Peptide-based hydrogels and dual system thereof

Abstract

The present disclosure generally relates to peptide-based hydrogels, more specifically cross-linked peptide-based hydrogels. In particular, peptide-based hydrogels that can be tailored with on-demand switchable mechanical properties for therapeutic use. The present disclosure also relates to self-assembling peptides for hydrogel formation, wherein the self-assembling peptides show improved mechanical and stability properties.

Description

PEPTIDE-BASED HYDROGELS AND DUAL SYSTEM THEREOFFIELD

[0001] The present disclosure generally relates to peptide-based hydrogels, more specifically cross-linked peptide-based hydrogels. In particular, peptide-based hydrogels that can be tailored with on-demand switchable mechanical properties for therapeutic use.BACKGROUND

[0002] Hydrogels are widely used in therapeutics, experimentally and clinically. Different hydrogelators can form hydrogels, including natural biological molecules such as chitosan, hyaluronic acid, collagen, fibroin or acellular ECM, synthetic molecules such as polyethylene glycol, polyacrylamide, polymethacrylamide, and supramolecular hydrogelators such as self-assembling peptides, nucleobases or monosaccharide. Self-assembling peptide (SAP) hydrogels, as a supramolecular hydrogelator, have attracted much attention for their bioactivity, biocompatibility, biodegradability and tunable mechanical stability. More importantly, the structurally changeable properties of SAPs make them compatible to encapsulate cells, exosomes and growth factors.

[0003] With building blocks including the natural / unnatural amino acids , selfassembling peptides, as one of the supramolecular hydrogelators, can be self-organized into ordered nanofibers and further into well-ordered scaffolds in water. Since their discovery, diverse classes of short SAPs have been applied to a wide range of applications, including 3D tissue cell culture, reparative and regenerative medicine, tissue engineering and slow drug release.

[0004] As cross-linking is also useful in designing functional materials composed of non-covalently assembled peptide fibers, various cross-linked peptide fibers are known to have been developed. Disulfide bonds in cysteine residues and electrostatic interactions with lysine or aspartic acid residues have frequently been utilized as crosslinking points. Hydrogels formed after the cross-linking of peptide fibers may impactthe storage modulus (G’) of the chemical hydrogel. In general, cross-linking methods are classified into two categories: chemical cross-linking by covalent bonds and physical cross-linking by non-covalent bonds. Chemical cross-linking by strong covalent bonds is advantageous to form hydrogels with high mechanical strength, while physical cross-linking by reversible non-covalent bonds can provide unique functions, and so it will be apparent to the skilled person that there is an on-going need for improved hydrogels.SUMMARY

[0005] The present disclosure provides an alternative and improved peptide-based hydrogel, more specifically cross-linked peptide-based hydrogels. The present inventors have undertaken research and development to provide cross-linked peptide- based hydrogels that can be tailored with on-demand switchable mechanical properties for therapeutic use.

[0006] In one aspect there is provided a crosslinker of Formula 1-1 :Formula 1-1 wherein R1and R2are each independently selected from the group comprising or consisting of Ci-ealkyl, -O-Ci-ealkyl, Ci-ehaloalkyl, -O-Ci-ehaloalkyl, C2-ealkenyl, C2 ealkynyl, 3-10-membered carbocyclyl, and 3-10-membered heterocyclyl; wherein each 3-10-membered carbocyclyl and 3-10-membered heterocyclyl, is unsubstituted or substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence; wherein R3aand R3bare each independently selected from C=C-R4a, C=C-R4b, and SH, wherein when R3ais C=C-R4aor C=C-R4b, R3bis not SH, or when R3bis C=C- R4aor C=C-R4b, R3ais not SH; andwherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl.

[0007] In another aspect there is provided a hydrogel comprising a reaction product of (i) a compound comprising a thiol, an alkyne, or an alkene and (ii) a cross-linking compound of Formula II- 1 :Formula II-l wherein R1and R2are each independently selected from the group comprising or consisting of Ci-ealkyl, -O-Ci-ealkyl, Ci-ehaloalkyl, -O-Ci-ehaloalkyl, C2-ealkenyl, C2- ealkynyl, 3-10-membered carbocyclyl, and 3-10-membered heterocyclyl; wherein each 3-10-membered carbocyclyl and 3-10-membered heterocyclyl, is unsubstituted or substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence; wherein R3aand R3bare each independently selected from C=C-R4aand C=C- R4b, and SH, wherein when R3ais C=C-R4aor C=C-R4b, R3bis not SH, or when R3bis C=C-R4aor C=C-R4b, R3ais not SH; and wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl.

[0008] The thiol, alkyne, or alkene compound may be selected from the group comprising or consisting of a peptide, a protein, or an enzyme, wherein the peptide, protein or enzyme comprises at least one thiol, alkyne, or alkene group. The compound may be a peptide comprising at least one thiol, alkyne, or alkene group.

[0009] In another aspect there is provided a process for preparing a hydrogel, comprising (i) obtaining a thiol, alkyne, or alkene compound, (ii) obtaining a crosslinking compound of Formula II- 1, (iii) mixing aqueous solutions of the thiol, alkyne,or alkene compound of step (i) and the cross-linker of step (ii) to form an aqueous mixture.

[0010] In another aspect there is provided a method of controlled release of an active agent from a hydrogel, wherein the hydrogel is as defined herein or prepared according to the process defined herein, on contact with a diol containing molecule or polymer. In another aspect there is provided a method of storing an active agent supported by or encapsulated within a hydrogel, wherein the hydrogel is as defined herein or prepared according to the process defined herein.

[0011] In another aspect there is provided a use of a hydrogel as defined herein as an injectable, drug delivery vehicle, tissue engineering, or carrier in controlled release of an active agent. The release of the active agent from the hydrogel may be activated by exposure to a diol containing molecule or polymer.

[0012] In another aspect there is provided a hydrogel bioink composition for bioprinting, comprising the hydrogel as defined herein or the hydrogel prepared according to the process defined herein. In an embodiment, the hydrogel bioink composition may be laden with cells.

[0013] In another aspect there is provided a process for preparing a cell supportive structure, the process comprising: (i) preparing a bioink composition comprising the hydrogel as defined herein or the hydrogel prepared according to the process defined herein; and (ii) 3D-printing the composition to produce a support.BRIEF DESCRIPTION OF THE FIGURES

[0014] Preferred embodiments of the present disclosure will now be further described and illustrated, by way of example only, with reference to the accompanying figures in which:

[0015] Figure 1 is a comparison of the storage modulus of the modified peptide sequences comprising at least one P-amino acid with two known peptide sequences at 2wt.% solid content at 20 °C.

[0016] Figure 2 is a comparison of the shear-thinning behaviour of the peptide hydrogels comprising at least one P-amino acids with two known peptide sequences at 2 wt.% solid content at 20 °C.

[0017] Figure 3 is a comparison of the recoverability of the peptide hydrogels comprising at least one P-amino acids with known peptide sequences at 2 wt.% solid content at 20 °C.

[0018] Figure 4 shows the injectability of the peptide hydrogel (pep- 13) at 2 wt.% solid content at room temperature.

[0019] Figure 5 is a comparison of the stability of the modified peptide sequences comprising at least one P-amino acid with two known peptide sequences at 2wt.% solid content at 37 °C.

[0020] Figure 6 is a comparison of the percentage of released active agent (BSA- FIRC) encapsulated within a peptide hydrogel comprising at least one P-amino acids with known peptide sequences at 2 wt.% solid content at 37 °C.

[0021] Figure 7 is a comparison of the stability of crosslinked and un-crosslinked peptide hydrogel with 2wt.% solid content at 37 °C in IX PBS. The molar ratio of peptide and 8-arm PEG-Norbomene were 8: 1. The peptide sequence is FEFQFKGGC. The crosslinked hydrogel were exposed to UV light (365 nm, intensity of 174 mW) for 1 min.

[0022] Figure 8 shows the changes in the rheological behaviour of peptide / 8-arm PEG-Norbornene 2wt.% solid content at 37 °C in IX PBS. The ratio of peptide and 8- arm PEG-Norbornene were 8: 1. The peptide sequence is FEFQFKGGC. The crosslinked hydrogel were exposed to UV light (365 nm, intensity of 174 mW) for 1 min.

[0023] Figure 9 shows the partial NMR spectrum (6 6 to 5 8 ppm) of a mixture of SEQ ID 41 and 42 in D2O at pH 4.3 and pH 7 in comparison to the NMR spectrum of SEQ ID 41 alone and SEQ ID 42 alone. At pH4 the NMR spectrum is just a mixture of the two sequences, i.e. no reaction has occurred. At pH 7, the NMR spectrum hassignificantly changed from the mixture of the two sequences, i.e. a reaction has occurred confirming that the boronic ester containing crosslinker has been prepared.

[0024] Figure 10 shows the partial NMR spectrum (6 6.1 to 8 7.2 ppm) of the adduct of SEQ ID 41 and 42 in D2O without exposure to glucose and with exposure to 25 mg / ml and 75mg / ml glucose. The spectrum changes considerably with increasing exposure to glucose indicating exchange in the boronic ester, effectively cleaving the crosslinker.

[0025] Figure 11 shows the partial NMR spectrum of SEQ ID 43 and 44 alone in D2O alone with their mixtures at pH 3.61, 6.6 and 10.6. No reaction occurred at pH 3.61, whilst significant changes in the spectra at pH 6.6 and 10.6 (upon addition of NaOD) are evidence that the successful formation of a boronic ester between SEQ ID43 and 44 under neutral to basic conditions. The shifting of the peaks provides clear evidence of the structural changes occurring as the pH is adjusted, demonstrating the pH-dependent nature of the crosslinking reaction.

[0026] Figure 12 shows the reaction of the formed dual crosslinker (SEQ ID 43 with SEQ ID 44, 20 mg / ml) with 8-arm PEG-Norborene (50mg / ml) in D2O under UV irradiation in the presence of a photoinitiator (Irgacure 2959, 5mg / ml). After exposure to UV light, the peaks associated with the double bonds of NB almost disappeared, confirming the occurrence of the thiol-ene reaction. This disappearance of the double bond peaks indicates that the thiol groups on the crosslinker successfully reacted with the alkenes on the 8-arm PEG-NB, forming a covalent network. This successful thiolene reaction is crucial for creating a stable hydrogel network, as evidenced by the significant changes in the 'H NMR spectrum. The disappearance of the peaks not only confirms the reaction but also demonstrates the efficiency of the crosslinking process under UV light.

[0027] Figure 13 shows that the crosslinker formed by the adduct of SEQ ID 43 and44 can form a network via thiol ene reaction with 8-arm PEG Norborene upon UV light exposure (from 120 s) in the presence of a photoinitiator (Irgacure 2959). 8-arm PEG- Norborene cannot form a gel alone even upon UV light exposure. However, the increase in G’ and G” and crossover of them of the adduct of SEQ ID 43 and 44 with 8-arm PEG Norborene upon UV light exposure indicates the formation of a stable gel network, confirming the successful crosslinking between the dual crosslinker and 8-arm PEG-N orb orene .

[0028] Figure 14 shows the NMR spectrum of SEQ ID 45 with 8-arm PEG Norborene before and after exposure to UV light. The disappearance of the norbornene ethylenic protons indicate that the thiol-ene reaction has occurred.DETAILED DESCRIPTION

[0029] The present disclosure describes the following various non-limiting examples, which relate to investigations undertaken to identify alternative and improved peptide- based hydrogels, more specifically cross-linked peptide-based hydrogels. A growing area of interest for peptide hydrogels is that of tissue engineering, which involves the use of living cells as building blocks to repair or replace portions of or whole tissues, e.g. bone, cartilage, blood vessels, bladder, skin, muscle, etc. Cells are typically implanted or 'seeded' into an artificial structure capable of supporting three-dimensional tissue formation. These structures, called scaffolds, serve as support while mimicking the in vivo environment of the cells. Scaffolds can be made of different materials, which can be of natural or synthetic sources and can be biodegradable or not. Examples of natural materials include collagen and fibrin, and polysaccharidic materials, like chitosan or glycosaminoglycans (GAGs), while synthetic materials include biodegradable polyesters such as PLGA and its analogues. Hydrogel based scaffolds have been developed of which peptide-based hydrogels have gained particular interest because of the properties mentioned above and in particular their good cell compatibility.DefinitionsGeneral Definitions and Terms

[0030] In the following description, reference is made to the accompanying figures which form a part hereof, and which is shown, by way of illustration, several embodiments. It is understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present disclosure.

[0031] With regards to the definitions provided herein, unless stated otherwise, or implicit from context, the defined terms and phrases include the provided meanings. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired by a person skilled in the relevant art. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0032] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0033] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0034] Throughout this disclosure, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

[0035] Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the examples, steps, features, methods, compositions,coatings, processes, and coated substrates, referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0036] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0037] Unless otherwise indicated, the terms “first,” “second,” etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and / or a higher-numbered item (e.g., a “third” item).

[0038] As used herein, the phrase “at least one of’, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

[0039] It is to be appreciated that certain features that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0040] Throughout the present specification, various aspects and components of the invention can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to havespecifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 5, 5.5 and 6, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0041] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0042] Any embodiment of the present disclosure herein shall be taken to apply mutatis mutandis to any other embodiment of the disclosure unless specifically stated otherwise.

[0043] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for embodiments, in organic synthetic chemistry, cell culture, molecular genetics, immunology, peptide chemistry, protein chemistry, and biochemistry).

[0044] As used herein, the term "peptide" refers to a short truncated protein generally comprising at least 4 naturally occurring or synthetic amino acids which can also be further modified including covalently linking the peptide bonds of the alpha carboxyl group of a first amino acid and the alpha amino group of a second amino acid by eliminating a molecule of water. The amino acids can be either those naturally occurring amino acids or chemically synthesized variants of such amino acids or modified forms of these amino acids which can be altered from their basic chemical structure by addition of other chemical groups which can be found to be covalently attached to them in naturally occurring compounds.

[0045] As used herein, the term "protein" refers to a polypeptide made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. Generally a protein is a large polypeptide such as a polypeptide comprising at least 30 amino acids. Generally, a protein will have a secondary structure due to the folding of the polypeptide chain.

[0046] As used herein, the term "hydrogelator" refers to a peptide that is capable of forming a hydrogel. The hydrogelator according to present invention is a peptide hydrogelator. The hydrogel described herein is preferably self-assembling.

[0047] As used herein, the term “controlled release” refers to the presentation or delivery of active agents in response to stimuli or time. Controlled release can be achieved by, for example, use of an injectable drug carrier such as gels, implants and devices. In present invention, hydrogels are preferably used for controlled release of one or more active agents. Controlled release may be sustained delivery for periods varying from days (such as one day, two, three, four, five, six or seven days), to weeks (such as one week, two, three or four weeks) or even months (such as one month, or two or three months).

[0048] As used herein, the term “crosslinker” and “cross-linking compound” may be used interchangeablyChemical Definitions and Terms

[0049] The term "alkyl" includes straight-chained, branched, and cyclic alkyl groups and includes both unsubstituted and substituted alkyl groups. Unless otherwise indicated, the alkyl groups typically contain from 1 to 20 carbon atoms. The alkyl groups may for example contain carbon atoms from 1 to 20, 1 to 10, or 1 to 60. The alkyl groups may for example contain carbon atoms from 2 to 20, 2 to 15, 2 to 10, or 2 to 6. Examples of “alkyl” as used herein include, but are not limited to, methyl, ethyl, n-propyl. n-butyl, n-pentyl, isobutyl, t-butyl, isopropyl, n-octyl, n-heptyl, ethylhexyl, cyclopentyl, cyclohexyl, cyclo heptyl, adamantyl, norbornyl, decanyl, pentadecanyl, eicosanyl, pentacontanyl, and the like. Unless otherwise noted, alkyl groups may be mono- or polyvalent.

[0050] As used herein, the terms "halo" or "halogen", whether employed alone or in compound words such as haloalkyl, means fluorine, chlorine, bromine or iodine.

[0051] As used herein, the term "haloalkyl" means an alkyl group having at least one halogen substituent, the terms "alkyl" and "halogen" being understood to have the meanings outlined above. Similarly, the term "monohaloalkyl” means an alkyl group having a single halogen substituent, the term “dihaloalkyl” means an alkyl group having two halogen substituents and the term “trihaloalkyl” means an alkyl group having three halogen substituents. Examples of monohaloalkyl groups include fluoromethyl, chloromethyl, bromomethyl, fluoromethyl, fluoropropyl and fluorobutyl groups; examples of dihaloalkyl groups include difluoromethyl and difluoroethyl groups; examples of trihaloalkyl groups include trifluoromethyl and trifluoroethyl groups.

[0052] As used herein, the term "alkenyl" encompasses both straight and branched chain unsaturated hydrocarbon groups with at least one carbon-carbon double bond. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl and hexenyl. Further examples of alkenyl groups include ethenyl, 1 -propenyl, 2-propenyl, but-2- enyl, decenyl, pentadecenyl, eicosenyl, pentacontenyl, and the like.

[0053] As used herein, the term "alkynyl" encompasses both straight and branched chain unsaturated hydrocarbon groups with at least one carbon-carbon triple bond. Examples of alkynyl groups include ethynyl, propynyl, butynyl, pentynyl and hexynyl. Further examples of alkynyl groups include ethynyl, 1 -propynyl, 2-propynyl, decynyl, pentadecynyl, eicosynyl, pentacontynyl, and the like.

[0054] The term "heteroalkyl" includes both straight-chained, branched, and cyclic alkyl groups interrupted with one or more heteroatoms (e.g. 1-3) independently selected from S, O, and N. The heteroalkyl may be unsubstituted or substituted. Unless otherwise indicated, the heteroalkyl groups typically contain from 1 to 50 carbon atoms. The heteroalkyl groups may for example contain carbon atoms from 1 to 40, 1 to 20, or 1 to 10. The heteroalkyl groups may for example contain carbon atoms from 2 to 40, 2 to 20, or 2 to 10. Examples of "heteroalkyl" as used herein include, but are not limited to, methoxy, ethoxy, propoxy, decoxy, pentadecoxy, eicosoxy, pentacontoxy,and the like. Unless otherwise noted, heteroalkyl groups may be mono-, di- or polyvalent.

[0055] The term “heteroarylalkyl” means a group comprising a heteroaryl and alkyl according to the meanings and any examples independently thereof as described herein.

[0056] The term "heteroalkenyl” includes both straight-chained, branched, and cyclic alkenyl groups as described herein with one or more heteroatoms (e.g. 1-3) independently selected from S, O, and N with both unsubstituted and substituted alkenyl groups. Unless otherwise indicated, the heteroalkenyl groups typically contain from 1 to 50 carbon atoms. The heteroalkenyl groups may for example contain carbon atoms from 1 to 40, 1 to 20, or 1 to 10. Unless otherwise noted, heteroalkenyl groups may be mono- or polyvalent.

[0057] The term “heteroalkynyl” includes both straight-chained, branched, and cyclic alkynyl groups as described herein with one or more heteroatoms (e.g. 1-3) independently selected from S, O, and N with both unsubstituted and substituted alkenyl groups. Unless otherwise indicated, the heteroalkynyl groups typically contain from 1 to 50 carbon atoms. The heteroalkynyl groups may for example contain carbon atoms from 1 to 40, 1 to 20, or 1 to 10. Unless otherwise noted, heteroalkynyl groups may be mono- or polyvalent.

[0058] As used herein, the terms “carbocyclic” and “carbocyclyl” represent a ring system wherein the ring atoms are all carbon atoms, e.g., from 3 to 20, or 3 to 10 carbon ring atoms, and which may be aromatic, non-aromatic, saturated, or unsaturated. The terms encompass single ring systems, e.g. cycloalkyl groups such as cyclopentyl and cyclohexyl, aromatic groups such as phenyl, and cycloalkenyl groups such as cyclohexenyl, as well as fused-ring systems such as naphthyl and fluorenyl.

[0059] As used herein, the terms “heterocyclic’ and “heterocyclyl” represent an aromatic or a non-aromatic cyclic group of carbon atoms wherein from one to three of the carbon atoms is / are replaced by one or more heteroatoms independently selected from nitrogen, oxygen or sulfur. A heterocyclyl group may, for example, be monocyclic or polycyclic, and contain for example from 3 to 20 or 3 to 10 ring atoms. In a bicyclic heterocyclyl group there may be one or more heteroatoms in each ring, oronly in one of the rings. Examples of heterocyclyl groups include piperidinyl, tetrahydrofuranyl, tetrahydropyranyl, pyridyl, pyrimidinyl and indolyl.

[0060] As used herein, the term “cycloalkyl” represents a ring system wherein the ring atoms are all carbon atoms, e.g., from 3 to 20 carbon ring atoms, and which is saturated. A cycloalkyl group can be monocyclic or polycyclic. A bicyclic group may, for example, be fused or bridged. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl and cyclopentyl. Other examples of monocyclic cycloalkyl groups are cyclohexyl, cycloheptyl and cyclooctyl. Examples of bicyclic cycloalkyl groups include optional substituted indenes.

[0061] As will be understood, an “aromatic” group means a cyclic group having 4m+2 Ti electrons, where m is an integer equal to or greater than 1. As used herein, "aromatic" is used interchangeably with "aryl" to refer to an aromatic group, regardless of the valency of aromatic group.

[0062] As used herein, the terms “aromatic carbocyclyl” or “aromatic carbocycle” represent a ring system which is aromatic and in which the ring atoms are all carbon atoms, e.g. having from 6-14 ring atoms. An aromatic carbocyclyl group may be monocyclic or polycyclic. Examples of aromatic carbocyclyl groups include phenyl, naphthyl and fluorenyl. Polycyclic aromatic carbocyclyl groups include those in which only one of the rings is aromatic, such as for example indanyl.

[0063] The term “aryl” or “aromatic” group or moiety includes 6-18 ring atoms and can contain optional fused rings, which may be saturated or unsaturated. Examples of aromatic groups include phenyl, naphthyl, biphenyl, phenanthryl, and anthracyl. The aromatic group may optionally contain 1-3 heteroatoms such as nitrogen, oxygen, or sulfur and can contain fused rings. Examples of aromatic group having heteroatoms include pyridyl, furanyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, and benzthiazolyl. Unless otherwise noted the aromatic group may be mono- or polyvalent. In an example, the “aromatic” group may be a monocyclic aromatic group, for example a benzene group that may be unsubstituted or substituted.

[0064] The term “arylalkyl” means a group comprising an aryl and an alkyl according to the meanings and any examples independently thereof as described herein.

[0065] The term “arylalkenyl” means a group comprising an aryl and an alkenyl according to the meanings and any examples independently thereof as described herein.

[0066] The term “arylalkynyl” means a group comprising an aryl and an alkynyl according to the meanings and any examples independently thereof as described herein.

[0067] As used herein, the terms “aromatic heterocycle” or “aromatic heterocyclyl” represent an aromatic cyclic group of carbon atoms wherein from one to three of the carbon atoms is / are replaced by one or more heteroatoms independently selected from nitrogen, oxygen or sulphur, e.g. having from 5-14 ring atoms. The term “aromatic heterocyclyl” is used interchangeably with ‘heteroaryl”. An aromatic heterocyclyl group may be monocyclic or polycyclic. Examples of monocyclic aromatic heterocyclyl groups (also referred to as monocyclic heteroaryl groups) include furanyl, thienyl, pyrrolyl, imidazolyl, pyridyl and pyrimidinyl. Examples of polycyclic aromatic heterocyclyl groups (also referred to as bicyclic heteroaryl groups) include benzimidazolyl, quinolinyl and indolyl. Polycyclic aromatic heterocyclyl groups include those in which only one of the rings is an aromatic heterocycle.

[0068] As used herein, the term “hydroxyl” represents a -OH moiety.

[0069] As used herein, the term “alkoxy” represents an -O-alkyl group in which the alkyl group is as defined supra. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, and the different butoxy, pentoxy, hexyloxy and higher isomers.

[0070] As used herein, the term “aryloxy” represents an -O-aryl group in which the aryl group is as defined supra. Examples include, without limitation, phenoxy and naphthoxy.

[0071] As used herein, the term “carboxyl” represents a -CO2H moiety.

[0072] Affixing the suffix “-ene” to a group indicates the group is a divalent moiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene is the divalent moiety of alkenyl, alkynylene is the divalent moiety of alkynyl, heteroalkylene is the divalent moiety of heteroalkyl, heteroalkenylene is the divalent moiety of heteroalkenyl, heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl, heterocyclylene is thedivalent moiety of heterocyclyl, arylene is the divalent moiety of aryl, and heteroarylene is the divalent moiety of heteroaryl.

[0073] As used herein, the term “PEG” refers to a polymer or oligomer of ethylene glycol. For example, O(CH2CH2O)n, where n greater or equal to 1.

[0074] As used herein, the term “saturated” refers to a group where all available valence bonds of the backbone atoms are attached to other atoms. Representative examples of saturated groups include, but are not limited to, butyl, cyclohexyl, piperidine, and the like.

[0075] As used herein, the term “unsaturated” refers to a group where at least one valence bond of two adjacent backbone atoms is not attached to other atoms. Representative examples include, but are not limited to, alkenes (e.g., -CH2- CH2CH=CH), phenyl, pyrrole, and the like.

[0076] As used herein, the term “optionally substituted” refers to a group being unsubstituted or substituted as described herein.

[0077] As used herein, the term “substituted” refers to a group having one or more hydrogens or other atoms removed from a carbon or suitable heteroatom and replaced with a further group (i.e., substituent).

[0078] As used herein, the term “unsubstituted” refers to a group that does not have any further groups attached thereto or substituted therefore.

[0079] As used herein, a bond is denoted by aand is to be understood to be a single bond between two atoms.Crosslinker

[0080] It will be appreciated that the crosslinker, as described herein, is designed to cross-link with the self-assembling peptides as described herein, however it will be understood by a person skilled in the art that the crosslinker may also reversibly crosslink other hydrogels containing alkyne, alkene and thiol groups known in the literature.

[0081] In an embodiment, the present disclosure provides a crosslinker of Formula I- 1 :Formula 1-1 wherein R1and R2are each independently selected from the group comprising or consisting of Ci-ealkyl, -O-Ci-ealkyl, Ci-ehaloalkyl, -O-Ci-ehaloalkyl, C2-ealkenyl, C2- ealkynyl, 3-10-membered carbocyclyl, and 3-10-membered heterocyclyl; wherein each 3-10-membered carbocyclyl and 3-10-membered heterocyclyl, is unsubstituted or substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence; wherein R3aand R3bare each independently selected from C=C-R4a, C=C-R4b, and SH, wherein when R3ais C=C-R4aor OC-R4b, R3bis not SH, or when R3bis C=C- R4aor C=C-R4b, R3ais not SH; and wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl.

[0082] In an embodiment, R1and R2may each be independently selected from the group comprising or consisting of 3-10-membered carbocyclyl and 3-10-membered heterocyclyl; wherein each 3-10-membered carbocyclyl and 3-10-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence. In an embodiment, R1and R2may each be independently selected from a 5- or 6-membered carbocyclyl and 5- or 6-membered heterocyclyl; wherein each 5- or 6-membered carbocyclyl and 5- or 6-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence. In an embodiment, R1and R2may each be independently selected from a 6-membered carbocyclyl and 6-membered heterocyclyl; wherein each 6-membered carbocyclyl and 6-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence. In one example, R1may be a 6-membered carbocyclyl substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or apeptide sequence and R2may be a 6-membered heterocyclyl substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence.

[0083] In an embodiment, the present disclosure provides a crosslinker of Formula I- 2:Formula 1-2 wherein R5a, R5b, R5c, R5d, R5e, R5f, R5g, are each independently selected from CH and N; wherein R6aand R6bare each independently selected from a PEG, a glycine, or a peptide sequence; and wherein R7aand R7bare each independently selected from C=C-R4a, C=C-R4b, and SH, wherein when R3ais C=C-R4aor C=C-R4b, R3bis not SH, or when R3bis C=C- R4aor C=C-R4b, R3ais not SH; and wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl.

[0084] In an embodiment or example, each one of R5a, R5b, R5c, R5d, R5e, R5f, R5gmay be CH. In another embodiment or example, at least one of R5a, R5b, R5c, R5d, R5e, R5f, R5gmay be N. In other embodiments, at least two of R5a, R5b, R5c, R5d, R5e, R5f, R5gmay be N. In an embodiment, at least one of R5a, R5b, R5c, R5dmay be N. In another embodiment, at least two of R5a, R5b, R5c, R5dmay be N. In one example, each one of R5a, R5C, R5d, R5e, R5f, R5gmay be CH and R5bmay be a N.

[0085] In embodiments or examples of any one of the crosslinkers of Formula 1-1 and Formula 1-2, the groups R7aand R7bare each independently selected from C=C-R4a,C=C-R4b, and SH. In another example, R7aand R7bmay each be C=C-R4awherein R4ais selected from hydrogen and Ci-ealkyl. In another example, R7aand R7bmay each be C=C-R4a, wherein R4ais hydrogen. In another example, R7aand R7bmay each be C=C- R4b, wherein R4bis selected from hydrogen and Ci-ealkyl. In another example, R7aand R7bmay each be C=C-R4b, wherein R4bis hydrogen. In another example, R7aand R7bmay each be SH. In another example, R7amay be C=C-R4a, wherein R4ais selected from hydrogen and Ci-ealkyl, and R7bmay be C=C-R4b, wherein R4bis selected from hydrogen and Ci-ealkyl. In another example, R7amay be C=C-R4b, wherein R4bis selected from hydrogen and Ci-ealkyl, and R7bmay be C=C-R4a, wherein R4ais selected from hydrogen and Ci-ealkyl. In another example, R7amay be C=C-R4a, wherein R4ais hydrogen, and R7bmay be C=C-R4b, wherein R4bis hydrogen. In another example, R7amay be C=C-R4b, wherein R4bis hydrogen, and R7bmay be C=C-R4a, wherein R4ais hydrogen.

[0086] The following Table 1 provides some further examples of crosslinkers, which may be provided with any other hydrogel, or with any one of the self-assembling peptides as described herein, for forming a hydrogel of the present disclosure:Table 1: Crosslinker examplesPeptide-Based Hydrogels

[0087] In embodiments, the hydrogels of the present disclosure may provide for advantages over other select prior art hydrogels including one or more of: improved mechanical properties, improved controllability, improved stability, minimal negative effects on living cells, and minimal impact of cell viability.

[0088] In an embodiment, the present disclosure provides a hydrogel comprising a reaction product of (i) a compound comprising a thiol, an alkyne or an alkene and (ii) a cross-linking compound of Formula II- 1 as described herein. In an embodiment, the thiol, alkyne or alkene compound may be selected from the group comprising or consisting of a peptide, a protein, or an enzyme. It will be appreciated that the peptide, protein or enzyme comprises at least one thiol, alkyne or alkene group. In a preferred embodiment, the compound is a peptide comprising at least one thiol, alkyne or alkene group.

[0089] In an embodiment, the cross-linking compound of Formula II- 1, as described herein, may be a diol-responsive cross-linker. It will be appreciated that the hydrogel, as described herein, is formed by covalent cross-linking of the peptide and crosslinking compound of Formula II- 1, as described herein, through reactive thiols, alkenes or alkynes. In a preferred embodiment the hydrogel comprises a reaction product of (i) a peptide comprising at least one thiol, alkyne or alkene group and (ii) a diol-responsive cross-linking compound of Formula II- 1, as described herein.

[0090] Covalent chemical cross-linking is an important strategy for designing biomaterials because covalently connected peptide fiber networks exhibit high mechanical strengths. In general, it is difficult to make hydrogels with high mechanical strength using peptide-based supramolecular fibers due to reversible non-covalent bonds, as compared with hydrogels composed of covalent polymers. In this sense, covalent chemical cross-linking is an effective strategy to design peptide-based hydrogels with increased mechanical strength. For cross-linking of peptides, reactions of amino acid side chains can be utilized by introducing thiol groups capable of forming covalent bonds. One or more advantages of the present disclosure according to at least some embodiments or examples as described herein is that owing to the diol-responsiveness of the cross-linking compound (e.g. boronic acid derivative), described herein, the hydrogel can undergo a change in mechanical properties when exposed to sugars, allowing reprogramming of the mechanical properties of peptide-based hydrogels. Furthermore, the degree of change in mechanical properties of the hydrogel may be altered by longer light exposure stiffening the hydrogel or alternatively exposure to a higher concentration of a diol which will soften the hydrogel. In this way the hydrogel mechanical properties can be altered upon demand, which may be useful for 3D printing scaffolds for cells as initially stiff for optimal print resolution but then soften post printing for optimum cell viability.

[0091] In an embodiment, the hydrogel may be a compound of Formula III- 1 :Formula III-l wherein R5a, R5b, R5c, R5d, R5e, R5f, R5g, are each independently selected from CH and N; wherein R6aand R6bare each independently selected from a PEG, a glycine, or a peptide sequence; wherein R7aand R7bare each independently selected from are each independently selected from SH, C=C-R4a, and C=C-R4b, and C-C-R4c; wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl; andX is selected from the group comprising or consisting of a peptide sequence, a protein, or an enzyme, wherein the peptide, protein or enzyme comprises at least one thiol, alkyne or alkene group.

[0092] In another embodiment, the hydrogel may be a compound of Formula III-2 :Formula III-2 wherein R5a, R5b, R5c, R5d, R5e, R5f, R5g, are each independently selected from CH and N; wherein R6aand R6bare each independently selected from a PEG, a glycine, or a peptide sequence; wherein R7aand R7bare each independently selected from are each independently selected from C=C-R4a, and C=C-R4b, and C-C-R4c; wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl; and wherein each peptide comprises at least one thiol group.

[0093] In another embodiment, the hydrogel may be a compound of Formula III- 3 :Formula III-3 wherein R5a, R5b, R5c, R5d, R5e, R5f, R5g, are each independently selected from CH and N; wherein R6aand R6bare each independently selected from a PEG, a glycine, or a peptide sequence; wherein R7aand R7bare SH;wherein each peptide independently comprises at least one group selected from C=C-R4aand C=C-R4b; and wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl.

[0094] It will be appreciated that the hydrogels of Formula III-2, and Formula III-3, as described herein, are formed by covalent cross-linking of the peptide and crosslinking compound of Formula II- 1 and Formula II-2 through reactive thiols (of the peptide) and alkenes / alkynes (of the cross-linking compound of Formula II- 1 ) or the alkene or alkyne group of the peptide reacts with a terminal thiol of the diol-responsive cross-linking compound of Formula II- 1 or Formula II-2.

[0095] It will be appreciated that the mechanical properties of the hydrogels, as described herein, can be advantageously tuned. To evaluate the mechanical properties of the hydrogels, as described herein, in a more quantitative manner, rheological experiments were performed. The viscoelastic properties of the peptide hydrogels were measured by dynamic rheometry at 20°C. This technique measures the storage modulus G' (corresponding to the material's stiffness or rigidity), loss modulus G" (corresponding to the viscous properties of the material) and loss factor tan 6 (ratio of G" over G'). After cross-linking, the storage modulus of the hydrogel, as described herein, may be 30-fold higher than that of the hydrogel before polymerization.

[0096] In an embodiment, the hydrogel has a storage modulus of greater than about 1000 Pa, for example, ranging from about 1000 Pa to about 200,000 Pa. The storage modulus (in Pa) may be at least about 1000, 2000, 4000, 8000, 10,000, 20,000, 40,000, 80,000, 100,000, 120,000, 140,000, 160,000, 180,000 or 200,000. The storage modulus (in Pa) may be less than about 200,000, 180,000, 160,000, 140,000, 120,000, 100,000, 80,000, 40,000, 20,000, 10,000, 8000, 4000, 2000, or 1000. Combinations of these amounts are possible, for examples the storage modulus may be between about 1000 Pa to about 180,000, between about 2000 Pa to about 140,000 Pa, or between about 4000 Pa to about 120,000 Pa.

[0097] The inventors have surprisingly found that the hardness and softness of the hydrogels can be controlled because of the diol-responsiveness of the cross-linker (e.g.boronic acid derivative) making the hydrogels suitable for use in a number of different biological applications. For example, the inventors have found that the hardness of the hydrogel, as described herein, can be increased by exposure to UV-light, i.e. increase in the thiol-ene cross-linking, thus finding potential application in, for example, bone repair. On the other hand, the softness of the hydrogel, as described herein, can be increased by exposure to diols, decrease or breaking of the boronic ester cross-linking, thus finding potential application in, for example, soft tissue regeneration.

[0098] In an embodiment, the hydrogel may comprises between about 0.05 mol % to about 5 mol % of the diol-responsive cross-linking compound of Formula II- 1, as described herein. The amount (in mol %) of diol-responsive cross-linking compound of Formula II-l, as described herein, may be at least about 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5. The amount (in mol %) of diol-responsive cross-linking compound of Formula II-l, as described herein, may be less than about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.1, or 0.05. The amount (in mol %) of diol-responsive cross-linking compound of Formula I, as described herein, may be in a range provided by any two of these upper and / or lower values, for example the amount (in mol %) of diol-responsive crosslinking compound of Formula II-l, as described herein may be in a range between 0.05 mol and 1 mol.

[0099] In an embodiment, the ratio of peptide to diol-responsive cross-linking compound of Formula II-l, as described herein, may be in a range between 1 : 1 and 10: 1. In an embodiment, the ratio of peptide to diol-responsive cross-linking compound of Formula II-l, as described herein, may be at least 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10:1. In an embodiment, the ratio of peptide to diol-responsive cross-linking compound of Formula II-l, as described herein, may be less than 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, or 1 : 1. The ratio may be in a range provided by any two of these upper and / or lower values, for example the ratio of peptide to diol-responsive crosslinking compound of Formula II-l, as described herein, may be in a range between 1 : 1 and 2: 1.

[0100] There is also provided a process for preparing the cross-linked hydrogel, as described herein. The process comprising (i) obtaining a thiol, alkyne or alkene compound, (ii) obtaining a cross-linking compound of Formula II-l, as describedherein, (iii) mixing aqueous solutions of the thiol, alkyne or alkene compound of step (i) and the cross-linking compound of step (ii) to form an aqueous mixture. The thiol, alkyne or alkene compound may preferably be a peptide comprising at least one thiol, alkyne or alkene group (e.g. a cysteine group) and the cross-linking compound of Formula II- 1, as described herein, may be a diol -responsive cross-linker. It will be appreciated that the hydrogel, as described herein, is formed by covalent cross-linking of the peptide and cross-linking compound of Formula II- 1 and Formula II-2 through reactive thiols (of the peptide) and alkenes / alkynes (of the cross-linking compound of Formula II- 1) or the alkene or alkyne group of the peptide reacts with a terminal thiol of the diol-responsive cross-linking compound of Formula II- 1 or Formula II-2.

[0101] In an embodiment, the aqueous mixture may be exposed to light. For example, the light source may be visible light or ultra-violet light. In one example, ultra-violet light for at least 5 seconds in the presence of a photo-initiator. An amount of photoinitiator may be added to the aqueous mixture before exposure to ultra-violet light. In an embodiment, step (iii) may further include (a) drying the aqueous mixture to form hydrogel powder. In an example, the hydrogel powder may be reconstituted in an different medium or aqueous solution (e.g. saline solution) to prepare an aqueous mixture depending on the intended application.

[0102] The process may further comprises step (iv) mixing an aqueous solution comprising an active agent with the aqueous mixture of step (iii) or the hydrogel powder of step (iii)(a) to form a hydrogel encapsulating an active agent.

[0103] Depending on the intended application of hydrogels, as described herein, it may be desirable that the hydrogels be formulated to incorporate an active agent within the hydrogel matrix. In an embodiment, the hydrogel may encapsulate or incorporate an active agent. For example, the active agent may be selected from primary cells, stem cells, tissues, biological agents such as growth factors, extracellular vesicles, therapeutic agents, or combinations thereof. In some embodiments the hydrogel may be formulated for use as an injectable (e g. filler with or without an active agent), drug delivery vehicle, tissue engineering, or carrier in controlled release of an active agent.

[0104] In an embodiment, there may be provided a method of controlled release of an active agent from a hydrogel, wherein release of the active agent may occur on contact with a diol containing molecule or polymer, in one example, the diol containing molecule or polymer may be a sugar molecule, such as glucose. The sugar compound may be in the form of a sugar solution. It will be appreciated that the rate of release of the active agent may be dependent on the concentration of the sugar solution. In an embodiment, the concentration of the sugar solution may be in a range of between about 0.1 mM to about 50 mM. It will be appreciated that the sugar solution acts to de- cross-link or dissociate the cross-linking compound of Formula I, as described herein, from the peptide.Cross-linking compound

[0105] In an embodiment, the cross-linker may be a diol -responsive cross-linker. It will be appreciated that the hydrogel, as described herein, is formed by covalent crosslinking of the peptide and cross-linker through reactive thiols and alkenes. In a preferred embodiment the hydrogel comprises a reaction product of (i) a peptide comprising at least one thiol, alkyne, or alkene group and (ii) a diol-responsive crosslinker. The diol-responsive cross-linker may be a boronic acid derivative. It will be understood that one or more advantages of the present disclosure according to at least some embodiments or examples as described herein is that the crosslinking compound has been found to independently control the mechanical properties (e.g. stiffness) of the hydrogel. For example, the crosslinking reaction can be used to stiffen the hydrogels to make it "3D printable". However, this is often too stiff for optimal cell viability. The inventors have surprisingly shown that once the hydrogel scaffold is 3D printed it can be softened by exposing the scaffold to diol containing molecules or polymers (e.g. such as glucose or a sugar) which undergoes competitive reversible bonding with the boronic acid component of the cross-linking compound of Formula I, as described herein, leading to cleavage of the boronic acid which results in softening of the hydrogel scaffold.

[0106] In an embodiment, the cross-linking compound may be a compound ofFormula II- 1 :Formula II-l wherein R1and R2are each independently selected from the group comprising or consisting of Ci-ealkyl, -O-Ci-ealkyl, Ci-ehaloalkyl, -O-Ci-ehaloalkyl, C2-ealkenyl, C2- ealkynyl, 3-10-membered carbocyclyl, and 3-10-membered heterocyclyl; wherein each 3-10-membered carbocyclyl and 3-10-membered heterocyclyl, is unsubstituted or substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence; wherein R3aand R3bare each independently selected from C=C-R4a, C=C-R4b, and SH, wherein when R3ais C=C-R4aor C=C-R4b, R3bis not SH, or when R3bis C=C- R4aor C=C-R4b, R3ais not SH; and wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl.

[0107] In an embodiment, R1and R2may each be independently selected from the group comprising or consisting of 3-10-membered carbocyclyl and 3-10-membered heterocyclyl; wherein each 3-10-membered carbocyclyl and 3-10-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence. In an embodiment, R1and R2may each be independently selected from a 5- or 6-membered carbocyclyl and 5- or 6-membered heterocyclyl; wherein each 5- or 6-membered carbocyclyl and 5- or 6-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence. In an embodiment, R1and R2may each be independently selected from a 6-membered carbocyclyl and 6-membered heterocyclyl; wherein each 6-membered carbocyclyl and 6-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence. In oneexample, R1may be a 6-membered carbocyclyl substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence and R2may be a 6-membered heterocyclyl substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence.

[0108] In another embodiment, the cross-linking compound of Formula II- 1 may be a compound of Formula II-2:Formula II-2 wherein R5a, R5b, R5c, R5d, R5e, R5f, R5g, are each independently selected from CH and N; wherein R6aand R6bare each independently selected from a PEG, a glycine, or a peptide sequence; and wherein R7aand R7bare each independently selected from are each independently selected from C=C-R4a, C=C-R4band SH, wherein when R3ais C=C-R4aor C=C-R4b, R3bis not SH, or when R3bis C=C-R4aor C=C-R4b, R3ais not SH; and wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl.

[0109] In an embodiment or example, each one of R5a, R5b, R5c, R5d, R5e, R5f, R5gmay be CH. In another embodiment or example, at least one of R5a, R5b, R5c, R5d, R5e, R5f, R5gmay be N. In other embodiments, at least two of R5a, R5b, R5c, R5d, R5e, R5f, R5gmay be N. In an embodiment, at least one of R5a, R5b, R5c, R5dmay be N. In another embodiment, at least two of R5a, R5b, R5c, R5dmay be N. In one example, each one of R5a, R5C, R5d, R5e, R5f, R5gmay be CH and R5bmay be a N.

[0110] In embodiments or examples of any one of the crosslinkers of Formula 1-1 and Formula 1-2, and the cross-linking compounds of Formula II- 1 and Formula II-2, the groups R7aand R7bare each independently selected from C=C-R4a, C=C-R4b, and SH. In another example, R7aand R7bmay each be C=C-R4awherein R4ais selected from hydrogen and Ci-ealkyl. In another example, R7aand R7bmay each be C=C-R4a, wherein R4ais hydrogen. In another example, R7aand R7bmay each be C=C-R4b, wherein R4bis selected from hydrogen and Ci-ealkyl. In another example, R7aand R7bmay each be C=C-R4b, wherein R4bis hydrogen. In another example, R7amay be C=C- R4a, wherein R4ais selected from hydrogen and Ci-ealkyl, and R7bmay be C=C-R4b, wherein R4bis selected from hydrogen and Ci-ealkyl. In another example, R7amay be C=C-R4b, wherein R4bis selected from hydrogen and Ci-ealkyl, and R7bmay be C=C- R4a, wherein R4ais selected from hydrogen and Ci-ealkyl. In another example, R7amay be C=C-R4a, wherein R4ais hydrogen, and R7bmay be C=C-R4b, wherein R4bis hydrogen. In another example, R7amay be C=C-R4b, wherein R4bis hydrogen, and R7bmay be C=C-R4a, wherein R4ais hydrogen.

[0111] Table 1 above provides some further examples of crosslinking compounds, which may be provided with any one of the self-assembling peptides as described herein for forming a hydrogel of the present disclosure.Peptides

[0112] Challenges in developing hydrogel-forming peptides are the selection of appropriate amino acids from the great diversity of available residues, their proneness to degradation by proteolytic enzymes, as well as the need to provide biocompatible, biodegradable and functional materials. A balance between hydrophobicity and hydrophilicity needs to be maintained when designing oligopeptide hydrogels in order to obtain a self-assembled system under suitable conditions. In an embodiment, the peptide, as described herein, is resistant to proteolytic cleavage. It will be appreciated that there are no protease side reactions to allow degradation by proteases. In some embodiments, the amino acid sequence may comprise natural and unnatural amino acids. For example, the modified self-assembling peptide may comprise at least one [3- amino acid that is resistant to proteolytic cleavage.

[0113] The biological stability, mechanical strength and release properties can be tuned by structural modifications in the amino acid sequence of the peptide sequence. The peptide possesses tuneable storage moduli and forms a self-supporting hydrogel. In an embodiment, the storage modulus (G1) of the modified self-assembling peptide may be greater than the loss modulus (G") of the self-assembling peptide. In an embodiment the storage modulus (G1) of the modified self-assembling peptide is greater than 1000 Pa. In an embodiment the storage modulus (G1) of the modified self-assembling peptide is greater than 1500 Pa. In an embodiment, the storage modulus (G1) of the modified self-assembling peptide may be greater than 2000 Pa. In an embodiment, the storage modulus (G1) of the modified self-assembling peptide may be greater than 4000 Pa. The storage modulus (G1) of the modified self-assembling peptide may be greater than 1000, 2000, 4000, 6000, 8000, 10,000, 15,000, 20,000, 25,000, 30,000 or 40,000 Pa. The storage modulus (G1) of the modified self-assembling peptide may be less than 80,000, 70,000, 60,000, 50,000, 40,000, 30,000, 20,000, 10,000, 5000, or 2000 Pa. The storage modulus (G1) of the modified self-assembling peptide may be in a range provided by any two of these upper and / or lower values. It will be appreciated that a high storage modulus (G1) can be achieved with a low solids content of the modified self-assembling peptide. In an embodiment, the solids content of the modified self-assembling peptide may be in a range between about 0.1 wt.% and about 50 wt.%, preferably between about 0.25 wt.% and about 30 wt.%, more preferably between about 0.5 wt.% and about 10 wt.%. The solids content (in wt.%) of the modified self-assembling peptide may be less than about 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, 1, 0.5, 0.25, or 0.1. The solids content (in wt.%) of the modified self-assembling peptide may be greater than about 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50. The solids content (in wt.%) of the modified self-assembling peptide may be in a range provided by any two of these upper and / or lower values. For example, the storage modulus (G1) of the modified self-assembling peptide may be greater than lOOOPa when the solids content of the modified self-assembling peptide is less than about 50 wt.% and greater than about 0.1 wt.%. In one example, the storage modulus (G) of the modified selfassembling peptide may be greater than 1000 Pa when the solids content of themodified self-assembling peptide is between about 0.5 wt.% and about 10 wt.%, preferably between about 1 wt.% and about 5 wt.%.

[0114] Short peptide sequences are preferred as they are simpler to produce, have improved tunable and lower production costs because it simplifies the synthesis and purification of larger quantities of peptides. When the peptide sequences are too short (two or three amino acids), structural variations might immediately lead to loss of hydrogelating ability. Therefore, a peptide sequence of intermediate length, i.e. between 4 and 30 amino acids, preferably between 6 and 30 amino acids, is preferred. In some embodiments, the peptide sequence may comprise less than 30 amino acids. In a preferred embodiment, the peptide may comprise between 6 and 20 amino acids in the amino acid sequence. The peptide sequence may comprise less than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids. In some embodiments, the peptide sequence may comprise at least 6 amino acids. The peptide sequence may comprise at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. The number of amino acids in the peptide sequence may be in a range provided by any two of these upper and / or lower values.

[0115] It will be appreciated that conformational change or modification of amino acids and peptides is the most common way to increase the stability of the hydrogels. The inventors have surprising found that the peptide sequences, as described herein, have an improved stability. In an embodiment, the peptide may be stable for at least 30 days. The peptide may be stable for at least 90 days.

[0116] In some embodiments, the thiol compound is a peptide comprising at least one thiol group. It will be appreciated that the crosslinking compound of Formula I, as described herein, can reversibly crosslink any other hydrogel containing thiols.

[0117] One or more advantages of the hydrogel forming peptides described herein may include the introduction of multiple peptide sequences with (i) the capability of forming hydrogels, (ii) exhibiting enhanced mechanical strength and stability, (iii) form self-assembled hydrogels with shear / storage modulus (G1) >1000 Pa, and / or one or more of the following (iv) are prepared from inherently biodegradable components(amino acids) found in nature, yet can be synthesised with high purity and low poly dispersity, unlike insolating natural biopolymers which are difficult to purify and often have a broad poly dispersity, (v) have high stability in aqueous solutions at near neutral pH (under biologically relevant conditions), (vi) have shear thinning behaviour and high recoverability (ability to return quickly to the gel state after reduction of shear forces) giving them the ability to be injectable hydrogels (meaning they can be introduced into the body via minimally invasive injection or can be used a 'inks' in 3D printing or bioprinting, (vii) they are inherently biodegradable and can biodegrade over time so they don't need to be surgically removed from the body, (viii) can be used to deliver payloads (cells, bioactive, contrast agents, radioactive particles etc), (ix) can be used as fillers or bulking agents for cosmetic or therapeutic applications.

[0118] In some embodiments, there is provided a modified self-assembling peptide for hydrogel formation, comprising a self-assembling peptide comprising an amino acid sequence according to Formula III:(Al)n(Bl)m(Xl)p(A2)n(Y)q(X2)p(A3)n(X3)p(B2)m Formula III wherein Ai to A3 each represent a hydrophobic amino acid residue; Bi and B2 represent a charged amino acid residue;Y represents an uncharged or hydrophobic amino acid residue; Xi to X3 each represent one or more amino acids or is absent; n is an integer of 1 to 4; m is an integer of 1 to 4; p is an integer of 0 to 4; and q is an integer of 1 to 4; wherein the storage modulus (G) of the modified self-assembling peptide is greater than 1000 Pa, and wherein Bi or B2 is optionally interchanged with Y within the amino acid sequence such that Bi or B2 takes the position of Y and Y takes the position of Bi or B2. For example, Formula III can be anyone of,(Al)n(Bl)m(Xl)p(A2)n(Y)q(X2)p(A3)n(X3)p(B2)m Formula III; or(Al)n(Y)q (Xl)p(A2)n(B l)m(X2)p(A3)n(X3)p(B2)mFormula Illa where Bi takes the position of Y and Y takes the position of Bi; or (Al)n(Bl)m(Xl)p(A2)n(B2)m(X2)p(A3)n(X3)p(Y)qFormula Illb where B2 takes the position of Y and Y takes the position of B2.

[0119] It will be appreciated that the term “interchanged” may be also referred to as “swapped” such that the amino acid represented by Bi may be swapped with the amino acid represented by Y, and vice versa. It will also be appreciated that the amino acid represented by B2 may be swapped with the amino acid represented by Y, and vice versa.

[0120] For example, when Bi is swapped with Y, the amino acid sequence may be according to Formula Illa: (Ai)n(Y)q(Xi)p(A2)n(Bi)m(X2)p(A3)n(X3)p(B2)m.Alternatively, when B2 is swapped with Y, the amino acid sequence may be according to Formula Illb: (Ai)n(Bi)m(Xi)p(A2)n(B2)m(X2)P(A3)n(X3)P(Y)q.

[0121] In some embodiments, there is provided a modified self-assembling peptide for hydrogel formation, comprising a self-assembling peptide comprising an amino acid sequence according to Formula IV:(Al)n(Bl)m(Xl)p(A2)n(Y)q(X2)p(A3)n(X3)p(B2)mFormula IV wherein Ai to A3 each represent a hydrophobic amino acid residue;Bi and B2 represent a charged amino acid residue;Y represents an uncharged or hydrophobic amino acid residue;Xi to X3 each represent one or more amino acids or is absent; n is an integer of 1 to 4; m is an integer of 1 to 4; p is an integer of 0 to 4; and q is an integer of 1 to 4; wherein the storage modulus (G1) of the modified self-assembling peptide is greater than 1000 Pa.

[0122] In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 2, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is 1, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 3, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 2, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is 1, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 3 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 2 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 1 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is absent, and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is 1.

[0123] In some embodiments, there is provided a modified self-assembling peptide for hydrogel formation, comprising a self-assembling peptide comprising an amino acid sequence according to Formula IVa:(Al)n(Bl)m(Xl)p(A2)n(Y)q(X2)p(A3)n(X3)p(B2)m Formula IVa wherein Ai to A3 each represent a hydrophobic amino acid residue; Bi and B2 represent a charged amino acid residue;Y represents an uncharged or hydrophobic amino acid residue; Xi to X3 each represent one or more amino acids or is absent; n is an integer of 1 to 3; m is an integer of 1 to 3;p is an integer of 0 to 3; and q is an integer of 1 to 3; wherein the storage modulus (G1) of the modified self-assembling peptide is greater than 1000 Pa.

[0124] In some embodiments, n is an integer of 1 to 2, m is an integer of 1 to 3, p is an integer of 0 to 3 and q is an integer of 1 to 3. In some embodiments, n is 1, m is an integer of 1 to 3, p is an integer of 0 to 3 and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 2, p is an integer of 0 to 3 and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 3, m is 1, p is an integer of 0 to 3 and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 3, p is an integer of 0 to 2 and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 3, p is an integer of 0 to 1 and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 3, p is absent and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 3, p is an integer of 0 to 3 and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 3, p is an integer of 0 to 3 and q is 1.

[0125] In some embodiments, there is provided a modified self-assembling peptide for hydrogel formation, comprising a self-assembling peptide comprising an amino acid sequence according to Formula IVb:(Al)n(Bl)m(Xl)p(A2)n(Y)q(X2)p(A3)n(X3)p(B2)mFormula IVb wherein Ai to A3 each represent a hydrophobic amino acid residue;Bi and B2 represent a charged amino acid residue;Y represents an uncharged or hydrophobic amino acid residue;Xi to X3 each represent one or more amino acids or is absent; n is an integer of 1 to 2; m is an integer of 1 to 2; p is an integer of 0 to 2; and q is an integer of 1 to 2;wherein the storage modulus (G1) of the modified self-assembling peptide is greater than 1000 Pa.

[0126] In some embodiments, n is 1, m is an integer of 1 to 2, p is an integer of 0 to 2 and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 2, m is 1, p is an integer of 0 to 2 and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 2, m is an integer of 1 to 2, p is an integer of 0 to 1 and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 2, m is an integer of 1 to 2, p is absent and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 2, m is an integer of 1 to 2, p is an integer of 0 to 2 and q is 1.

[0127] In embodiments there is provided a modified self-assembling peptide for hydrogel formation, comprising a self-assembling peptide comprising an amino acid sequence according to Formula IVc:A1B1X1A2YX2A3X3B2Formula IVc wherein Ai to A3 each represent a hydrophobic amino acid residue;Bi and B2 represent a charged amino acid residue;Y represents an uncharged or hydrophobic amino acid residue; andXi to X3 each represent one or more amino acids or is absent; wherein the storage modulus (G1) of the modified self-assembling peptide is greater than 1000 Pa.

[0128] In an embodiment of any one of Formulas III, Illa, Illb, IV, IVa, IVb and IVc, Ai to A3 may be each independently selected from a hydrophobic aromatic amino acid. For example, Ai to A3 may be each independently selected from phenylalanine, tyrosine, and tryptophan. In one example, when n is 1, Ai to A3 may be each phenylalanine or tryptophan.

[0129] In an embodiment of any one of Formulas III, Illa, Illb, IV, IVa, IVb and IVc, Bi and B2 may be each independently selected from a positive or negative amino acid residue. For example, Bi and B2 may be each independently selected from arginine, histidine, lysine, aspartic acid or glutamic acid.

[0130] In an embodiment of any one of Formulas III, Illa, Illb, IV, IVa, IVb and IVc, Xi to X3, if present, may be each independently selected from a hydrophobic, charged, or uncharged amino acid residue.

[0131] In some embodiments, there is provided a modified self-assembling peptide for hydrogel formation, comprising a self-assembling peptide comprising an amino acid sequence according to Formula V:(Al)n(Bl)m(Xl)p(A2)n(Y)q(X2)p(A3)n(X3)p(B2)mFormula V wherein Ai to A3 each represent an aromatic hydrophobic amino acid residue selected from phenylalanine, tyrosine, and tryptophan;Bi and B2 represent a charged amino acid residue;Y represents an uncharged or hydrophobic amino acid residue;Xi to X3 each represent one or more amino acids or is absent; n is an integer of 1 to 4; m is an integer of 1 to 4; p is an integer of 0 to 4; and q is an integer of 1 to 4; wherein the storage modulus (G1) of the modified self-assembling peptide is greater than 1000 Pa.

[0132] In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 2, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is 1, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 3, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 2, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is 1, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 3 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 2 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is aninteger of 1 to 4, p is an integer of 0 to 1 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is absent and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 4. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 4, m is an integer of 1 to 4, p is an integer of 0 to 4 and q is 1.

[0133] In some embodiments, there is provided a modified self-assembling peptide for hydrogel formation, comprising a self-assembling peptide comprising an amino acid sequence according to Formula Va:(Al)n(Bl)m(Xl)p(A2)n(Y)q(X2)p(A3)n(X3)p(B2)mFormula Va wherein Ai to A3 each represent an aromatic hydrophobic amino acid residue selected from phenylalanine, tyrosine, and tryptophan;Bi and B2 represent a charged amino acid residue;Y represents an uncharged or hydrophobic amino acid residue;Xi to X3 each represent one or more amino acids or is absent; n is an integer of 1 to 3; m is an integer of 1 to 3; p is an integer of 0 to 3; and q is an integer of 1 to 3; wherein the storage modulus (G1) of the modified self-assembling peptide is greater than 1000 Pa.

[0134] In some embodiments, n is an integer of 1 to 2, m is an integer of 1 to 3, p is an integer of 0 to 3 and q is an integer of 1 to 3. In some embodiments, n is 1, m is an integer of 1 to 3, p is an integer of 0 to 3 and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 2, p is an integer of 0 to 3 and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 3, m is 1, p is an integer of 0 to 3 and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 3, p is an integer of 0 to 2 and q is an integer of 1 to 3.In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 3, p is an integer of 0 to 1 and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 3, p is absent, and q is an integer of 1 to 3. In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 3, p is an integer of 0 to 3 and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 3, m is an integer of 1 to 3, p is an integer of 0 to 3 and q is 1.

[0135] In some embodiments, there is provided a modified self-assembling peptide for hydrogel formation, comprising a self-assembling peptide comprising an amino acid sequence according to Formula Vb:(Al)n(Bl)m(Xl)p(A2)n(Y)q(X2)p(A3)n(X3)p(B2)mFormula Vb wherein Ai to A3 each represent an aromatic hydrophobic amino acid residue selected from phenylalanine, tyrosine, and tryptophan;Bi and B2 represent a charged amino acid residue;Y represents an uncharged or hydrophobic amino acid residue;Xi to X3 each represent one or more amino acids or is absent; n is an integer of 1 to 2; m is an integer of 1 to 2; p is an integer of 0 to 2; and q is an integer of 1 to 2; wherein the storage modulus (G1) of the modified self-assembling peptide is greater than 1000 Pa.

[0136] In some embodiments, n is 1, m is an integer of 1 to 2, p is an integer of 0 to 2 and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 2, m is 1, p is an integer of 0 to 2 and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 2, m is an integer of 1 to 2, p is an integer of 0 to 1 and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 2, m is an integer of 1 to 2, p is absent, and q is an integer of 1 to 2. In some embodiments, n is an integer of 1 to 2, m is an integer of 1 to 2, p is an integer of 0 to 2 and q is 1.

[0137] In embodiments there is provided a modified self-assembling peptide for hydrogel formation, comprising a self-assembling peptide comprising an amino acid sequence according to Formula Vc:A1B1X1A2YX2A3X3B2Formula Vc wherein Ai to A3 each represent an aromatic hydrophobic amino acid residue selected from phenylalanine, tyrosine, and tryptophan;Bi and B2 represent a charged amino acid residue;Y represents an uncharged or hydrophobic amino acid residue; andXi to X3 each represent one or more amino acids or is absent; wherein the storage modulus (G1) of the modified self-assembling peptide is greater than 1000 Pa.

[0138] In an embodiment of any one of Formulas V, Va, Vb and Vc, when n is 1, Ai to A3 may be each phenylalanine or tryptophan.

[0139] In an embodiment of any one of Formulas V, Va, Vb and Vc, Bi and B2 may be each independently selected from a positive or negative amino acid residue. For example, Bi and B2 may be each independently selected from arginine, histidine, lysine, aspartic acid or glutamic acid.

[0140] In an embodiment of any one of Formulas V, Va, Vb and Vc, Xi to X3, if present, may be each independently selected from a hydrophobic, charged, or uncharged amino acid residue.

[0141] In an embodiment, the modified self-assembling peptide for hydrogel formation may comprise a self-assembling peptide comprising an amino acid sequence according to Formula VI:A1B1X1A2YX2A3X3B2Formula VI wherein Ai to A3 are each phenylalanine or tryptophan;Bi and B2 represent a charged amino acid residue;Y represents an uncharged or hydrophobic amino acid residue; and Xi to X3 each represent one or more amino acids or is absent; wherein the storage modulus (G1) of the modified self-assembling peptide is greater than 1000 Pa.

[0142] In an embodiment, the modified self-assembling peptide for hydrogel formation may comprise a self-assembling peptide comprising an amino acid sequence according to Formula Via:A1B1X1A2YX2A3X3B2Formula Via wherein Ai to A3 are each phenylalanine;Bi and B2 represent a charged amino acid residue;Y represents an uncharged or hydrophobic amino acid residue; and Xi to X3 each represent one or more amino acids or is absent; wherein the storage modulus (G1) of the modified self-assembling peptide is greater than 1000 Pa.

[0143] In an embodiment of Formulas VI, Via, Bi and B2 may be each independently selected from a positive or negative amino acid residue. For example, Bi and B2 may be each independently selected from arginine, histidine, lysine, aspartic acid or glutamic acid.

[0144] In an embodiment of Formulas VI, Via, Xi to X3, if present, may be each independently selected from a hydrophobic, charged, or uncharged amino acid residue.

[0145] The amino acid residues of the peptide sequence can be a-amino acid residues and / or P-amino acid residues. In an embodiment, the peptide may comprises at least one P-amino acid. Further advantages may be provided wherein the peptide sequence comprises at least one P-amino acid. The inventors have surprisingly found that introduction of P-amino acid(s) may increase stability of the hydrogel. For example, at least one P-amino acid may be introduced into the amino acid sequence. In some examples, at least one of Ai to A3 may be a P-amino acid.

[0146] In an embodiment, at least one amino acid residue comprising a thiol group may be introduced into the amino acid sequence at the N- or C-terminus of the peptide.In another embodiment, at least one amino acid residue comprising a thiol group may be introduced into the amino acid sequence at the N- and C-terminus of the peptide. For example, at least one cysteine may be introduced or substituted into the amino acid sequence.

[0147] In some embodiments, at least one of Xi to X3 may be a cysteine or any one ofAi to A3, Bi to B2 and Y may be substituted with a cysteine. In an embodiment, at least one of Ai, Xi to X3 may be a cysteine or any one of Bi to B2 and Y may be substituted with a cysteine. In an embodiment, at least one of Ai and B2 may be substituted with a cysteine. In another embodiment, Ai and B2 may be substituted with a cysteine.

[0148] The following table provides some further examples of self-assembling peptide sequences as described herein for forming a hydrogel of the present disclosure:Table 2: Self-assembling peptide sequence examplesBiological Applications

[0149] The hydrogels as described herein are useful in a variety of applications, and are particularly suited for biological applications, including 3D bioprinting, drug delivery, tissue engineering, and as a therapeutic depot. Depending on the intended application of hydrogels, as described herein, it may be desirable that the hydrogels be formulated to incorporate an active agent within the hydrogel matrix. In an embodiment, the hydrogel may encapsulate or incorporate an active agent. For example, the active agent may be selected from primary cells, stem cells, tissues, biological agents such as growth factors, extracellular vesicles, therapeutic agents, or combinations thereof. In some embodiments the hydrogel may be formulated for use as an injectable (e.g. filler with or without an active agent), drug delivery vehicle, tissue engineering, or carrier in controlled release of an active agent.Biofabrication

[0150] It will be appreciated that the hydrogel as defined herein may be applied to effectively integrate into living tissue systems. This may include the incorporation of biologically -appropriate cells into biologically-compatible gels and the processing of these and / or biologically active material scaffolds (without cells) into structures for functional remodelling of failing or otherwise dysfunctional tissue systems. Technologies such as wet-spinning, hot-melt extrusion, reactive gelation, electrospinning and drop and cast moulding are some of the main methods used for biofabricating hydrogels into biologically active and compatible structures. Additive Biofabrication technologies utilise additive technologies such as 3D bioprinting for the formation of structures to be used for biological applications in living systems (e.g. neo-genesis of bone, muscle and other tissues).3D Bioprinting

[0151] The term “bioink” as used herein refers to a hydrogel or a mixture of cells with a hydrogel used for bioprinting. Therefore, a bioink composition may be applied for bioprinting. In addition, the bioink composition may be a hydrogel, wherein the hydrogel is as defined herein.

[0152] In one embodiment, the bioink composition may be a cell-laden composition. It will be appreciated that cells can grow within the hydrogel matrix. It will also be appreciated that cells can be introduced into the hydrogel matrix before or after the hydrogel has been cross-linked. In addition, the bioink composition may further include a growth factor, extracellular vesicles and / or a differentiation factor. The cells included in the bioink composition may include cells to be cultured in the hydrogel composition, tissues, or cells to be differentiated into other cells, or cells to be used for tissue regeneration. Examples of the cells are stem cells, sensory cells, brain cells, germ cells, epithelial cells, immune cells, cartilage cells, bone cells, cancer cells, or combinations thereof. The stem cells may refer to cells having differentiation potency, and examples thereof are blast cells, hepatocytes, fibroblasts, myoblasts, adult stem cells, mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow- derived mesenchymal stem cells, nerve-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, cord blood stem cells or combinations thereof. The growth factor may refer to a substance capable of regulating the growth and function of a cell. The differentiation factor may refer to a substance that induces differentiation of cells into tissues or other cells. For example, the growth factor or the differentiation factor may include a transformed growth factor (TGF), a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), an epithelial growth factor (EGF), a platelet-derived growth factor (PDGF), a hepatocyte growth factor (HGF), an insulinlike growth factor (IGF), a cytokine, a chemokine, or combinations thereof.Delivery Vehicle

[0153] In one embodiment, the hydrogel, as described herein, can be employed to deliver agents such as active agents. Active agents can be noncovalently associated with the hydrogel compositions by exposing the hydrogel to the active agent before or during cross-linking, or by subsequently contacting a hydrogel with the active agent. Active agents can also be covalently incorporated into the hydrogels either through direct linkage to the hydrogel, or indirectly through a crosslinking agent. Direct and indirect linkages may be formed through, for example, interactions with one or more functional groups of the hydrogel, or at a terminus thereof.

[0154] Active agents can also be incorporated into the hydrogel through a combination of covalent and noncovalent interactions.

[0155] The hydrogels, as described herein, may also be used to deliver a broad variety of diagnostic agents. The hydrogel, as described herein, can also be used to deliver living cells to a desired site of administration. The delivered cells may be used for a variety of purposes including the release hormones and growth factors or the formation of new tissue. In order to entrap the cells within a formed hydrogel, the cells can be suspended in a suitable medium and then mixed with the hydrogel. The components forming the hydrogel can be added in variety of ways including but not limited to addition as: a dry powder or as a reconstituted solution which has not yet gelled. Alternatively, the components forming the cross-linked hydrogel can be prepared as a mildly buffered solution or suspension at a pH that inhibits hydrogel formation, and the cells are prepared in a buffered medium sufficient to maintain the viability of the cellsupon admixture with the hydrogel components. Formation of the hydrogel at the pH of the resultant combined hydrogel and cell culture medium will result in the entrapping the cells within the hydrogel.

[0156] When used as a delivery means, the hydrogels, as described herein, can provide a depot for sustained release of materials, such as active agents, associated with or entrapped within the hydrogels, such that they may be released under physiological conditions. In an embodiment there is provided a method of controlled release of an active agent from a hydrogel , wherein the hydrogel is as defined by any one or more embodiments or examples herein. In some embodiments, the controlled release may occur under physiological conditions. In alternative embodiments, the controlled released may occur under other conditions, such as acidic conditions. It will be appreciated that the controlled release may include release of an active agent from the hydrogel for application in wound healing including a sprayable hydrogel for wound healing or antibacterial release, cartilage repair, etc.

[0157] In some embodiment, there is provided a method of storing an active agent supported by or encapsulated within a hydrogel, wherein the hydrogel is as defined by any one or more embodiments or examples herein. The hydrogels, as described herein, can be delivered to a patient by a variety of means including but not limited to subdermally (subcutaneously), orally, intravenously, intraperitoneally, dermally (transdeimal delivery), intradermally, intratumorily, intraocularly, intravicscerally, intraglandularly, intravaginally, intrastromally, intrasynovially intrasinus, intraventricullarly, intrathecally, intramuscularly, and intrarectally.Tissue Engineering

[0158] The hydrogel, as described herein, can also be used for tissue engineering purposes, such as the formation of scaffold materials and tissue augmentation, including soft and hard tissue augmentation.

[0159] A general method for effecting augmentation of tissue involves injecting solutions or suspensions of the hydrogel within the tissue in need of augmentation. The pH of the hydrogel may be adjusted through use of appropriate physiologically compatible buffer solutions. When the hydrogel contains bioactive agents such ascollagen, the collagen can be incorporated into the matrix by reacting with the hydrogel as discussed above.

[0160] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.EXAMPLES

[0161] The present disclosure is further described by the following examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.Example 1: General peptide synthesisExample la: Materials

[0162] All natural Fmoc-protected a-amino acids, HATU, Fmoc-Rink Amide AM, and Oxyme were purchased from Chemlmpex. P-amino acids were purchased from ChemScene. HPLC grade acetonitrile, and N,N-dimethylformamide (DMF) were obtained from Merck (Australia). 8-arm PEG Norbomene (MW 20000) were obtained from JenKem Technology Inc (USA). All other reagents were obtained from Sigma- Aldrich (Australia).Example lb: Method

[0163] The peptides that chosen were synthesized on LibertyBlue synthesizer using Fmoc-Rink-amide AM resin at a scale of 0.25 mmol. The resin was loaded into a syringe reactor equipped with a porous polyethylene frit for the synthesis. To initiate the synthesis, the resin was pre-swollen in DCM for 10 minutes and then filtered and washed three times with dimethylformamide. Then, the swollen resin mounted on Liberty blue. The Fmoc-deprotection process has been conducted using deprotection solution (piperidine and DMF 20:80, v / v). For the coupling steps in this solid-phase peptide synthesis (SPPS) protocol.

[0164] After removal of the last protecting group and washing steps, the resin was placed in a desiccator overnight. Afterwards, the peptide resin was exposed to a cleavage mixture consisting of TFA, TIS, and water in a ratio of 95:2.5:2.5 (v / v / v) for 90 minutes at room temperature (RT). The cleavage solution was separated from the resin by filtration, and the resulting peptides were precipitated by adding chilled diethyl ether, followed by centrifugation and washing twice with diethyl ether. The obtained peptide left under vacuum for 30 minutes to remove residual solvents. Finally, the peptides were dissolved in water and subjected to lyophilization.

[0165] To prepare the peptide hydrogel, the peptide powder (2 wt.%) was added to 450 pL of MilliQ water and subjected to ultrasonic bath treatment for ten minutes to ensure complete dissolution. Following this, 50 pL of lOx PBS (Gibco, 70011044) was swiftly added to the solution and mixed using a spatula. The mixture was then centrifuged at a gradually increasing speed, reaching 2000 rpm, for a duration of ten seconds. The samples were left to rest overnight before any further experiments.

[0166] The peptide sequences were synthesized using solid-phase peptide synthesis (SPPS). This technique is used to produce short peptides.Example 1c: HPLC and LCMS characterisation of the peptide sequences

[0167] Analytical and Preparative HPLC was performed on Cl 8 column (21.2 3 250 mm, 10 Im, Phenomenex, Torrance, CA) in a model Agilent 1260 Infinity system (Agilent Technologies, Santa Clara, CA). Solvents A and B were 0.1% TFA (v / v) in water and acetonitrile, respectively, and Elution was undertaken with linear gradients of solvent B into A over 15 min at 1 mL / min flow rate, with UV detection at 215 nm. Preparative fractions of satisfactory purity by analytical HPLC were pooled and lyophilized. Finally, the purified peptides were characterized by LCMS.Example Id: Rheological measurements of the peptide sequences

[0168] The rheological characteristics of the peptide-based hydrogel were evaluated using a Physica MCR302 rheometer (Anton Paar, Ostfildern, Germany) equipped with a plate-plate accessory (with a diameter of 25 mm and a gap of 0.4 mm). The hydrogelswere prepared at 2 wt.% in previously and subsequently placed on the Peltier module, where they were left undisturbed for a duration of 10 minutes.

[0169] The oscillatory frequency sweep test was performed with a frequency range of 0.01 to 100 Hz, and a constant 1% strain, to monitor changes in the storage modulus (G1) and loss modulus (G"). The viscosity of the hydrogels was also measured by subjecting them to a shear rate ramp ranging from 0.01 to 1000 s'1. To evaluate the recoverability of the hydrogels, changes in G' and G" were monitored during two repetitive steps. First, the samples were subjected to a 1% strain for 1 minute, followed by a 100% strain for another minute, simulating low and high strain conditions. This cycle of alternating low and high strains was repeated for 3 cycles, allowing for observation of any changes in G' and G" and evaluating the ability of the hydrogels to recover their mechanical properties after repeated strain cycles. Table 3 presents a summary of the storage modules of the hydrogels prepared using novel peptide sequences at 100 Hz.

[0170] The inventors have shown that having P-amino acids within the peptide sequence could improve the stability of the peptide hydrogels. By doing so, the rheological behaviour and stability of the self-assembled peptide was improved. Figure 1-3 compares the rheological behaviour of the modified peptide sequences comprising at least one P-amino acid with two known peptide sequences at 2wt.% solid content at 20 °C in terms of storage modules, shear-thinning behaviour and recoverability. Figure 4 shows the injectability of the peptide hydrogel (SEQ ID NO: 13) at 2 wt.% solid content at room temperatureTable 3: Storage modulus of the peptide hydrogels with 2 wt. % solid content at 100Hz

[0171] Comparative peptides Pep-Cl and C2 were prepared following the method outlined in Example 1 and the peptides sequences are reported in:• Pep-Cl : (1) Martin, C., et al. (2016). “Injectable peptide hydrogels for controlled release of opioids.” MedChemComm 7(3): 542-549. (2) Martin, C., et al. (2017). "Injectable peptide-based hydrogel formulations for the extended in vivo release of opioids." Materials Today Chemistry 3: 49-59.• Pep-C2: Mangelschots, J., et al. (2016). "Mixed a / p-Peptides as a Class of Short Amphipathic Peptide Hydrogelators with Enhanced Proteolytic Stability." Biomacromolecules 17(2): 437-445Example le: Peptide stability study

[0172] For the stability study, triplicate of 2 wt. % SEQ ID NO: 13, SEQ ID NO: 14, and pep-Cl hydrogels were prepared in 15-mL Falcon tubes and left to rest for two hours. The hydrogels were weighed (including the Falcon tubes) before 4 mL of 1 x PBS (Gibco, 70011044) was added and then placed in an orbital mixer incubator (Ratek) at 37 degrees and 100 rpm. After different intervals, the supernatant was removed and the hydrogels were weighed. Weight loss due to degradation was calculated using equation 1.where Mo: hydrogel weight at time 0 [g] and Mt: hydrogel weight at time t [g]

[0173] Table 4 provides a summary of the weight loss results for the hydrogels prepared using novel peptide sequences after a 45-day period. Figure 5 shows the stability of the modified peptide sequences comprising at least one P-amino acid with two known peptide sequences at 2wt.% solid content at 37 °C.Table 4: Weight loss of the peptide hydrogels with 2 wt.% solid content after 45 daysExample If: Drug release study

[0174] For the drug release study, BSA-FITC (Sigma Aldrich, A9771) was dissolved in MilliQ water in the first preparation step of the hydrogels before adding the peptide powders. Then, the standard protocol for the hydrogel preparation (Example lb) was followed. Hydrogels were prepared in 15-mL Falcon tubes and allowed to rest for two hours before adding 4 mL of PBS. Samples were stored in an orbital mixer incubator (Ratek) at 37 °C and 100 rpm. The entire volume of supernatant was carefully removedand replaced with fresh PBS at distinct time points, 0 (for the determination of the drug incorporation efficiency), 18, 24, 48, and 72 hours, and then once a week. Supernatant samples were stored at 4 °C until measured. BSA-FITC concentration was quantified with a plate reader (BMG Labtech, CLARIOstar Plus) by measuring the fluorescence intensity at 530 nm (excitation wavelength: 483 nm). A calibration curve between the signal intensity and the BSA-FITC concentration was constructed for each measurement. Table 5 provides a summary of the weight percentage of the drug released from the prepared hydrogels after a 21 -day period. Figure 6 demonstrates the percentage of released active agent (BSA-FIRC) encapsulated within a peptide hydrogel at 2 wt.% solid content at 37 °C.Table 5: Weight loss of the peptide hydrogels with 2 wt.% solid content after 90 daysExample 2: General cross-linker synthesisExample 2a: Part A

[0175] Generally, five steps are involved in the Bromoacetylation and propargylation of the resin as follows and shown below in Scheme 1. Representative crosslinkers and their building block components are shown in Table 6.

[0176] Step (a) In a dry and Argon flushed reactor 10 equivalents of bromoacetic acid 1 and 5 equivalents of N,N’diisopropylcarbodiimide (DIC) were dissolved in 5 mL of dry DCM.

[0177] Step (b) The mixture was stirred for 30 min and then transferred to another vessel containing previously DCM swollen and Fmoc deprotected resin.

[0178] Step (c) The mixture was shaken for 2 h, filtered off, washed with DCM and DMF four times.

[0179] Step (d) The resin-bound bromide was then displaced with propargylamine (10 equivalents in 5 mL of DMF), shaken for 8 h at room temperature, filtered and washed three times with DMF, methanol, chloroform, and diethyl ether respectively.

[0180] Step (e) The peptide spacer and the diol (or boronic acid) was then added.Scheme 1: Synthesis of the two part cross-linkerTable 6: Representative Crosslinkers and their building block componentsExample 2b: Part B

[0181] The final cross-linker was prepared as follows and as shown in Scheme 2 below. The equivalent crosslinkers can also be prepared with thiol end groups instead of alkyne groups.

[0182] Peptide-boronic acid and peptide-diol were added to a flask under an inert atmosphere. The mixture was then stirred in tetrahydrofuran (THF) for approximately 30 minutes until it was completely dissolved. Sodium sulphate was then slowly added to the solution and stirred for 16 hours at room temperature. The resulting mixture was then filtered to remove the sodium sulphate and the filtrate was concentrated under reduced pressure to yield the pure solid product as an off-white solid in a yield that is equal to the amount of product that was theoretically possible to obtain.

[0183] Figures 9 and 11 show NMR spectra of starting materials and their corresponding final products (boronic ester crosslinkers) containing either alkyne (Figure 9) or thiol (Figure 11) end groups made using similar reactions. The changes in the NMR spectra compared to the starting material support the successful formation of the desired boronic ester containing crosslinkers.Scheme 2: Conjugation of the cross-linkerExample 3: Crosslinking and de-crosslinking of the hydrogel network

[0184] After synthesis of cross-linker and the peptide sequences, the resulting selfassembling peptide and cross-linker and photo-initiator will be mixed in aqueous solution and react under UV to form the strong hydrogel, to dissociate the hydrogel, a diol containing molecule or polymer, such as sugar, was added to disrupt the boronic ester bond. Figure 7 illustrates the stability of crosslinked and un-crosslinked peptide hydrogel with 2wt.% solid content at 37 °C in IX PBS. The molar ratio of peptide and 8-arm PEG-Norbornene were 8:1. The peptide sequence is FEFQFKGGC. The crosslinked hydrogel were exposed to UV light (365 nm, intensity of 174 mW) for 1 min. As seen, unlike un-crosslinked hydrogel, the crosslinked hydrogel remain stable over the experiment period. Figure 8 shows the changes in the rheological behaviour of peptide / 8-arm PEG-Norbornene 2wt.% solid content at 37 °C in IX PBS. The ratio of peptide and 8-arm PEG-Norbornene were 8: 1. The peptide sequence is FEFQFKGGC. The crosslinked hydrogel were exposed to UV light (365 nm, intensity of 174 mW) for 1 min. Accordingly, the storage modulus of the peptide hydrogel jump to higher values immediately after being exposed to UV light.

[0185] Further examples of crosslinking and de-crosslinking the hydrogels networks are shown in Figures 10, 12, 13 and 14. Figure 10 shows that the boronic estercontaining crosslinker formed by the adduct of SEQ ID No:41 with SEQ ID No:42 can be cleaved (de-crosslinked) upon exposure to a diol, in this case glucose. The degree of cleavage can be controlled by the diol (glucose) concentration. Figure 12 shows the NMR spectrum of a thiol containing crosslinker formed by the adduct of SEQ ID No:43 with SEQ ID No:44 (20 mg / ml) which can form a crosslinked network (hydrogel) by reaction with an 8-arm PEG-Norborene (50mg / ml) in D2O under UV irradiation in the presence of a photoinitiator (Irgacure 2959, 5mg / ml). The disappearance of the ethylenic bonds of the 8-arm PEG-Norborene support the crosslinking reaction occurring. Further evidence of successful crosslinked network (hydrogel) of the adduct of SEQ ID No:43 with SEQ ID No:44 upon reaction with an 8-arm PEG-Norborene is shown in Figure 13 which shows the change in shear modulus upon UV exposure whereas the 8-arm PEG-Norborene alone shows no change in mechanical properties upon UV exposure. Figure 14 also shows successful crosslinking of thiol containing crosslinker component SEQ ID No:45 with an 8-arm PEG-Norborene upon UV exposure in the presence of a photoinitiator (Irgacure 2959) by disappearance of the ethylenic bonds of the 8-arm PEG-Norborene.

Claims

CLAIMS:

1. A crosslinker of Formula 1-1:Formula 1-1 wherein R1and R2are each independently selected from the group comprising or consisting of Ci-ealkyl, -O-Ci-ealkyl, Ci-ehaloalkyl, -O-Ci-ehaloalkyl, C2-ealkenyl, C2- ealkynyl, 3-10-membered carbocyclyl, and 3-10-membered heterocyclyl; wherein each 3-10-membered carbocyclyl and 3-10-membered heterocyclyl, is unsubstituted or substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence; wherein R3aand R3bare each independently selected from C=C-R4a, C=C-R4b, and SH, wherein when R3ais C=C-R4aor C=C-R4b, R3bis not SH, or when R3bis C=C- R4aor C=C-R4b, R3ais not SH; and wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl.

2. The crosslinker of claim 1, wherein R1and R2are each independently selected from the group comprising or consisting of 3-10-membered carbocyclyl and 3-10- membered heterocyclyl; wherein each 3-10-membered carbocyclyl and 3-10-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence.

3. The crosslinker of claim 1 or claim 2, wherein R1and R2are each independently selected from a 5- or 6-membered carbocyclyl and 5- or 6-membered heterocyclyl; wherein each 5- or 6-membered carbocyclyl and 5- or 6-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence.

4. The crosslinker of any one of the preceding claims, wherein R1and R2are each independently selected from a 6-membered carbocyclyl and 6-membered heterocyclyl; wherein each 6-membered carbocyclyl and 6-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence.

5. The crosslinker of any one of the preceding claims, wherein R1is a 6- membered carbocyclyl substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence and R2is a 6-membered heterocyclyl substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence.

6. The crosslinker of any one of the preceding claims, wherein the crosslinker of Formula 1-1 is a compound of Formula 1-2:Formula 1-2 wherein R5a, R5b, R5c, R5d, R5e, R5f, R5g, are each independently selected from CH and N; wherein R6aand R6bare each independently selected from a PEG, a glycine, or a peptide sequence; and wherein R7aand R7bare each independently selected from C=C-R4a, C=C-R4b, and SH, wherein when R3ais C=C-R4aor C=C-R4b, R3bis not SH, or when R3bis C=C- R4aor C=C-R4b, R3ais not SH; and wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl.A hydrogel comprising a reaction product of (i) a compound comprising a thiol, an alkyne or an alkene and (ii) a cross-linking compound of Formula II-l :Formula II-l wherein R1and R2are each independently selected from the group comprising or consisting of Ci-ealkyl, -O-Ci-ealkyl, Ci-ehaloalkyl, -O-Ci-ehaloalkyl, C2-ealkenyl, C2- ealkynyl, 3-10-membered carbocyclyl, and 3-10-membered heterocyclyl; wherein each 3-10-membered carbocyclyl and 3-10-membered heterocyclyl, is unsubstituted or substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence; wherein R3aand R3bare each independently selected from C=C-R4aand C=C- R4b, and SH, wherein when R3ais C=C-R4aor C=C-R4b, R3bis not SH, or when R3bis C=C-R4aor C=C-R4b, R3ais not SH; and wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl.The hydrogel of claim 7, wherein the thiol, alkyne, or alkene compound is selected from the group comprising or consisting of a peptide, a protein, or an enzyme, wherein the peptide, protein or enzyme comprises at least one thiol, alkyne, or alkene group.The hydrogel of claim 7 or claim 8, wherein the thiol, alkyne, or alkene compound is a peptide comprising at least one thiol, alkyne, or alkene group.

10. The hydrogel of any one claims 7 to 9, wherein the cross-linking compound of Formula II-l is a diol -responsive cross-linker.

11. The hydrogel of any one of claims 7 to 10, comprising a reaction product of (i) a peptide comprising at least one thiol, alkyne, or alkene group and (ii) a diolresponsive cross-linking compound of Formula II-l.

12. The hydrogel of any one of claims 7 to 11, wherein R1and R2are each independently selected from the group comprising or consisting of 3-10-membered carbocyclyl and 3-10-membered heterocyclyl; wherein each 3-10-membered carbocyclyl and 3-10-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence.

13. The hydrogel of any one of claims 7 to 12, wherein R1and R2are each independently selected from a 5- or 6-membered carbocyclyl and 5- or 6-membered heterocyclyl; wherein each 5- or 6-membered carbocyclyl and 5- or 6-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence.

14. The hydrogel of any one of claims 7 to 13, wherein R1and R2are each independently selected from a 6-membered carbocyclyl and 6-membered heterocyclyl; wherein each 6-membered carbocyclyl and 6-membered heterocyclyl, is substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence.

15. The hydrogel of any one of claims 7 to 14, wherein R1is a 6-membered carbocyclyl substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence and R2is a 6- membered heterocyclyl substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence.

16. The hydrogel of any one of claims 7 to 15, wherein the cross-linking compound of Formula II- 1 is a compound of Formula II-2:Formula II-2 wherein R5a, R5b, R5c, R5d, R5e, R5f, R5g, are each independently selected from CH and N; wherein R6aand R6bare each independently selected from a PEG, a glycine, or a peptide sequence; and wherein R7aand R7bare each independently selected from are each independently selected from C=C-R4aand C=C-R4b, and SH, wherein when R3ais C=C- R4aor C=C-R4b, R3bis not SH, or when R3bis C=C-R4aor C=C-R4b, R3ais not SH; and wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl.

17. The hydrogel of any one of claims 7 to 16, wherein the thiol group of the peptide reacts with a terminal alkene or alkyne group of the diol-responsive crosslinking compound of Formula II- 1 or Formula II-2 or the alkene or alkyne group of the peptide reacts with a terminal thiol of the diol-responsive cross-linking compound of Formula II- 1 or Formula II-2.

18. The hydrogel of any one of claims 7 to 19, wherein the reaction product is a compound of Formula III-l :Formula III-l wherein R5a, R5b, R5c, R5d, R5e, R5f, R5g, are each independently selected from CH and N; wherein R6aand R6bare each independently selected from a PEG, a glycine, or a peptide sequence; wherein R7aand R7bare each independently selected from are each independently selected from SH, C=C-R4a, and C=C-R4b, and C-C-R4c; wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl; andX is selected from the group comprising or consisting of a peptide sequence, a protein, or an enzyme, wherein the peptide sequence, protein or enzyme comprise at least one thiol, alkyne, or alkene group.

19. The hydrogel of any one of claims 9 to 18, wherein the peptide comprises less than 30 amino acids.

20. The hydrogel of any one of claims 9 to 18, wherein the peptide comprises at least 6 the amino acids.

21. The hydrogel of any one of claims 9 to 18, wherein the peptide comprises between 6 and 20 amino acids in the amino acid sequence.

22. The hydrogel of any one of claims 9 to 21, wherein the peptide is stable for at least 30 days.

23. The hydrogel of any one of claims 9 to 22, wherein the peptide is stable for at least 90 days.

24. The hydrogel of any one of claims 9 to 23, wherein the ratio of peptide to cross-linking compound is at least 2: 1.

25. The hydrogel of any one of claims 7 to 24 encapsulating an active agent.

26. A process for preparing a hydrogel, comprising(i) obtaining a thiol, alkyne or alkene compound,(ii) obtaining a cross-linking compound of Formula II-l :Formula II-l wherein R1and R2are each independently selected from the group comprising or consisting of Ci-ealkyl, -O-Ci-ealkyl, Ci-ehaloalkyl, -O-Ci-ehaloalkyl, C2-ealkenyl, C2- ealkynyl, 3-10-membered carbocyclyl, and 3-10-membered heterocyclyl; wherein each 3-10-membered carbocyclyl and 3-10-membered heterocyclyl, is unsubstituted or substituted with one or more substituents selected from the group comprising or consisting of a PEG, a glycine, or a peptide sequence; wherein R3aand R3bare each independently selected from C=C-R4a, C=C-R4b, and SH, wherein when R3ais C=C-R4aor C=C-R4b, R3bis not SH, or when R3bis C=C- R4aor C=C-R4b, R3ais not SH; and wherein R4aand R4bare each independently selected from hydrogen and Ci- ealkyl, and(iii) mixing aqueous solutions of the thiol, alkyne or alkene compound of step (i) and the cross-linker of step (ii) to form an aqueous mixture.

27. The process of claim 26, wherein the aqueous mixture is exposed to light.

28. The process of claim 26 or claim 27, wherein the aqueous mixture is exposed visible light.

29. The process of claim 26 or claim 27, wherein the aqueous mixture is exposed ultra-violet light.

30. The process of any one of claims 26 to 29, wherein the cross-linking compound is according to any one of claims 7 to 16.

31. The process of any one of claims 26 to 30, wherein the thiol, alkyne or alkene compound is selected from the group comprising or consisting of a peptide, a protein, or an enzyme, wherein the peptide, protein or enzyme comprises at least one thiol, alkyne or alkene group.

32. The process of any one of claims 26 to 31, wherein the thiol, alkyne or alkene compound is a peptide comprising at least one thiol, alkyne or alkene group.

33. A method of controlled release of an active agent from a hydrogel, wherein the hydrogel is according to any one of claims 7 to 25 or prepared according to any one of claims 26 to 32, on contact with a diol containing molecule or polymer.

34. The method of claim 33, wherein the diol containing molecule or polymer is selected from a sugar.

35. The method of claim 34, wherein the sugar is in the form of a solution and present in the solution at a concentration of between about 0.1 mM to about 50 mM.

36. A method of storing an active agent supported by or encapsulated within a hydrogel, wherein the hydrogel is according to any one of claims 7 to 25 or prepared according to any one of claims 26 to 32.

37. Use of a hydrogel of any one of claims 7 to 25 as an injectable, drug delivery vehicle, tissue engineering, or carrier in controlled release of an active agent.

38. The use of claim 37, wherein the release of the active agent from the hydrogel is activated by exposure to a diol containing molecule or polymer.

39. The use of claim 38, wherein the hydrogel softens on exposure to a diol containing molecule or polymer.

40. A hydrogel bioink composition for bioprinting, comprising the hydrogel according to any one of claims 7 to 25 or the hydrogel prepared according to any one of claims 26 to 32.

41. The hydrogel bioink composition of claim 40, wherein the hydrogel bioink composition is laden or seeded with cells.

42. A process for preparing a cell supportive structure, the process comprising:(i) preparing a bioink composition comprising the hydrogel according to any one of claims 7 to 25 or the hydrogel prepared according to any one of claims 26 to 32; and(ii) 3D-printing the composition to produce a support.

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