peptides.

[0186] While the physicochemical properties are largely unchanged, sequence inversion of ultrashort self-assembling peptides can have significant effect on its biological properties. Most notably, when used as synthetic 3D stem cell culture substrates, it was observed that Ac-KGAVLI-NH.sub.2 prompted enhanced cell migration and consistency in generating single colonies for organoid derivation, compared to Ac-ILVAGK-NH.sub.2. In view of its reproducibility and printability, this new subset of self-organizing nanobiomaterials is well-positioned to facilitate the bioengineering of scalable and customizable in vitro tissue models.