Peptides, hydrogels, photoactivatable hydrogel precursors, methods of preparation and devices for delivery thereof, and methods of treatment therewith

Abstract

Provided herein are self-assembling collagen-like peptide (CLP) comprising a plurality of amino acid trimer repeats having the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine. Also provided herein are compositions, scaffolds, photoactivatable hydrogel precursors and hydrogels comprising the CLP and a multi-arm polyethylene glycol (PEG) polymer. Methods of preparing photoactivatable hydrogel precursors and hydrogels are also described. Methods of treating a wound, of treating a corneal disease, of treating a myocardial infarct, of promoting cell growth and of treating cellular degeneration using the compositions, scaffolds, photoactivatable hydrogel precursors and / or hydrogels are also described. Also provided is a device for delivering a peptide-based material. The device comprises an on / off button or switch for initiating and stopping delivery of the peptide-based material in the subject; a chamber for containing the peptide-based material; an aperture through which the peptide-based material exits the chamber, wherein the aperture is adapted for receiving a nozzle adapter; a camera port for securing a camera thereto; and a digital control display.

Description

Peptides, hydrogels, photoactivatable hydrogel precursors, methods of preparation and devices for delivery thereof, and methods of treatment therewith FIELD OF INVENTION

[0001] The present invention relates generally to peptides, hydrogels, photoactivatable hydrogelprecursors (PHP), methods of preparing hydrogels and PHP, and methods of treatment using the hydrogels and / or the PHP. More specifically, the present invention relates to peptides for the preparation of hydrogels and PHP, methods of preparing hydrogels and PHP for treating diseases related to cellular degeneration and / or acute injuries. BACKGROUND

[0002] Historically, naturally occurring polymers have been widely used as building blocks for thesynthesis of materials with therapeutic potential for organ and tissue repair.1While some of these materials are already in clinical use,2-6the lack of precise chemical tunability, batch-to-batch variability, generally weak mechanical properties, and potential immunogenicity stemming from impurities remain barriers to biomedical innovation.7-9Further, the concept of “one size fits all” is unrealistic when considering the intrinsic differences in physical and mechanical properties amongst organs and tissues, as well as the variance between individuals in the shape and extent of tissue damage in need of repair.10Thus, in recent years, the use of synthetic materials,11whose properties can be finely tuned, has gained considerable interest, with successful examples in soft organ repair.12Amongst the many synthetic materials, short peptide-based materials (<50 amino acids long) present several advantages, which include relatively low cost of manufacturing at clinical grade and the flexibility for further chemical modification. However, the use of external chemical crosslinkers to assemble 3D structures, which are longitudinally used for most therapeutic biomaterials, can be detrimental for their use in applications such as stem cell delivery and has largely limited their application to pre-crosslinked implants where side-products have been washed out.13Further, the precise design of peptide sequences must encompass self-assembly of the chains in the crosslinked state to strengthen the resulting mechanical properties of the material. Some examples in the literature report bio-orthogonal assembly of peptide-based hydrogels, mainly using single cysteine containing peptides.14-16However, the application of this chemistry with collagen-like peptides (CLPs) remains for the most part underdeveloped.17CLPs mimic the tertiary structure of collagen, a structural protein that provides strength to tissues, and are often composed of Glycine-Proline-Proline (GPP) or Glycine-Proline-Hydroxyproline (GPO) amino acid triplets.18

[0003] Wound healing is a natural process that occurs in the body to repair damaged tissue. This responsehighlights an evolutionary trade-off that practises functionality over aesthetic results and thus restricts the range of wounds that can be fully restored to their original state. For large or chronic wounds, intervention is often necessary to prevent infection and promote local healing. As a result, considerable resources have been devoted towards the development of effective wound closure methods and materials for this therapeutic use1–3.

[0004] Medical sutures have remained the gold-standard treatment for wound closure, primarily due totheir exceptional tensile strength and versatility in accommodating different type of wounds4. Despite their effectiveness, the intrusive nature of sutures has several disadvantages, namely the labor-intensive process, the need for highly qualified professionals, lengthy application time, the potential for further tissue injury and infection, as well as unsatisfactory aesthetic outcomes3While alternative methods, such as staples, offer advantages in terms of time-efficiency and wound closure strength, they possess several limitations that restrict their applicability. For instance, the use of staples in surgical procedures has been found to significantly reduce operation times, but due to their intrusive nature, they have been frequently linked with higher postoperative pain and worse cosmetic outcomes compared to sutures3,5. Alternatives such as bioadhesives have gained significant attention due to their fast and efficient wound closure while also providing superior aesthetic results in relation to conventional mechanical techniques3,6.

[0005] Tissue adhesives are far from a novel idea, in fact, they have been in development for the pastthree decades7. The fundamental premise consists of utilizing or developing a material capable of binding two or more fragmented tissues while maintaining the integrity of the surrounding tissue environment. Currently, a range of surgical adhesives are commercially available with the most common being fibrin, cyanoacrylate, and glutaraldehyde derivatives8,9. Taking BioGlue® as an example, this is a two- component solution capable of sealing the targeted region by utilizing bovine serum albumin (BSA) and glutaraldehyde10. When BSA and glutaraldehyde come into contact, the material polymerizes sealing the area while providing significant tensile strength to the adhesion target. However, despite offering quick application times and satisfactory adhesion, BioGlue® lacks the natural tunable elastic qualities of epithelial tissue. Additionally, the use of glutaraldehyde as a polymerizing molecule has been criticized for posing several safety concerns, as glutaraldehyde products have been documented to be cytotoxic for both in vitro and in vivo experimentations8,10. With few exceptions the benefits and limitations of BioGlue® outlined here are characteristics shared by most contemporary tissue adhesives11,12. As aresult, the next generation of tissue adhesives should address the toxicity of materials used and strive to emulate the mechanical properties of skin while maintaining the ease of use and time efficiency that are characteristic of current adhesives products.

[0006] Alternative, additional, and / or improved hydrogels, and methods for the production thereof, aretherefore desirable, particularly for the development of improved tissue adhesives. SUMMARY OF INVENTION

[0007] Provided herein are peptides, scaffolds, hydrogels, precursor hydrogels thereof, compositionsthereof and uses thereof. Photosensitive bulking agents (PBA) comprising peptides, scaffolds, hydrogels, and / or precursor hydrogels are provided herein. Also provided herein are methods of preparing hydrogels and methods of treating corneal diseases.

[0008] According to a first embodiment, there is provided herein a self-assembling collagen-like peptide(CLP) comprising a plurality of amino acid trimer repeats having the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0009] According to an embodiment, the plurality of amino acid trimer repeats is flanked at the N-terminal and the C-terminal by at least two amino acid residues.

[0010] According to an embodiment, the at least two amino acid residues are cysteine, glycine, valineor a combination thereof.

[0011] According to an embodiment, the at least two amino acid residues is defined by amino acidsequence GCG.

[0012] According to an embodiment, the collagen-like peptide (CLP) is defined by amino acid sequenceG-X-G(POG)nG-X-G, wherein X is G or C, and 3 ≤ n ≤ 20.

[0013] According to an embodiment, X is C and 6 ≤ n ≤ 10.

[0014] According to an embodiment, n = 8.

[0015] A second aspect of the present invention is directed to a hydrogel.

[0016] According to an embodiment, the hydrogel comprises a self-assembling collagen-like peptide(CLP) and a multi-arm polyethylene glycol (PEG) polymer, wherein the CLP comprises a plurality of amino acid trimer repeats having the amino acid sequence POG, wherein P is a proline, O is a hydroxyproline or a cysteine, and G is a glycine or a valine.

[0017] According to an embodiment, the multi-arm PEG polymer is crosslinked to the CLP.

[0018] According to an embodiment, the multi-arm PEG polymer is a 2‐Arms‐PEG acrylate, 4‐Arms‐PEG acrylate, 8‐Arms‐PEG acrylate, a 2‐Arms‐PEG maleimide, 4‐Arms‐PEG maleimide, 8‐Arms‐PEG maleimide or any combination thereof.

[0019] According to an embodiment, the PEG is an 8‐Arms‐PEG acrylate, an 8‐Arms‐PEG maleimideor any combination thereof.

[0020] According to an embodiment, the plurality of amino acid trimer repeats is flanked at the N-terminal and the C-terminal by at least two amino acid residues.

[0021] According to an embodiment, the at least two amino acid residues are cysteine, glycine, valine ora combination thereof.

[0022] According to an embodiment, the at least two amino acid residues is defined by amino acidsequence GCG.

[0023] According to an embodiment, the collagen-like peptide (CLP) is defined by amino acid sequenceG-X-G(POG)nG-X-G, wherein X is G or C, and 3 ≤ n ≤ 20.

[0024] According to an embodiment, X is C and 6 ≤ n ≤ 10.

[0025] According to an embodiment, n = 8.

[0026] According to an embodiment, the water content in the hydrogel is between about 60% to about90%.

[0027] According to an embodiment, the self-assembling CLP has a CMratio of between about 1.0 toabout 5.0.

[0028] A third aspect of the present invention is directed to a photoactivatable hydrogel precursor (PHP).

[0029] According to an embodiment, the PHP comprises a self-assembling collagen-like peptide (CLP),wherein the CLP comprises a plurality of amino acid trimer repeats having the amino acid sequence POG, wherein P is a proline, O is a hydroxyproline or a cysteine, and G is a glycine or a valine; and a multi-arm polyethylene glycol (PEG) polymer comprising at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator.

[0030] According to an embodiment, a hydrogel is obtained by photoactivating the hydrogel precursorwith visible light.

[0031] According to an embodiment, the visible light has a wavelength of between about 500 nm toabout 580 nm.

[0032] According to an embodiment, the visible light is green light.

[0033] According to an embodiment, the photoinitiator is Rose Bengal, Eosin Y, or any other photoactivemolecule.

[0034] According to an embodiment, the photoinitiator is Eosin Y.

[0035] According to an embodiment, the concentration of PEG-maleimide and / or PEG-acrylate isbetween about 1% w / v and about 25% w / v.

[0036] A fourth aspect of the present invention is directed to a bioadhesive.

[0037] According to an embodiment, the bioadhesive comprises the self-assembling CLP, the hydrogelor the photoactivatable hydrogel as described herein.

[0038] A fifth aspect of the present invention is directed to a scaffold.

[0039] According to an embodiment, the bioadhesive comprises the self-assembling CLP, the hydrogelor the photoactivatable hydrogel as described herein.

[0040] A sixth aspect of the present invention is directed to a composition.

[0041] According to an embodiment, the composition comprises the self-assembling CLP as describedherein and a multi-arm polyethylene glycol (PEG) polymer.

[0042] According to an embodiment, the PEG polymer comprises at least one acrylate group and / or atleast one maleimide group, and at least one photoinitiator.

[0043] A seventh aspect of the present invention is directed to method of preparing a hydrogel.

[0044] According to an embodiment, the method comprises mixing a self-assembling collagen-likepeptide (CLP) with a multi-arm polyethylene glycol (PEG) polymer, wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0045] According to an embodiment, a hydrogel precursor is first obtained by mixing the self-assemblingCLP with the multi-arm PEG polymer, wherein the hydrogel is obtained after the hydrogel precursor is incubated for a period of time to allow crosslinking of the self-assembling CLP to the multi-arm PEG.

[0046] According to an embodiment, the period of time is less than 5 minutes.

[0047] An eight aspect of the present invention is directed to a method of preparing a photoactivatablehydrogel precursor (PHP).

[0048] According to an embodiment, the method comprises mixing a self-assembling collagen-likepeptide (CLP) with a multi-arm polyethylene glycol (PEG) polymer, wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, and wherein the multi-arm PEG polymer comprises at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator.

[0049] A ninth aspect of the present invention is directed to a method of preparing a photoactivatedhydrogel.

[0050] According to an embodiment, the method comprises photoactivating the PHP as described herein.

[0051] According to an embodiment, photoactivating the PHP comprises irradiating the PHP with light.

[0052] According to an embodiment, the light is visible light.

[0053] According to an embodiment, the visible light has a wavelength of between about 500 nm toabout 580 nm.

[0054] According to an embodiment, the visible light is green light.

[0055] According to an embodiment, the photoinitiator is Rose Bengal or Eosin Y.

[0056] According to an embodiment, the photoinitiator is Eosin Y.

[0057] According to an embodiment, the concentration of PEG-maleimide and / or PEG-acrylate isbetween about 1% w / v and about 25% w / v.

[0058] A tenth aspect of the present invention is directed to a method of treating a wound.

[0059] According to an embodiment, the method comprises exposing a wound to a therapeuticallyeffective amount of a hydrogel precursor, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) with a multi-arm polyethylene glycol (PEG) polymer, wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0060] According to an embodiment, the method further comprises waiting for a period of time afterexposing the wound to allow crosslinking of the self-assembling CLP to the multi-arm PEG.

[0061] According to an embodiment, exposing the wound comprises covering the wound with thehydrogel precursor.

[0062] According to an embodiment, exposing the wound comprises injecting the therapeuticallyeffective amount of the hydrogel precursor in a subject in need thereof.

[0063] According to an embodiment, injecting is performed at or around a wound site.

[0064] According to an embodiment, the wound is in a soft tissue.

[0065] According to an embodiment, the soft tissue is muscle.

[0066] According to an embodiment, the soft tissue is cornea.

[0067] According to an embodiment, the soft tissue is skin.

[0068] An eleventh aspect of the present invention is directed to a method of treating a corneal diseasein a subject in need thereof.

[0069] According to an embodiment, the method comprises injecting a therapeutically effective amountof a hydrogel precursor in the subject, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0070] According to an embodiment, the method further comprises waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0071] According to an embodiment, the corneal disease comprises corneal thinning and / or a cornealwound.

[0072] A twelfth aspect of the present invention is directed to a method of treating a myocardial infarctin a subject in need thereof.

[0073] According to an embodiment, the method comprises injecting a therapeutically effective amountof a hydrogel precursor in the subject, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLPcomprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0074] According to an embodiment, the method further comprises waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0075] A thirteenth aspect of the present invention is directed to a method of promoting cell growth.

[0076] According to an embodiment, the method comprises treating a cell with a therapeutically effectiveamount of a hydrogel precursor in the subject, wherein the hydrogel precursor comprises a self- assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0077] According to an embodiment, the method further comprises waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0078] A fourteenth aspect of the present invention is directed to a method of treating cellulardegeneration in a subject in need thereof.

[0079] According to an embodiment, the method comprises injecting a therapeutically effective amountof a hydrogel precursor in the subject, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0080] According to an embodiment, the method further comprises waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0081] A fifthteenth aspect of the present invention is directed to a method of treating a wound.

[0082] According to an embodiment, the method comprises exposing a wound to a therapeuticallyeffective amount of a photoactivatable hydrogel precursor (PHP), wherein the PHP comprises a self- assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, and a multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator; and photoactivating the PHP.

[0083] According to an embodiment, the method further comprises waiting for a period of time afterphotoactivating to allow crosslinking of the PHP to the PEG polymer.

[0084] According to an embodiment, photoactivating the PHP comprises irradiating the wound withlight.

[0085] According to an embodiment, the light is visible light.

[0086] According to an embodiment, the visible light has a wavelength of between about 500 nm toabout 580 nm.

[0087] According to an embodiment, the visible light is green light.

[0088] According to an embodiment, the photoinitiator is Rose Bengal or Eosin Y.

[0089] According to an embodiment, the photoinitiator is Eosin Y.

[0090] According to an embodiment, the concentration of PEG-maleimide and / or PEG-acrylate isbetween about 1% w / v and about 25% w / v.

[0091] According to an embodiment, exposing the wound comprises covering the wound with the PHP.

[0092] According to an embodiment, exposing the wound comprises injecting the therapeuticallyeffective amount of the PHP in a subject in need thereof.

[0093] According to an embodiment, the wound is in a soft tissue.

[0094] According to an embodiment, the soft tissue is muscle.

[0095] According to an embodiment, the soft tissue is cornea.

[0096] According to an embodiment, the soft tissue is skin.

[0097] A sixteenth aspect of the present invention is directed to a method of treating a corneal disease ina subject in need thereof.

[0098] According to an embodiment, the method comprises injecting a therapeutically effective amountof a photoactivatable hydrogel precursor (PHP) in the subject, wherein the PHP comprises a self- assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, and a multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator; and photoactivating the hydrogel precursor.

[0099] According to an embodiment, the method further comprises waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0100] According to an embodiment, photoactivating the PHP comprises irradiating the subjectwith light.

[0101] According to an embodiment, the light is visible light.

[0102] According to an embodiment, the visible light has a wavelength of between about 500 nmto about 580 nm.

[0103] According to an embodiment, the visible light is green light.

[0104] According to an embodiment, the photoinitiator is Rose Bengal or Eosin Y.

[0105] According to an embodiment, the photoinitiator is Eosin Y.

[0106] According to an embodiment, the concentration of PEG-maleimide and / or PEG-acrylateis between about 1% w / v and about 25% w / v.

[0107] According to an embodiment, the PHP is injected in the cornea.

[0108] According to an embodiment, the corneal disease comprises corneal thinning and / or acorneal wound.

[0109] A seventeenth aspect of the present invention is directed to a method of treating amyocardial infarct in a patient in need thereof.

[0110] According to an embodiment, the method comprises injecting a therapeutically effectiveamount of a photoactivatable hydrogel precursor (PHP) in the patient, wherein the PHP comprises a self- assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine; and a multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator; and photoactivating the hydrogel precursor.

[0111] According to an embodiment, the method further comprises waiting for a period of timeafter injecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0112] According to an embodiment, photoactivating the PHP comprises irradiating the subjectwith light.

[0113] According to an embodiment, the light is visible light.

[0114] According to an embodiment, the visible light has a wavelength of between about 500 nmto about 580 nm.

[0115] According to an embodiment, the visible light is green light.

[0116] According to an embodiment, the photoinitiator is Rose Bengal or Eosin Y.

[0117] According to an embodiment, the photoinitiator is Eosin Y.

[0118] According to an embodiment, the concentration of PEG-maleimide and / or PEG-acrylateis between about 1% w / v and about 25% w / v.

[0119] According to an embodiment, PHP is injected in the infract or in a region proximal to theinfarct.

[0120] An eighteenth aspect of the present invention is directed to a method of promoting cellgrowth.

[0121] According to an embodiment, the method comprises treating a cell with a therapeuticallyeffective amount of a photoactivatable hydrogel precursor (PHP), wherein the PHP comprises a self- assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, and a multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator; and photoactivating the hydrogel precursor.

[0122] According to an embodiment, the method further comprises waiting for a period of timeafter injecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0123] According to an embodiment, photoactivating the PHP comprises irradiating the cell withlight.

[0124] According to an embodiment, the light is visible light.

[0125] According to an embodiment, the visible light has a wavelength of between about 500 nmto about 580 nm.

[0126] According to an embodiment, the visible light is green light.

[0127] According to an embodiment, the photoinitiator is Rose Bengal or Eosin Y.

[0128] According to an embodiment, the photoinitiator is Eosin Y.

[0129] According to an embodiment, the concentration of PEG-maleimide and / or PEG-acrylateis between about 1% w / v and about 25% w / v.

[0130] A nineteenth aspect of the present invention is directed to a method of treating cellulardegeneration in a subject in need thereof.

[0131] According to an embodiment, the method comprises treating the subject with atherapeutically effective amount of a photoactivatable hydrogel precursor (PHP), wherein the PHP comprises a self-assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, and a multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator; and photoactivating the hydrogel precursor.

[0132] According to an embodiment, the method further comprises waiting for a period of timeafter injecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0133] According to an embodiment, photoactivating the PHP comprises irradiating the subjectwith light.

[0134] According to an embodiment, the light is visible light.

[0135] According to an embodiment, the visible light has a wavelength of between about 500 nmto about 580 nm.

[0136] According to an embodiment, the visible light is green light.

[0137] According to an embodiment, the photoinitiator is Rose Bengal or Eosin Y.

[0138] According to an embodiment, the photoinitiator is Eosin Y.

[0139] According to an embodiment, the concentration of PEG-maleimide and / or PEG-acrylateis between about 1% w / v and about 25% w / v.

[0140] A twentieth aspect of the present invention is directed to a use of the hydrogel as describedherein, the PHP as described herein, the bioadhesive as described herein, or the scaffold as described herein, as a wound sealant, for improving wound closure, for treating a corneal disease, for treating a myocardial infarct, for treating a corneal disease, for promoting cell growth and / or for treating cellulardegeneration.

[0141] A twenty-first aspect of the present invention is directed to a device for delivering apeptide-based material to a subject.

[0142] According to an embodiment, the device comprises an on / off button or switch forinitiating and stopping delivery of the peptide-based material to the subject, a chamber for containing the peptide-based material, an aperture through which the peptide-based material exits the chamber, wherein the aperture is adapted for receiving a nozzle adapter, a camera port for securing a camera thereto; and a digital control display.

[0143] According to an embodiment, the chamber is adapted for receiving one or more peptidecartridge containing the peptide-based material.

[0144] According to an embodiment, the peptide cartridge is a preloaded syringe.

[0145] According to an embodiment, the camera allows live monitoring when delivering thepeptide-based material in the subject.

[0146] According to an embodiment, the digital control display allows adjusting at least onedelivery parameter.

[0147] According to an embodiment, the at least one delivery parameter comprises volume to bedelivered and speed at which the volume is delivered to the subject.

[0148] According to an embodiment, the nozzle adapter is adapted to receive a variety of nozzles.

[0149] According to an embodiment, the variety of nozzles comprise a nozzle for topicalapplication, a nozzle for cornea sealant delivery, and a nozzle for intratissue injection.

[0150] According to an embodiment, the device further comprises a handle.

[0151] According to an embodiment, the device is a handheld device.

[0152] Other and further aspects and advantages of the present invention will be betterunderstood upon the reading of the illustrative embodiments about to be described or will be indicated in the appended claims, and various advantages not referred to herein will occur to one skilled in the art upon employment of the invention in practice.BRIEF DESCRIPTION OF DRAWINGS

[0153] These and other features will become better understood with regard to the followingdescription and accompanying drawings, wherein:

[0154] FIGURE 1 shows the mass spectra illustrating detected ions from peptide 1 havingsequence NH2-GCG(POG)4GCG-OH.

[0155] FIGURE 2 shows the mass spectra illustrating detected ions from peptide 2 havingsequence NH2-GCG(POG)5GCG-OH.

[0156] FIGURE 3 shows the mass spectra illustrating detected ions from peptide 3 havingsequence NH2-GCG(POG)6GCG-OH.

[0157] FIGURE 4 shows the mass spectra illustrating detected ions from peptide 4 havingsequence NH2-GCG(POG)7GCG-OH.

[0158] FIGURE 5 shows the mass spectra illustrating detected ions from peptide 5 havingsequence NH2-GCG(POG)8GCG-OH.

[0159] FIGURE 6 shows the mass spectra illustrating detected ions from peptide 6 havingsequence NH2-GCG(POG)9GCG-OH.

[0160] FIGURE 7 shows the mass spectra illustrating detected ions from peptide 7 havingsequence NH2-GCG(POG)10GCG-OH.

[0161] FIGURE 8 shows the mass spectra illustrating detected ions from peptide 8 havingsequence NH2-GGG(POG)8GCG-OH.

[0162] FIGURE 9 shows the mass spectra illustrating detected ions from peptide 9 havingsequence NH2-GCG(POG)8GGG-OH.

[0163] FIGURE 10 shows the mass spectra illustrating detected ions from peptide 10 havingsequence NH2-GGG(POG)8GGG-OH.

[0164] FIGURE 11 shows the mass spectra illustrating detected ions from peptide 11 havingsequence NH2-GGG(POG)3(PCG)2(POG)3GGG-OH.

[0165] FIGURE 12 shows the mass spectra illustrating detected ions from peptide 12 havingsequence NH2-GGG(POG)2(PCG)(POG)5GCG-OH.

[0166] FIGURE 13 shows the mass spectra illustrating detected ions from peptide 13 havingsequence NH2-GCG(POG)5(PCG)(POG)2GGG-OH.

[0167] FIGURE 14 shows the mass spectra illustrating detected ions from peptide 14 havingsequence NH2-GGG(POG)2(PCG)(POG)2(PCG)(POG)2GGG-OH.

[0168] FIGURE 15 shows the mass spectra illustrating detected ions from peptide 15 havingsequence NH2-GCG(POG)4POV(POG)3GCG-OH.

[0169] FIGURE 16 shows the mass spectra illustrating detected ions from peptide 16 havingsequence NH2-GCG(PPG)8GCG-OH.

[0170] FIGURE 17 shows the mass spectra illustrating detected ions from peptide 17 havingsequence NH2-GC(POG)8CG-OH.

[0171] FIGURE 18 shows the mass spectra illustrating detected ions from peptide 18 havingsequence NH2-GCV(POG)8VCG-OH.

[0172] FIGURE 19 shows the mass spectra illustrating detected ions from peptide 19 havingsequence NH2-GCR(POG)8RCG-OH.

[0173] FIGURE 20 shows the mass spectra illustrating detected ions from peptide 20 havingsequence NH2-GCC(POG)8CCG-OH.

[0174] FIGURE 21 shows the mass spectra illustrating detected ions from peptide 21 havingsequence NH2-GCG(POG)2(PCG)(POG)2(PCG)(POG)2GCG-OH.

[0175] FIGURE 22 shows the mass spectra illustrating detected ions from the control peptidehaving sequence NH2-CG(PKG)4(POG)4(DOG)4-OH.

[0176] FIGURE 23 shows the relative abundance of Pep-5 monomer to disulfide dimer measuredthrough mass spectroscopy identification of [M*2+3H] signal for the disulfide dimer product in Hanks buffer saline solution (HBSS). Dimer formation creates a new peptide mass of 2,591*2 – 2H = 5,180 Da that produces a +3H ion signal of 1,728 Da.

[0177] FIGURE 24 shows the percent reactive thiol for Pep-5 (NH2-GCG(POG)8GCG-OH), Pep-10 (NH2-GGG(POG)8GGG-OH), and free cysteine (positive control). Reactivity was measured with Ellman’s assay via reaction with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). All solutions were freshly prepared in 100 mM sodium phosphate buffer pH 7.4 while samples (n=3) prepared at a concentrationof 0.5 mM for peptides (1.0 mM thiol concentration for Pep-5 containing two cysteines) and 1.0 mM free cysteine then diluted 21x with 0.5 mM DTNB and incubated for 30 minutes in the dark before measurement. The sequence containing cysteine (Pep-5) showed similar available cysteine compared to the control while the sequence with no cysteine residues (Pep-10) showed no available cysteine.

[0178] FIGURE 25 shows Top left: CD spectra for selected peptides (3, 4, and 5) measured at200 µM concentration in phosphate saline buffer pH 7.4. The other plots in the figure correspond to melting curve plots measured for the 21 peptides and CLP at 220 nm scanned from 20 to 75ºC. Curves were fitted using a sigmoidal model with R2> 0.99.

[0179] FIGURE 26 shows the folding (Rpn) of the top candidate peptide Pep-5 and the controlCLP at different concentrations. Experiments were carried out in phosphate saline buffer pH 7.4.

[0180] FIGURE 27 shows that modification of a single amino acid in peptide sequence affectsbonding capacity of the engineered hydrogel. A glycine spacer was removed from the effective sequence of Pep-5 to produce the sequence Pep-17 to investigate the effect of the glycine spacer. Tensile strength tests were performed with these two peptides. The spacer peptide sequence was significantly stronger p<0.05 (~45 kPa, n=5) than the sequence lacking a spacer (~18 kPA, n=5) despite similar folding propensity (Rpn0.087 vs 0.080 for Pep-5 and Pep-17, respectively) which could indicate a less efficient coupling reaction.

[0181] FIGURE 28 shows a schematic depiction of the strategy used in this study for the in situassembling of the materials. Assembly of the hydrogel takes place within minutes at room temperature. Some of the key chemical motifs of the material components are depicted at the bottom of the schematic..

[0182] FIGURE 29 shows a flow diagram of the tests used to screen the material formulations.This methodology was designed to narrow down the number of candidate formulations.

[0183] FIGURE 30 shows changes in absorption intensities at 300 nm (squares) and 260 nm(romboid) for maleimide solutions measured in carbonate buffer at three different temperatures (n=3). Changes in absorption intensities are indicative of losing maleimide group.

[0184] FIGURE 31 shows the ability of the top peptide candidate (Pep-5) to form hydrogels invarious common buffers (ammonium bicarbonate, MES, Tris, PBS) in a range of physiological pH values (6.4-7.4) and different ratios of peptide to PEG maleimide (CMratio). Representative pictures taken after 30 min hydrogel preparation. Hydrogel formation was visible in all buffers at the different CMratiotested.Note that for the 4.0 CMratio, the hydrogel has to be removed from the Eppendorf tube as it was a solid material. Scale bars for 1.0 and 3.0 CMratio are 5 mm. For the 4.0 CMratio scale bar is 2 cm.

[0185] FIGURE 32 shows (A) Heatmap displaying changes in gelation time (s) measured asdescribed herein. illustrating changes in the parameters of materials with different CMratio and peptide formulations (abbreviated as Pep, n=5) and (B) representative still images of two selected peptide formulations (50 µL, CMratioof 4.0) taken at 0, 30, and 45s after positioning the samples at a 45º inclination. Scale bar is 2 mm.

[0186] FIGURE 33 shows heatmaps displaying changes in (A) Transmittance (%); (B) refractiveindex; (C) water content (%); (D) denaturation temperature (ºC); (E) collagenase degradation (mg / h); measured as described herein illustrating changes in the parameters of materials with different CMratioand peptide formulations (abbreviated as Pep, n=5);

[0187] FIGURE 34 shows (A) ATR-FTIR (with the respective deconvoluted Gaussian fit)spectra of selected peptides at CMratio1.0; and (B) heatmap showing the change in the relative peak area of the double bond maleimide against Amide I peptide signals.

[0188] FIGURE 35 shows (A) 3D renders for the custom-designed hand-held device fordelivering the peptide-based materials designed in this study. The render displays the main features of the device that include: (1) Dispensing on / off control, (2) enclosed compartment for placing peptide- cartridges (preloaded syringes), (3) aperture for nozzle adapters, (4) camera port for live monitoring, and (5) digital controller screen for volume and speed settings. (B) Three representative nozzles designed for the hand-held device presented herein for topical application (left), cornea sealant delivery (middle), and intratissue injection (right).

[0189] FIGURE 36 shows a diagrammatic depiction for assembly instructions of the handle. (A)Right handle compartment with the locations for (1) the main driver board, (2) the USB-C port and (3) charge port. (B) Left handle compartment with the locations for (1) the camera control module, (2) DC- DC converter, (3) the rechargeable 9V battery and (4) actuation buttons. (C) Steps to put together the handle by combing both the left and right compartment and securing them using 2 bolts at the bottom and a plate connecting them.

[0190] FIGURE 37 shows a diagrammatic depiction for assembly of the extrusion chamber. (A)Installation of the Lead Screw, Driving Nut and DC motor in the lower main chassis. (B) Installation ofthe upper chassis rotating panel. (C) Process to assemble the main chassis using 2 bolt. (D) Installation of the power switch, adjusting knob and OLED display at the back of the main compartment. (E) Attachment of the extrusion chamber to the handle using 3 headless bolts.

[0191] FIGURE 38 shows a hand-held device for the delivery of the peptide-based material. (A)Side view of the hand-held device with the 2 cartridge install and side panels open with overall dimensions. (B) Back View of the device with the overall dimensions that include; (1) 1.3in OLED display, (2) Clickable Knob to navigate the device’s menu and adjust any of the device parameters, (3) An On / Off Switch. (C) Overview of the Extrusion Mechanism which includes; (1) The driven gear of the lead screw, (2) the bearing to stabilize the screw, (3) Driving Nut which attaches to the cartridge’s plungers, (4) Lead Screw, (5) Driving Gear of the Motor, (6) DC Motor with Magnetic Encoder. (D) Bottom-Isometric view of the Hand-Held Device which includes (1) The Hinged magnetic doors, (2) the charge port, (3) a USB-C port to update the microcontroller’s firmware and (4) the connector for the camera.

[0192] FIGURE 39 shows the injection mixer for cornea sealant delivery. (A) Front view of thenozzle with overall dimensions, (1) outlet and (2) Dual Inlet with Luer Lock Connector. (B) Close-up view of the nozzle tip that includes (1) the outlet, (2) the mixing system based on the Kenics micromixer and (3) contact point for the solutions. (C) Detailed look at the Kenics micromixer in the nozzle tip with overall dimensions, (1) counterclockwise helical piece, (2) clockwise helical piece.

[0193] FIGURE 40 shows the injection mixer Luer adapter for intratissue injection. (A) Frontview of the nozzle with overall dimensions showing (1) the outlet and (2) Dual-Inlet with Luer-Lock Connectors. (B) Close-up view of the nozzle tip showing (1) the mixing system based on the Kenics micromixer and (2) Male Luer lock adapter.

[0194] FIGURE 41 shows the topical application mixer. (A) Front view of the nozzle with overalldimensions. (B) Close-up view of the nozzle tip showing (1) the flexible brush for spreading the material and (2) the bi-layer outlet.

[0195] FIGURE 42 shows (A) a schematic depiction for the procedure followed to assess skinbonding in a full-thickness incision. Bonded tissue was assessed using uniaxial mechanical stretching tests, and (B) mechanical strength (kPa), also known as wound closure strength test (ASTM F2458), measured for murine skin tissue after application of peptide-based formulations CMratio4.0 (10 mm / min extension) for top peptide-based hydrogels prepared using peptide 3, 4, and 5. Values measured forapplications of BioGlue® and the collagen-like peptides (CLP) are also included in the plot.

[0196] FIGURE 43 shows that pep-5 is a good peptide sequence for tissue bonding. Tensilestrength in kPa for mice skin ex vivo bonding carried out using the different peptide variants (CMratio= 4.0, n=5). Samples measured in quintuplicate. P value calculated from t-test (two tails unpaired data).

[0197] FIGURE 44 shows (A) Mechanical strength (kPa) for murine skin tissue measured usingthe 4.0 ratio but at two different dilutions ½ and ¼ (n=3-5). (B) Changes in bond strength as a function of time post-application (10, 30, and 60 min). Samples were incubated at 37ºC in 100% humidity (n=3- 5). (C) Left: Illustration for leap-shearing tests using porcine skin. The adhesion surface was maintained constant at 50 mm2using a 10 mm / min extension. The adhesive (peptide-based formulation or BioGlue®) was applied within the two pieces of skin. Right: Adhesion force values measured for the peptide-based formulation CMratio 4.0 and BioGlue®. P-values were calculated using One-Way ANOVA and post-hoc Holmes test at 95% confidence interval.

[0198] FIGURE 45 shows (A) hydrogel samples of Pep-5 formulations that were either testedright after preparation or swollen overnight in phosphate saline buffer. Then, the hydrogel structure was evaluated using a TESCAN scanning electron microscope, Model VegaII XMU, at low-vacuum conditions. SEM images of hydrogels were captured at varying magnifications. Setting conditions included an acceleration voltage of 20 kV, a Cryo-Stage temperature of −50 °C and a chamber vacuum ≈35 Pa; (B) Quantification of pore diameter.

[0199] FIGURE 46 shows cell viability images measured using Live / Dead assay for fibroblastcells seeded on top of Pep-5 hydrogels or gelatin coated well plates (48h post seeding, n=3). (B) Data was not statistically different between the two groups.

[0200] FIGURE 47 shows a schematic of the experimental design used for the in vivo testing ofthe tissue bonding capabilities of the peptide-based materials using C57BL / 6 female mice (7-8 weeks).

[0201] FIGURE 48 shows representative images of the full-thickness wounds taken at 0-, 1-, 3,and 7-days post-treatment for the different experimental groups. Scale bars = 5.0 mm.

[0202] FIGURE 49 shows mechanical strength (kPa) for murine skin tissue measured 7 dayspost-operation (n=5-8) obtained for the different experimental groups. The Sham group corresponds to animals that underwent the same steps as those that received incisions, but the skin remained intact, p values were calculated using One-Way ANOVA and post-hoc Holmes test at 0.05 confidence interval.

[0203] FIGURE 50 shows (A) representative histological images of the skin at 7 days for the 3treatment groups. (B) and (C) Analysis of the histological sections obtained for the different treatmentgroups (n≥12) including collagen deposition (B), and epidermal thickness (C) p values were calculatedusing One-Way ANOVA and post-hoc Holmes test at 0.05 confidence interval.40-42

[0204] FIGURE 51 shows (A) images for tissue slides displaying CD206+ positive cells forsuture, BioGlue, and peptide (Pep-5) treated groups, and (B) quantification thereof Data was not statiscally different between all three groups.

[0205] FIGURE 52 shows Left: Burst pressure values measured for the peptide-based materialCMratio 4.0 or cyanoacrylate (n=3-5). Right: Illustration for the pig cornea perforation ex vivo model used in this study. The numbers in the Figure illustrate (1) initial perforation to create a wound bed, (2) inner full-thickness cornea perforation using a 1 mm biopsy puncher, (3) application of the treatment to seal the hole, and (4) formation of a “corneal” patch, student t-tests were used (unpaired unequal variance).

[0206] FIGURE 53 shows cell viability measured using Live / Dead assay for human epithelialcorneal cells seeded on top of Pep-5 hydrogels or gelatin coated well plates (48h post seeding, n=3). Data was not statistically different between the two groups.

[0207] FIGURE 54 shows changes in implant thickness that were obtained using 30 µL of theCMratio4.0 peptide material or Viscoat (n=3-5), student t-tests were used (unpaired unequal variance).

[0208] FIGURE 55 shows (A) Illustration of the ex vivo pig cornea pocket and cornea reshapingmodel used in this study. The numbers in the figure illustrate (1) initial surgical incision to create a wound bed, (2) insertion of needle (27G) to open a cavity, (3) injection of the peptide-based material as anintracorneal patch, and (4) positioning of a solid contact lens. (B) Representative corneal topographicaxial maps showing surface elevation of corneas injected with either CMratio 4.0 or Viscoat. The topographic maps are generated for corneas before injection, after injection, and injection in conjunction with a contact lens (Centracone). The scale on the right shows the height in mm, with warmer colors representing steeper areas and cooler colors marking flatter ones. (C) Fold change vs. empty pocket for K values of curvature (mm) obtained for corneas that have been injected with either CMratio 4.0 or Viscoat, with the placement of Centracone rigid contact lens in conjunction with the peptide-based material (n=4-5) with volumes ≈30µL, student t-tests were used (paired data).

[0209] FIGURE 56 shows (A) a schematic of the experimental design for in vivo testing of thetissue bonding capabilities of the peptide-based materials. Experiments were carried out using C57BL / 6 female mice (7-8 weeks), and (B) Percentage of live human cardiac endothelial cells measured at 0 and 2 days after seeding on the CMratio 1.0 material or collagen-based hydrogel.

[0210] FIGURE 57 shows left ventricular ejection fraction (LVEF) fold change relative tobaseline measured 28 days after treatment with CMratio 1.0 material or PBS (n=4-5), p values were calculated using t-test (unpaired data unequal variance).

[0211] FIGURE 58 shows (A) Representative Masson trichrome staining images for heartstreated with CMratio 1.0 material or PBS. (B) Analysis of the scar size for the two treatment groups (n=4- 5).

[0212] FIGURE 59 shows the number of CD206+ cells counted in myocardial tissue sectionswithin the scar, border zone, and remote areas.

[0213] FIGURE 60 shows (A) Quantification for ex vivo fluorescence imaging (λexcitation= 570nm; λemission= 640 nm) of hearts injected with the Alexa-Fluor@594-labelled CMratio1.0 material at different days post-injection. (B) Representative IVIS images of the MI hearts harvested after 2- or 7- days post-injection (n=3-5).

[0214] FIGURE 61 shows FTIR analysis of Pep-5 and other components. (A) FTIR spectra forthe different components of the peptide-based material prepared using Pep-5. (B-E) Gaussian fit analysis for Pep-5 alone or mixed with PEG-Mal at different CMratio values.

[0215] FIGURE 62 shows FTIR analysis of Pep-10 and other components. (A) FTIR spectra forthe different components of the peptide-based material prepared using Pep-10. (B-C) Gaussian fit analysis for Pep-10 alone or mixed with PEG-Mal.

[0216] FIGURE 63 shows Gaussian fit analysis for Pep-3 alone or mixed with PEG-Mal atdifferent CMratiovalues.

[0217] FIGURE 64 shows Gaussian fit analysis for Pep-3 alone or mixed with PEG-Mal atdifferent CMratio values.

[0218] FIGURE 65 shows a schematic summary of the development of a comprehensive low-volume approach for fabrication of external crosslinker-free soft materials using custom-designed collagen-like peptides containing bifunctional thiols for in situ repair of skin, cornea, and cardiac tissue.

[0219] FIGURE 66 shows a schematic, dimensions and volumes used for wound closure testing:Sample was standardized into strips of 30 mm length, 5 mm with and 1 mm thickness, the sample was bisected and 30 µL of sample was placed between the ‘wound’ area, the sample was exposed to green light at varied dosage depending on the test, material wound closure strength was tested on an INSTRON that pulled the sample on a single axis at a rate of 5 mm / min where the force of the material was recorded until the point of mechanical failure.

[0220] FIGURE 67 shows the research methodology for the development and testing of tissueadhesives: Step 1: Optimization of light-activated formulation with respect to adhesion to skin tissue,Step 2: Selection of most effective material delivery method to ensure good adhesion strength andmaterial stability over time, Steps 3, 4 and 5: Application of tissue adhesive to skin incision and materialactivation in situ using green light to establish strong and stable wound closure.

[0221] FIGURE 68 shows mechanical and adhesive properties of peptide-based green lightactivated material with optimization of wound closure strength by varying the ratio of PEG-maleimide, PEG-acrylate and peptide in the formulations.

[0222] FIGURE 69 shows (A) concentrations of PEG-Maleimide, PEGAcrylate, and collagen-like peptide have a direct impact on the adhesion strength of the material, (B) average viscosity of top formulations, (C) impact of photoreactive molecule on formulation wound closure strength, (D) effect of green light irradiation time on material wound closure strength (E) highest adhesion strength attained before the adhesive-skin tissue junction fractures, (F) the maximum strain of the top hydrogel candidates before fracture, All tissue adhesives were irradiated with green light after application on the skin for 2 minutes (19 J / cm2) unless otherwise specified, statistical significance was set at *p <0.05 by one-way ANOVA lack of symbol indicates no significance. Error bars are ± SEM, n=5 except for figure B and C n=3.

[0223] FIGURE 70 shows physical characterization of the top tissue adhesive candidates, with(A) average swelling profile of the top two hydrogels in PBS over a 2-day period (B) water content of top formulations (C) effect of collagenase on hydrogel degradation over time. (D) average reduction in absorption peak intensity for materials exposed to higher amounts of green light. Statistical significance was set at *p <0.05 by one-way ANOVA. Error bars are ± SEM, n=5 except for figure D and C n=3.

[0224] FIGURE 71 shows optimization of individual elements for formulation P, with (A) effectof molecular weight on formulation, (B) effect of varied concentrations of PLL on strength of thematerial, (C) increasing concentrations of PEGDA effect on wound closure strength, (D) effect of the collagen-like peptide on formulation P, all tissue adhesives were irradiated with green light after application on the skin for 2 minutes (19 J / cm2) unless otherwise specified, statistical significance was set at *p <0.05 by one-way ANOVA lack of symbol indicates no significance. Error bars are ± SEM, n=5 except for figure A (n=4) and B (n=3); and

[0225] FIGURE 72 shows wound closure strength data of multiple formulations, all tissueadhesives were irradiated with green light after application on the skin for 2 minutes (19 J / cm2) unless otherwise specified, statistical significance was set at *p <0.05 by one-way ANOVA lack of symbol indicates no significance, error bars are ± SEM, n=4. DETAILED DESCRIPTION

[0226] Novel collagen-like peptides (CLP), compositions, scaffolds, photoactivatable hydrogelprecursors (PHP), hydrogels and methods of preparation thereof will be described hereinafter. Methods of treating a wound, a corneal disease, a myocardial infarct, of promoting cell growth and of treating cellular degeneration using the compositions, scaffolds, photoactivatable hydrogel precursors and / or hydrogels will also be described hereinafter. Uses of the collagen-like peptides (CLP), compositions, scaffolds, photoactivatable hydrogel precursors (PHP), hydrogels for treating a wound, a corneal disease, a myocardial infarct, of promoting cell growth and of treating cellular degeneration will also be described. A device for delivering a hydrogel or PHP will also be described. Although the invention is described in terms of specific illustrative embodiments, it is to be understood that the embodiments described herein are by way of example only and that the scope of the invention is not intended to be limited thereby.

[0227] The terminology used herein is in accordance with definitions set out below.

[0228] By "about", it is meant that the value can vary within a certain range depending on themargin of error of the method or device used to evaluate or measure. A margin of error of 10% is generally accepted.

[0229] The description which follows, and the embodiments described therein are provided byway of illustration of an example of particular embodiments of principles and aspects of the present invention. These examples are provided for the purposes of explanation and not of limitation, of thoseprinciples of the invention. In the description that follows, like parts and / or steps are marked throughout the specification and the drawing with the same respective reference numerals.

[0230] Poly (ethylene glycol), commonly abbreviated as PEG, is a synthetic polyether materialthat has garnered significant attention in the field of biomaterials13–15. Its extensive utilization can be attributed to its well-established biocompatibility and ability to modify its backbone with various groups suit specific needs13,14. For biomaterials, the fundamental structure of PEG is often adorned with active groups that can be used for different types of cross-linking reactions with photoreactive cross-linking being one of the most common due to its ability to control the time of the reaction9,16,17. In particular, the use of UV cross-linking has been the most widely employed method for the purpose of creating bioadhesive materials18. All UV cross-linking processes are analogous, using the PEG adhesive FocalSeal® as an example, the process of hydrogel formation begins when the material’s photoinitiator is activated by 450-500 nm light, causing the formation of free radicals that in turn react with acrylic groups resulting in the polymerization of the material and binding of fragmented tissues18,19. Despite FocalSeal®’s efficiency in sealing air lung leaks and retinal brakes, other parts of the body cannot be treated with UV-crosslinking due to the UV lights low penetration beyond 5 mm, limiting its application to superficial tissue repair18. Additionally, it is worth nothing that while the Food and Drug Administration (FDA) has granted approval for certain procedures involving the use of UV-light, it is important to consider the potential safety concerns associated with this method. Specifically, the risk of local tissue damage occurring during the usage of UV-light20,21. Furthermore, it is crucial to highlight that the surgical community has found FocalSeal® to have limited adaptability, which has ultimately resulted in the discontinuation of this product22. A potential avenue for addressing the limitations associated with photoactivated tissue sealants, such as FocalSeal®, could involve exploring approaches that utilize a lower energy wavelength such as visible light or through the addition of alternative cross- linking methods that enable a broader application. Regarding the use of lower energy light, recent research into photoinitiators indicate that either Rose Bengal or Eosin Y molecules could be feasible alternatives as these have been reported to exhibit comparable free-radical production when exited by visible light in the range of 500 -580 nm (green light), and also for potentially deeper tissue penetration and thus addressing some of the restrictions presented by systems that use UV-light23–25.

[0231] Self-assembling peptide structures bearing a cysteine to each end of the peptide ends,were first combined with maleimide-baring multi-arm polyethylene glycol (PEG); an FDA approvedsynthetic polymer13to rapidly produce 3D-structures with tunable mechanical properties via Michael- addition at physiological pH. This material design combines the dual mechanisms of chemical bonding and molecular assembly that are often used in nature to provide tissues with mechanical stability.14 Using Michael-addition chemistry minimizes potential cross-reactions when used in vivo compared to other chemical groups such as acrylamides,15and renders stable bonds (>2 years)16. While in principle, the concept of peptide-PEG on-the-spot assembly sounds simple, there are many structural and chemical considerations, which in practice involves challenging peptide design and synthesis. In this study, we developed a library of +20 peptide sequences to identify the best candidate peptide for on-the-spot tissue repair. Top peptide sequences were classified as those that: (1) Formed a triple helix in solution and showed stability at physiological pH and temperature; (2) showed suitable gelation times, e.g., 15-120s; (3) formed 3D materials with suitable physical properties for use in soft tissue repair (e.g., transmittance, refractive index, denaturation temperature, water content, and resistance to collagenase degradation).

[0232] Development, optimization, and characterization of a PEG-based hydrogel comprisingthe top performing peptides was then performed to obtain a hydrogel or a photoactivatable hydrogel precursor (PHP) suitable as a photoactivated adhesive formulation for the purpose of suture-free wound closure. The formulations presented here took advantage of click-chemistry through the utilization of a thiolated collagen-like peptide (CLP), multi-arm PEGs functionalized with acrylate and maleimide groups and a photo-initiator to form stable networks that cause adhesion between local tissue and the hydrogel24,26–33.The cross-linking process of the material was made possible by exposure to green light, with the adhesive properties being modulated by varying the time of exposure24,34. We tested our material’s adhesiveness ex vivo and demonstrated our top tissue adhesive candidates could adhere to mouse skin more effectively than BioGlue after only 2 minutes of light activation. The comprehensive research strategy employed in this study is concisely summarized in Figure 1.

[0233] A first aspect of the present invention is directed to self-assembling collagen-like peptides(CLP).

[0234] In an embodiment, there is provided herein a self-assembling collagen-like peptide (CLP)comprising a plurality of amino acid trimer repeats having the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0235] In an embodiment, the plurality of amino acid trimer repeats is flanked at the N-terminaland the C-terminal by at least two amino acid residues, which may be cysteine, glycine, valine or acombination thereof.

[0236] In an embodiment, the at least two amino acid residues is defined by amino acid sequenceGCG.

[0237] In an embodiment, the collagen-like peptide (CLP) is defined by amino acid sequence G-X-G(POG)nG-X-G, wherein X is G or C, and 3 ≤ n ≤ 20. X may be C and 6 ≤ n ≤ 10, for example, n = 8.

[0238] A second aspect of the present invention is directed to a hydrogel comprising a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, wherein the CLP comprises a plurality of amino acid trimer repeats having the amino acid sequence POG, wherein P is a proline, O is a hydroxyproline or a cysteine, and G is a glycine or a valine.

[0239] In an embodiment, the multi-arm PEG polymer may be crosslinked to the CLP.

[0240] In an embodiment, the multi-arm PEG polymer is a 2‐Arms‐PEG acrylate, 4‐Arms‐PEGacrylate, 8‐Arms‐PEG acrylate, a 2‐Arms‐PEG maleimide, 4‐Arms‐PEG maleimide, 8‐Arms‐PEG maleimide or any combination thereof.

[0241] In an embodiment, the PEG is an 8‐Arms‐PEG acrylate, an 8‐Arms‐PEG maleimide orany combination thereof.

[0242] In an embodiment, the plurality of amino acid trimer repeats is flanked at the N-terminaland the C-terminal by at least two amino acid residues.

[0243] In an embodiment, the at least two amino acid residues are cysteine, glycine, valine or acombination thereof.

[0244] In an embodiment, the at least two amino acid residues is defined by amino acid sequenceGCG.

[0245] In an embodiment, the collagen-like peptide (CLP) is defined by amino acid sequence G-X-G(POG)nG-X-G, wherein X is G or C, and 3 ≤ n ≤ 20.

[0246] In an embodiment, X is C and 6 ≤ n ≤ 10.

[0247] In an embodiment, n = 8.

[0248] In certain embodiments, the water content in the hydrogel is between about 60% to about90%.

[0249] A third aspect of the present invention is directed to a photoactivatable hydrogel precursor(PHP) comprising a self-assembling collagen-like peptide (CLP), wherein the CLP comprises a plurality of amino acid trimer repeats having the amino acid sequence POG, wherein P is a proline, O is a hydroxyproline or a cysteine, and G is a glycine or a valine. The PHP also comprises a multi-arm polyethylene glycol (PEG) polymer comprising at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator.

[0250] In an embodiment, a hydrogel is obtained by photoactivating the hydrogel precursor withvisible light.

[0251] In an embodiment, the visible light has a wavelength of between about 500 nm to about580 nm..

[0252] In an embodiment, the visible light is green light.

[0253] In an embodiment, the photoinitiator is Rose Bengal, Eosin Y, or any other photoactivemolecule.

[0254] In an embodiment, the photoinitiator is Eosin Y.

[0255] In an embodiment, the concentration of PEG-maleimide and / or PEG-acrylate is betweenabout 1% w / v and about 25% w / v.

[0256] A fourth aspect of the present invention is directed to a bioadhesive comprising the self-assembling CLP, the hydrogel or the photoactivatable hydrogel as described herein.

[0257] A fifth aspect of the present invention is directed to a scaffold comprising the self-assembling CLP, the hydrogel or the photoactivatable hydrogel as described herein.

[0258] A sixth aspect of the present invention is directed to a composition comprising the self-assembling CLP, and a multi-arm polyethylene glycol (PEG) polymer.

[0259] In an embodiment, the PEG polymer comprises at least one acrylate group and / or at leastone maleimide group, and at least one photoinitiator.

[0260] A seventh aspect of the present invention is directed to method of preparing a hydrogel.

[0261] In an embodiment, the method comprises mixing a self-assembling collagen-like peptide(CLP) with a multi-arm polyethylene glycol (PEG) polymer, wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is anhydroxyproline or a cysteine, and G is a glycine or a valine.

[0262] In an embodiment, a hydrogel precursor is first obtained by mixing the self-assemblingCLP with the multi-arm PEG polymer, wherein the hydrogel is obtained after the hydrogel precursor is incubated for a period of time to allow crosslinking of the self-assembling CLP to the multi-arm PEG.

[0263] In an embodiment, the period of time is less than 5 minutes.

[0264] In an embodiment, the self-assembling CLP has a CMratio of between about 1.0 to about5.0.

[0265] An eight aspect of the present invention is directed to a method of preparing aphotoactivatable hydrogel precursor (PHP).

[0266] In an embodiment, the method comprises mixing a self-assembling collagen-like peptide(CLP) with a multi-arm polyethylene glycol (PEG) polymer, wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, and wherein the multi-arm PEG polymer comprises at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator.

[0267] A ninth aspect of the present invention is directed to a method of preparing aphotoactivated hydrogel comprising photoactivating the PHP as described herein.

[0268] In an embodiment, photoactivating the PHP comprises irradiating the PHP with light.

[0269] In an embodiment, the light may be visible light. The visible light may have a wavelengthof between about 500 nm to about 580 nm and may be green light.

[0270] In an embodiment, the photoinitiator may be Rose Bengal or Eosin Y.

[0271] In an embodiment, the photoinitiator may be Eosin Y.

[0272] In an embodiment, the CLP may have a CMratio of between about 1.0 to about 5.0.

[0273] A tenth aspect of the present invention is directed to a method of treating a wound. themethod comprising:

[0274] In an embodiment, the method comprises exposing a wound to a therapeutically effectiveamount of a hydrogel precursor, wherein the hydrogel precursor comprises a self-assembling collagen- like peptide (CLP) with a multi-arm polyethylene glycol (PEG) polymer, wherein the CLP comprises aplurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0275] In an embodiment, the method further comprises waiting for a period of time afterexposing the wound to allow crosslinking of the self-assembling CLP to the multi-arm PEG.

[0276] In an embodiment, exposing the wound comprises covering the wound with the hydrogelprecursor.

[0277] In an embodiment, exposing the wound comprises injecting the therapeutically effectiveamount of the hydrogel precursor in a subject in need thereof.

[0278] In an embodiment, injecting is performed at or around a wound site.

[0279] In an embodiment, the wound is in a soft tissue such as muscle, cornea, or skin.

[0280] An eleventh aspect of the present invention is directed to a method of treating a cornealdisease in a subject in need thereof.

[0281] In an embodiment, the method comprises injecting a therapeutically effective amount ofa hydrogel precursor in the subject, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0282] In an embodiment, the method may further comprise waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0283] In an embodiment, the corneal disease comprises corneal thinning and / or a cornealwound.

[0284] A twelfth aspect of the present invention is directed to a method of treating a myocardialinfarct in a subject in need thereof.

[0285] In an embodiment, the method comprises injecting a therapeutically effective amount ofa hydrogel precursor in the subject, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0286] In an embodiment, the method may further comprise waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0287] A thirteenth aspect of the present invention is directed to a method of promoting cellgrowth.

[0288] In an embodiment, the method comprises treating a cell with a therapeutically effectiveamount of a photoactivatable hydrogel precursor (PHP), wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0289] In an embodiment, the method may further comprise waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0290] A fourteenth aspect of the present invention is directed to a method of treating cellulardegeneration in a subject in need thereof.

[0291] In an embodiment, the method comprises injecting a therapeutically effective amount ofa hydrogel precursor in the subject, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

[0292] In an embodiment, the method may further comprise waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0293] A fifthteenth aspect of the present invention is directed to method of treating a wound.

[0294] In an embodiment, the method comprises exposing a wound to a therapeutically effectiveamount of a photoactivatable hydrogel precursor (PHP). The PHP comprises: a self-assembling collagen- like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine; a multi- arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group; and at least one photoinitiator. The method further comprises photoactivating the PHP.

[0295] In an embodiment, the method further comprises waiting for a period of time afterphotoactivating to allow crosslinking of the PHP to the PEG polymer.

[0296] In an embodiment, photoactivating the PHP comprises irradiating the wound with light.The light may visible light and may have a wavelength of between about 500 nm to about 580 nm. The visible light may be green light.

[0297] In an embodiment, the photoinitiator may be Rose Bengal or Eosin Y.

[0298] In an embodiment, the photoinitiator is Eosin Y.

[0299] In an embodiment, the concentration of PEG-maleimide and / or PEG-acrylate is betweenabout 1% w / v and about 25% w / v.

[0300] In an embodiment, the CLP may have a CMratio of between about 1.0 to about 5.0.

[0301] In an embodiment, exposing the wound comprises covering the wound with the PHP.

[0302] In an embodiment, exposing the wound comprises injecting the therapeutically effectiveamount of the PHP in a subject in need thereof.

[0303] In an embodiment, the wound is in a soft tissue such as muscle, cornea, or skin.

[0304] A sixteenth aspect of the present invention is directed to a method of treating a cornealdisease in a subject in need thereof.

[0305] In an embodiment, the method comprises injecting a therapeutically effective amount ofa photoactivatable hydrogel precursor (PHP) in the subject. The PHP comprises a self-assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine; and a multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group; and at least one photoinitiator. The method further comprises photoactivating the hydrogel precursor.

[0306] In an embodiment, the method may further comprise waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0307] In an embodiment, photoactivating the PHP comprises irradiating the subject with light.The light may visible light and may have a wavelength of between about 500 nm to about 580 nm. The visible light may be green light.

[0308] In an embodiment, the photoinitiator may be Rose Bengal or Eosin Y.

[0309] In an embodiment, the photoinitiator is Eosin Y.

[0310] In an embodiment, the concentration of PEG-maleimide and / or PEG-acrylate is betweenabout 1% w / v and about 25% w / v.

[0311] In an embodiment, the CLP may have a CMratio of between about 1.0 to about 5.0.

[0312] In an embodiment, the PHP is injected in the cornea.

[0313] In an embodiment, the corneal disease comprises corneal thinning and / or a cornealwound.

[0314] A seventeenth aspect of the present invention is directed to a method of treating amyocardial infarct in a patient in need thereof.

[0315] In an embodiment, the method comprises injecting a therapeutically effective amount ofa photoactivatable hydrogel precursor (PHP) in the subject. The PHP comprises a self-assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine; and a multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group; and at least one photoinitiator. The method further comprises photoactivating the hydrogel precursor.

[0316] In an embodiment, the method may further comprise waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0317] In an embodiment, photoactivating the PHP comprises irradiating the subject with light.The light may visible light and may have a wavelength of between about 500 nm to about 580 nm. The visible light may be green light.

[0318] In an embodiment, the photoinitiator may be Rose Bengal or Eosin Y.

[0319] In an embodiment, the photoinitiator is Eosin Y.

[0320] In an embodiment, the concentration of PEG-maleimide and / or PEG-acrylate is betweenabout 1% w / v and about 25% w / v.

[0321] In an embodiment, the CLP may have a CMratio of between about 1.0 to about 5.0.

[0322] In an embodiment, the PHP is injected in the infract or in a region proximal to the infarct.

[0323] An eighteenth aspect of the present invention is directed to a method of promoting cellgrowth.

[0324] In an embodiment, the method comprises treating the subject with a therapeuticallyeffective amount of a photoactivatable hydrogel precursor (PHP) in the subject. The PHP comprises a self-assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine; and a multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group; and at least one photoinitiator. The method further comprises photoactivating the hydrogel precursor.

[0325] In an embodiment, the method may further comprise waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0326] In an embodiment, photoactivating the PHP comprises irradiating the subject with light.The light may visible light and may have a wavelength of between about 500 nm to about 580 nm. The visible light may be green light.

[0327] In an embodiment, the photoinitiator may be Rose Bengal or Eosin Y.

[0328] In an embodiment, the photoinitiator is Eosin Y.

[0329] In an embodiment, the concentration of PEG-maleimide and / or PEG-acrylate is betweenabout 1% w / v and about 25% w / v.

[0330] In an embodiment, the CLP may have a CMratio of between about 1.0 to about 5.0.

[0331] A nineteenth aspect of the present invention is directed to a method of treating cellulardegeneration in a subject in need thereof.

[0332] In an embodiment, the method comprises treating the subject with a therapeuticallyeffective amount of a photoactivatable hydrogel precursor (PHP), wherein the PHP comprises a self- assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, and a multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator; and photoactivating the hydrogel precursor.

[0333] In an embodiment,, the method further comprises waiting for a period of time afterinjecting to allow crosslinking of the self-assembling CLP to the multi-arm PEG polymer.

[0334] In an embodiment, photoactivating the PHP comprises irradiating the subject with light.

[0335] In an embodiment, the light is visible light.

[0336] In an embodiment, the visible light has a wavelength of between about 500 nm to about580 nm.

[0337] In an embodiment, the visible light is green light.

[0338] In an embodiment, the photoinitiator is Rose Bengal or Eosin Y.

[0339] In an embodiment, the photoinitiator is Eosin Y.

[0340] In an embodiment, the concentration of PEG-maleimide and / or PEG-acrylate is betweenabout 1% w / v and about 25% w / v.

[0341] A twentieth aspect of the present invention is directed to a use of the hydrogel as describedherein, the PHP as described herein, the bioadhesive as described herein, or the scaffold as described herein, as a wound sealant, for improving wound closure, for treating a corneal disease, for treating a myocardial infarct, for treating a corneal disease, for promoting cell growth and / or for treating cellular degeneration.

[0342] A twenty-first aspect of the present invention is directed to a device for delivering apeptide-based material in a subject.

[0343] In an embodiment, the device comprises an on / off button or switch for initiating andstopping delivery of the peptide-based material in the subject, a chamber for containing the peptide-based material, an aperture through which the peptide-based material exits the chamber, wherein the aperture is adapted for receiving a nozzle adapter, a camera port for securing a camera thereto; and a digital control display.

[0344] In an embodiment, the chamber is adapted for receiving one or more peptide cartridgecontaining the peptide-based material.

[0345] In an embodiment, the peptide cartridge is a preloaded syringe.

[0346] In an embodiment, the camera allows live monitoring when delivering the peptide-basedmaterial in the subject.

[0347] In an embodiment, the digital control display allows adjusting at least one deliveryparameter.

[0348] In an embodiment, the at least one delivery parameter comprises volume to be deliveredand speed at which the volume is delivered in the subject.

[0349] In an embodiment, the nozzle adapter is adapted to receive a variety of nozzles.

[0350] In an embodiment, the variety of nozzles comprise a nozzle for topical application, anozzle for cornea sealant delivery, and a nozzle for intratissue injection.

[0351] In an embodiment, the device further comprises a handle.

[0352] In an embodiment, the device is a handheld device.

[0353] The present invention will be further illustrated in the following examples.Examples MATERIALS AND METHODS

[0354] The following material and methods were performed in the examples described below.Materials and Methods for Examples 1 and 2 Peptide Synthesis Fmoc protected amino acids and low-loading Wang resin were purchased from CEM. All peptides were synthesized using microwave assisted Fmoc solid phase peptide synthesis (SPPS)67in a Liberty Blue automated system. Briefly, the required amount of resin was swelled in DMF for 5 min. Next, Fmoc deprotection was carried out with 20% piperidine at 90°C for 60s. Standard coupling cycles using DIC / Oxyma Pure were run at 90°C for 240s in each amino acid. For difficult couplings, the temperature was increased to 100°C and additional reagent equivalents / double couplings were used along with an increased reaction time and addition of urea as a chaotropic agent. Peptides were cleaved from the resin and deprotected with TFA / TIS / EDT / H2O (92.5 / 2.5 / 2.5 / 2.5 %v / v) at 42°C for 30 min, and thenprecipitated in -20°C diethyl ether. Peptide crude products were then dried under vacuum overnight and purified by RP-HLPC in a Waters 1525EF semi-preparative system with a 21.6 x 250 mm C18 column at 20 mL / min. Peptide purity and identity was confirmed via RP-UPLC-UV / MS in a Waters Acquity UPLC Xevo TQD using a 2.1x100 mm UPLC BEH C8 column. A purity of >95% was determined through HPLC peak analysis. Infrared spectroscopy Fourier-transform infrared (FTIR) spectra were recorded using a Nicolet iS5 FTIR with an Attenuated Total Reflectance (ATR) iD7 accessory and with a diamond crystal. All spectra were taken at 4 cm-1of resolution, and 64 scans in the 4000-500 cm-1range at room temperature. Before measurements, the prepared peptides and hydrogels were freeze-dried. The FTIR spectra of the Amide I region (1750-1550 cm-1) were then fitted by multiple Gaussian peaks, see further details in Figures 61-64. Circular Dichroism Circular dichroism (CD) spectra were collected on a JASCO J-810 CD spectrometer with a bandwidth of 1.0 nm in the ultraviolet (UV) region (190–260 nm) using a 0.5 mm quartz cuvette, at 20 °C using a MPTC-490S accessory. To measure Rpn and Tm, all samples were dissolved at a concentration of 200 µM using phosphate buffer 1 mM (pH 7.4). Then, different dilutions were prepared to obtain 6.25, 12.5, 25, 50, 100, and 200 µM concentrations of each sample to measure folding at different concentrations. The spectra were obtained with solvent background subtraction and were collected with 10 scans. Hydrogel preparation Hydrogels were prepared from three components: peptide, 8-Arm PEG-Maleimide MW 40K (Creative PEGworks, USA), and a buffer solvent. Reagents were freshly dissolved in buffer before use and kept on ice until application. Three different formulations of peptide to PEG-Mal were used: CMratio 1.0, 3.0 and 4.0. Solutions were loaded into syringes and administered to the target site for in-situ gelation via our custom applicators. Peptide concentrations ranged from 3.5 mM to 16 mM for the peptide. PEG concentrations were kept ≈4.0±0.5 mM in all cases. Reagents were freshly dissolved in buffer and kept on ice until use. All hydrogels were prepared with freshly made solutions.Gelation time testing Gelation time was calculated using fluid mechanics on an inclined plane. As fluid viscosity increases, the velocity of the flow reduces until it becomes static, indicating that the substance has transitioned from liquid to solid. The gelation time was measured using a 22.5° inclined plane with 60 µL samples placed on the glass inclined plane. Sliding time and distance recorded for up to 3 minutes. Peptides 1 to 21 and CLP were evaluated at concentration ratios of 1.0, 3.0, and 4.0. Transmittance The absorbance and transmittance of the hydrogels were evaluated using a SpectraMax M2 / M2e microplate reader (Molecular Devices, USA). The hydrogels (20 μL) were placed on a 96-well plate containing 150 μL of PBS, the absorbance was measured at a wavelength of 550 nm. As a control, the absorbance of wells containing 150 µL of PBS was measured. The method for calculating percent transmittance was: % Transmittance =10(2 - absorbance). The peptides studied were 1 to 21, as well as CLP at the CMratios of 1.0, 3.0, and 4.0. Refractive index measurement The refractive index was measured with an Abbemat 300 refractometer (Anton Paar, Canada) at a temperature of 37 °C. The hydrogel samples were made on a glass slide and transferred into the refractometer sensor where the nD index was recorded and reported. Collagenase and water content The stability of the hydrogels was assessed by soaking them in collagenase from Clostridium histolyticum (Sigma-Aldrich, USA) at 5 Unit / mL in 0.1 M Tris-HCL Buffer containing 5 mM CaCl2 and measuring their loss of mass over time. Percentage of residual weight was calculated by residual mass (%) = W(total) / W(initial). The calculation of water content was done by comparing the mass of the casted hydrogel against the lyophilized mass. Lap-shear test A modified ASTM F2255-05 standard was utilised to conduct this test. This was performed at a pace of 10 mm / min on an Instron 3342 universal testing machine (Instron, USA) and analysed using INSTRON Series IX / S (Instron, USA) software. The samples consisted of applying the hydrogel to a 0.5 cm2sectionof pig skin. The force and strain of each sample was recorded as it was pulled to failure. Viscosity The viscosity of each formulation was measured using a Brookfield rheometer with an RCT-25-1 spindle. The micrometer ring of the rheometer was first set to zero and then the measuring head was lowered to the zero position. The spindle was tightened and then the micrometer was set to the measuring point (M) allowing for 50 µm of space between the spindle and the bottom plate. Equal volumes of peptide-5 solution and 8-arm PEG maleimide (100 µL total) were mixed and added to the center of the plate. The spindle was fully lowered, and excess sample was removed. The viscosity was measured between 0–200 s−1shear rate at 25 °C using Rheo3000 v2 software. Denaturation Temperature The thermal properties of the gel were measured via differential scanning calorimetry analysis with a DSC-Q200 (TA instruments, USA). From sequences Pep-4 and 5, 8 – 10 mg of sample of the various CMratiowere evaluated. The samples were then equilibrated at 20 °C, and then ramped up at 20 °C min-1to 100 °C, and the total heat flow was recorded. The calibration standard used was Indium. Material porosity Hydrogel samples were initially swollen in 1X PBS solution for 24h unless specified otherwise. Then, the microstructure of each hydrogel was evaluated using a TESCAN scanning electron microscope, Model VegaII XMU, at low-vacuum conditions. SEM images of hydrogels were captured at different magnifications. Setting conditions included an acceleration voltage of 20 kV, cryo-stage temperature ≈−50 °C and chamber vacuum ≈35 Pa. Hydrogel pore diameter was calculated from the average pore area observed in different regions of the gel using Fiji software. Wound closure and mechanical strength A modified ASTM F2458-05 test measured tissue adhesive and sealant wound closure strength77. All animal care and procedures were performed in strict compliance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Ottawa Heart Institute. Skin samples were obtained from a cohort of male and female C57BL / 6 mice (Charles Rivers Laboratories, Canada). The mouse skin was partitioned into strips of 30 ± 5 mm length, 5 ± 0.5 mm width, and 0.7 ± 0.2 mm thickness. A surgical blade was then used to completely sever the samples at mid-length and simulate a wound. The simulated wound was treated with 30 uL of hydrogel sample to bond the severedskin pieces back together. A few drops of PBS were applied to the ends of the skin strips every 5 minutes until testing to prevent the mouse skin from drying out. The hydrogel-bonded mouse skin was clamped on an Instron 3342 universal testing machine (Instron, USA) at a 5 mm distance from the simulated wound. The Instron transductor sensor Model 2519 – 101 (Instron, USA) was used to record the formulation’s resistance force during the tensile test at a crosshead speed of 5 mm / min until material failure. For the control group, the same clamping procedure was used but using an uncut skin sample. The software used was the INSTRON Series IX / S (Instron, USA). Young’s modulus and strain % were obtained from the stress-strain graphs. Design and 3D printing of the hand-held device and nozzles All 3D printed components were designed using Solidworks (Dassault Système) and printed on a Form 3B Stereolithography 3D printer (Formlabs). The structural components for the handheld device were printed using Black Resin V4 (RS-F2-GPBK-04) while the mechanical components were printed using Durable Resin V1 (RS-F2-DUCL-02). For the nozzles, the topical mixer was printed using Flexible 80A Resin V1 (RS-F2-FL80-01) while both injection mixers were printed using Clear Resin V4 (RS-F2- GPCL-04). All components for the hand-held device were printed at a resolution of 100um while the nozzles were printed at a resolution of 50 μm with automatically generated supports then washed using isopropanol and cured with a combination of UV light and heat treatment. Design and 3D printing of the hand-held device components and nozzles All 3D printed components were design using Solidworks (Dassault Système) and printed on a Form 3B Stereolithography 3D printer (Formlabs). The structural components for the handheld device were printed using Black Resin V4 (RS-F2-GPBK-04) while the mechanical components were printed using Durable Resin V1 (RS-F2-DUCL-02). As for the nozzles, the Topical mixer was printed using Flexible 80A Resin V1 (RS-F2-FL80-01) while both injection mixer were printed using Clear Resin V4 (RS-F2- GPCL-04). Prior to printing, all the nozzles were oriented vertically on the printing platform while the hand-held components were oriented parallel to the printing platform’s surface. Supports were automatically generated using the default settings for each specific nozzles. All components for the hand-held device were printed at a resolution of 100 µm while the nozzles were printed at a resolution of 50 µm. All printed components were washed using isopropanol and cured with a combination of UV light andheart treatment. The wash and curing time / temperature were dependent on the resin type and manufacturer specifications and can be found in the table below. Specifically for the nozzles, a second washing step was added before curing where the nozzles were subsequently flushed with isopropanol and dried with nitrogen gas and was repeated twice. Black / Clear Resin Durable Resin Flexible 80A Resin Wash 30 min 20 min 15 minCure 30min@60°C 60 min@60°C 10 min@60°CLocatioResin-Type Left Handle Compartment Handheld-Device_Handle_Left- C t tSTL Black 0AHand-held device assembly The assembly instructions for the handheld device are summarized in the step-by-step figures provided herein. Briefly, the device is assembled in 2 distinct sub-assembly which are then attached together: the extrusion chamber and the handle. For the handle, the main microcontroller board, charge port, microcontroller port, battery, camera moduleand buttons are installed and wired inside the handle and the handle is assembled. As for the extrusion chamber, the lead screw, driving nut, motor, power switch and display were secured and wired inside the casing. Once both sub-assemblies were built, the screen and motors were connected to the microntroller and both chambers were secured together. Finally, the code for the microcontroller was uploaded and the device was charged before use. Injection mixer for cornea sealant delivery The injection nozzle for cornea sealant delivery is based on the Kenics micromixer which uses a series of twisting helical element to split and reorient the flow at each mixing stage. The current design uses 2 helical pieces with a twisting angle of 180 degree and they alternate between clockwise and counterclockwise to induce chaotic mixing and improve the uniformity of the gel at the output. Looking at the overall nozzle design, the inlets have 2 female luer connectors to securely attach both syringes to the nozzle and are spaced 24mm apart to fit at the tip of the handheld device. The inlets then lead to a 6.5mm length channel with a gradually decreasing internal diameter to generate back pressure and generate a uniform flow right before reaching the solutions contact point Once the solutions reach the mixing chamber, the helical element cut in half the stream of each solution and force it to mix with the other solution and that process is repeated again where the flow is splited again and recombined to mix until it exit the mixing chamber. At that point, the diameter decrease by half in order to optain the desired gel size and the fully mixed solution is extruded. The dimensions of the nozzles and mixing chamber were optimized such that it can be printed on a consumer grade SLA 3D printer as a single part. Injection mixer Luer adapter for intratissue injection The injection mixer Luer Adapter for intratissue injection is built around the same kenics micromixer design as the topical nozzle but integrates a male luer adapter in order to connect any compatible female luer syringe, enable the use of small gauge subcutaneous injection needle or any commercial injection tip with a luer adapter. This approach was chosen as opposite to direflty printing the needle at the tip of the nozzle for multiple reason. First, a single nozzle can be use with a wide variety of needle which allows for a greater level ofcustomization and can be easilty adapter for different application. Then, the minimum size that could be achieve with a 3D printed needle was similar to a 22 gauge needle but it resulted in a weak tip that wasn’t strong enough to puncture tissue and was extremely susceptible to bending / shearing. On the opposite, commercial injection needles are significantly stronger and are available in significantly smaller gauge. The mixer was integrated inside the tip of the male luer adapter and further detailed about the mixing mechanics can be found in the topical cornea nozzle of the ESI above. Like for the the cornea nozzle, this nozzle can be coupled to the handheld device to control the injection process. Topical application mixer Compared to the injection and cornea nozze which employ a chaotic micromixer to mix the 2 solution into an homogeneous gel with a circular shape at the ouput, the topical mixer produces 2 flat liquid streams at the out which are stack on each other, generating a bi-layer gel solution. The slits are 4mm wide by 0.5mm, thin enough to maximize the contact area between the 2 solution while being wide enough to cover the entire wound area. In addition to the bi-layer output, the nozzle inegrate a flexible brush at the tip used to flatten the bi-layer solution and spread it more evenly across the targeted area. One important design of the nozzle was the used of a flexible stereolithographic resin to manufacture the nozzle as oppose to the rigid one used on the other 2 applicators. This was done in order for the tip to be directly in contact with the surface of the skin without any risk of further damage or scratches on the tissue. Furtermore, the use of a flexible polymer means that both the nozzle tip and brush can freely bend when in contact with the skin, allowing for maximal contact area and improved versatility. Finally, the flexibility of the nozzle also allowed the use of a slip-on tip at the input where the syringe can attached by simply inserting it into the inlet. Infrared characterization A comparison of the FTIR spectra of reactants and Pep-5 hydrogel is shown in Figure 61. The spectrum of PEG-MAL shows characteristic signals at 1095 cm-1, attributed to st C–O vibration, 1710 and 1738 cm-1, corresponding to NC=O cyclic imide modes (symmetric and asymmetric), 1670 cm-1attributed to the C=C of maleimide, and 2880 cm-1, attributed to st C-H of PEG polymer chain. Moreover, the Pep-5 peptide shows the characteristic proteins and peptide signals: st N-H at 3310 cm-1, Amide I at 1630 cm-1, Amide II at 1544 cm-1, and Amide III at 1444 cm-1, among others. FTIR was also used to analyze the obtained hydrogel from Pep-5 and PEG-MAL. Characteristic signals of PEG and Pep-5 can be observed.Although the S-H bond of the thiol signal from Cys residue typically observed at 2300 – 2500 cm-1cannot be elucidated, the shifting of the maleimide carbonyl signal in PEG from 1710 to 1698 cm-1in the Pep-5 hydrogel could indicate the cross-linking reaction. Furthermore, the change in the Amide I region (1750-1550 cm-1) could be understood as changes in the backbone conformation of the peptide- based hydrogel.1-3Figure 61B-E shows the Gaussian fits of the Amide I region of Pep-5 and Pep-5 hydrogel at different CMratiovalues. In the same region of the spectra of the Pep-5 hydrogel, a change in the intensity of the folding peaks is observed, indicating a change in the arrangement due to the reaction with PEG-MAL. Besides, a new peak appeared at 1705 cm-1, which could be attributed to the carbonyl signal of the now-reacted maleimide residue (red peaks). The comparison of PEG-MAL and Pep-5 hydrogel spectra at CMratio4.0 denotes the absence of the C=C of maleimide groups, which indicates a complete functionalization of PEG-Maleimide with Pep-5. The appearance of a signal around 1650 cm-1for CMratio 3.0 and 1.0 (violet peak) attributed to C=C stretching indicates the presence of unreacted maleimide groups. These results are expected due to a lower amount of thiol than maleimide groups. Considering that the S-H vibrational mode is not observed in the IR spectra of the peptides and the Cys thiol group is a highly reactive functional group, the presence and relative intensity of the C=C maleimide peak could indirectly indicate the efficiency of the reaction, giving lower values when the reaction is more efficient. Comparing the overall area of the Gaussian fit peak of the Amide signal (green, yellow and blue peaks) with the peak area of C=C maleimide, the results indicate that in a 4:1 ratio, the reaction is more efficient due to a 100% amide area, as explained before. For the Pep-5 hydrogel at CMratio3.0, a relative peak area of the maleimide double bond is evidenced. This peak increases at CMratio1.0, indicating more C=C groups in the hydrogels with a lower ratio. The same analysis was carried out using Pep-10, corresponding to a sequence without thiol groups (Figure S19), to corroborate this point. The Gaussian fit in the Amide I region of the mixing of PEG-MAL and Pep-10 (Figure 62 B-C) shows the characteristic signals of maleimide at 1738, 1707 and 1670 cm-1, indicating the non-reaction between maleimide groups and Pep-10. In summary, comparing the hydrogels prepared with Pep-3 (Figure 63), Pep-4 (Figure 64) and Pep-5 at different CMratio values, it is possible to rationalize the ability of each peptide to react with PEG-MAL, giving a higher relative area percent of Amide I / C=C signal for those efficient hydrogels, as shown in Figure 34A and 34B on the main manuscript. Skin incision sealing modelC57BL6 female mice (12-14 weeks, weighing ~22–25 g; Charles River Laboratories) were housed in groups of five until surgery. All animal care and procedures were performed in strict compliance with protocols approved by the Institutional Animal Care and Use Committee at the University of Ottawa Heart Institute. Before surgery, mice were given an epidural injection of buprenorphine and put under using general anesthesia (2.5% isoflurane). Dorsal hair was shaved, and the skin was prepped using ethanol swabs. Then, 1-cm full-thickness incisions were made on the back of each mouse using sterile scalpel blades (#15; Integra Miltex).78For suture-closed incisions, four evenly spaced simple interrupted knots were used to close a 1-cm incision using 7-0 nylon suture (#SN5699G; Medtronic; Monosof Black 18″ P-13 cutting). For BioGlue control, 48 μl of bovine serum albumin (BSA) and 12 µL of glutaraldehyde were topically applied to the incision site as specified in the manual. For the peptide / PEG- maleimide formulation, 30 µL peptide-5 and 30 µL of PEG-maleimide were mixed and topically applied to the incision site using a custom-made nozzle tip for a final concentration of 17.5 mM peptide and 4.38 mM PEG-maleimide (CMratio4). The mice were allowed to recover on heating pads until mobile and were housed individually. Incisions were assessed on days 1, 3, and 7 post-surgery for any signs of infection, suture removal, or bleeding, and mice with any of these conditions were removed from the study. Histology Skin tissue from the wound site was fixed in 4% paraformaldehyde for 72h. Then, skin samples were washed and embedded in paraffin. Hematoxylin and eosin (H&E) staining was done according to the standard protocol provided in a Leica Autostainer XL (Leica Biosystems, Buffalo Grove, IL). Skin epidermal thickness was measured at six different points along the wound site using Fiji and the average thickness was calculated for each mouse wound.41Masson’s trichrome stains were done manually using a standard protocol with the Trichrome stain kit (Sigma-Aldrich, St. Louis, MO). To determine the area of blue stained collagen in Masson Trichrome images, a 400 µm x 400 µm region was selected inside the wound region using Fiji. Then, the collagen area for each skin section was calculated by taking the blue stained area and dividing it by the total selected area.79For immunohistochemistry, tissue sections were deparaffinized and washed with PBS and citric acid. Sections were blocked with goat serum and incubated at 4.0 °C with the specific primary antibody: Ab64693 (1:200) for CD206. Further labeling was performed with specific secondary antibodies: AF488 anti-rabbit secondary (ThermoFisher, Cat#A- 11008 at 1:500 dilution). The fixed cell nuclei were subsequently stained with DAPI. Images of thesamples were obtained under a microscope, with each section taken at 10× and 40× magnification, respectively. Ex vivo pig cornea model mounting Pig eyes were obtained from certified local farms. Freshly harvested eyes were transported in an ice bucket and processed the same day. Eyes were disinfected in Iodine-PVP (10%) for 2 minutes and then transferred to sterile 0.1% sodium thiosulphate solution for 1 minute before immersion in sterile saline solution for 2 minutes. Using a scalpel blade, a small (5-8 mm) incision was made at the equatorial and carefully extended by 360° around the entire eyeball while avoiding perforation of the underlying choroid layer. Once the cut was made, the ciliary body-choroid was pulled downwards using forceps to avoid touching the corneoscleral disk. The corneoscleral disk was held in place using a pair of tweezers and the retina was peeled off the inside of the corneoscleral disk using another pair of tweezers. Then, the corneoscleral was rinsed with PBS and placed in DMEM media containing 10% FBS, 1% Gentamycin antibiotics (ANTI-ANTI), and HEPES 15mM. The corneoscleral disk was placed on the base of the perfusion chamber developed in our lab (size 18 mm, 45° angle).80The chamber was twisted clockwise while maintaining downwards pressure on the chamber base to prevent sliding of the cornea and to tightly seal it in place. The chamber was then filled with DMEM media with 5% dextran using two syringes attached to two irrigation ports that were then attached to maintain an intracorneal pressure within the physiological range. Injection of material into pig cornea Following the dissection and mounting of the cornea on the ex vivo perfusion chamber, a 6 mm biopsy punch marked with hydrophobic pen ink was used to gently mark the central corneal surface (apex). A 2 mm incision was then made at the edge of this mark to initiate the dissection of an intrastromal tunnel, which was carefully enlarged using a crescent knife to create an intrastromal pocket under the marked corneal surface. For intrastromal injections, each cornea was slightly deflated and 30 μL of the peptide- based material or Viscoat injected into the stromal pockets. Optical coherent tomography (OCT) imaging of injected corneas at 3 timepoints (before gel injection, after gel injection, and at 24h) was done to assess the ability of the gel to remain constant in the corneal stroma. This was done by measuring the gel thickness in FIJI. For the cornea reshaping experiments, corneal topographic images were taken using the CT-1000 optical coherence topographer and used to measure the change in corneal curvature before and after the injectionof the peptide-based material or Viscoat. Measurements of corneal curvature were taken from two average K values of steepest meridian and the one perpendicular to it. Images were taken of the natural eye curvature before injection, with a pocket, after the injection, and of the injection with a rigid contact lens (Centracone). Intraocular pressure was held at a maximum across all images and the natural lubrication of the eyes was mimicked with saline and glycerol. Burst pressure testing of treated pig corneas The same procedure to dissect and mount the pig corneas described above was followed. A perforation was created using a 4 mm biopsy punch at the corneal apex going halfway through the cornea. Following this, a 2 mm biopsy punch was inserted in the middle of the 4 mm perforation to fully penetrate the cornea. Treatment was applied to fill the perforation and the Luer locks of the cornea chamber system were sealed. The flow rate on the syringe pump was set to a steady rate of 18 ml / hour, and the pressure was monitored until the pressure peaked due to the cornea bursting at the sealed perforation site. Windaq data acquisition software was used to monitor the pressure. In vitro human cardiac endothelial cell (HCEC) culture and viability assay HCECs were cultured in tissue culture plates for 24 h and maintained in HCEC media (Sigma, Cat# C22022). The injectable PEG-Pep-5 hydrogel CMratio1.5 formulation was pre-cast in two sterilized glass slides in a humidity box and in a 4 °C for 24 hours. HCECs (1× 104cells / well) were seeded on top ofPEG-Pep-5 hydrogel pieces after washing with PBS in a 96-well plate. After 24 hours, HCECs werestained using a Calcein-AM and Ethidium Homodimer-1 (CaAM / EthD-1) viability kit (ThermoFisher Scientific, Cat#L3224) according to the manufacturer’s instructions. Stained cells were imaged by fluorescence microscopy, and nine random images were taken for each experimental group. Total cell numbers were calculated using ImageJ software (NIH, USA). Each condition was performed in triplicate. Myocardial infarction model and intramyocardial injection MI was induced in 8-week-old C57BL / 6 female mice (Charles River) and treatment delivery was performed using a previously established intramyocardial injection protocol.43Briefly, mice were anesthetized (2.5% isoflurane), intubated, and the heart was exposed via a fourth intercostal thoracotomy. The left anterior descending coronary artery was then ligated just below its emergence from the left atrium. At 1-week post-MI (baseline), mice were randomly assigned to receive one of 2 treatments: 1) PBS (control) and 2) Pep-5-PEG hydrogel. The treatment was administered in five equivolumetricintramyocardial injections (10 μL each site, 50 μL total) through a 27-G needle using a minimally invasive echocardiography-guided closed-chest procedure. Mice were euthanized by terminal anesthesia at 4 weeks post-treatment and hearts were collected for histological analysis. Echocardiography and strain analysis Transthoracic echocardiography was performed on parasternal long-axis views using a Vevo3100 system in B mode with a MX400 series real-time microvisualization scanhead probe and analysis performed with its accompanying Vevo LAB 3.1.1 software (VisualSonics). The imaging was carried out at 7 days (right before the treatment) and 4 weeks post-MI. To assess contractility, cardiac strain analysis was performed through the longitudinal axis. The longitudinal endocardial strain at the time of AVC (representing end systole) in segment 6 (anterior apex segment), corresponding to the infarct zone area, was calculated. Results for both echocardiography and strain analyses were averaged from the analysis of two researchers blinded to the treatment groups. Ex vivo imaging of pep-5-PEG hydrogel The Pep-5-PEG hydrogel was labelled by adding Alexa-Fluor®594 dye (25 nmol of dye per hydrogel) to the mixture prior to injection to the infarcted mouse heart, as described above. Animals were sacrificed at 2 and 7 days after treatment, and hearts were harvested and imaged ex vivo by IVIS® Spectrum (PerkinElmer) to visualize Pep-5-PEG hydrogel retention within the hearts (λexcitation: 570 nm; λemission: 640 nm). Statistical analysis Statistical analysis was performed using KaleidaGraph and GraphPad Prism software. Statistically significant differences between multiple groups were determined by one-way ANOVA at a significance set at p<0.05. Statistical significance of data with two groups was performed via a two-tail unpaired t- test set at a p<0.05. Data was reported as mean ± SEM. EXAMPLE 1 – Peptide engineering and validation

[0355] Collagen is one of the most abundant proteins in the human body and is present in mosttissues and organs, spanning from bones to highly vascularized tissues such as the heart.23With >20 different types, collagen’s mechanical properties are as diverse as the tissues it supports.23The versatilityof collagen as a scaffold protein is a consequence of its unique nanosized triple helical structure formed by three collagen polypeptide strains, and how this supramolecular structure can further macro- assemble.24Mimicking the trimer assembly mechanism of collagen is possible by using CLPs.18In CLPs, the amino acid trimer sequence proline-hydroxyproline-glycine (POG) is well-known to yield a triple helical structure when >4 repeats are present.24,25However, triple helix formation is a dynamic process, which in some cases is favoured by attachment of a CLP to a polymeric structure.18

[0356] A peptide library including a total of 21 sequences plus a standard collagen like peptidestructure (see Table 1) was developed, which had the POG repetitive unit as a building block. Using POG as a repetitive unit advantageously decreased the synthetic costs and considerably simplified the peptide sequence synthesis, an important advantage for future translational purposes. The range of peptide sequences synthesized spanned from 18 to 38 amino acids (see Tables 1 and 2). Table 1. Sequences, molecular weight, number of residues, ellipticity, and denaturation temperature for peptide sequences prepared in this study. Further characterization for the peptides can be found in Table 2. To facilitate visualization of the structural changes carried out, POG repetitive sequences and C residues are bolded in black and underligned, respectively.Sample SequenceSequence structural featuresRpn* Tm (°C)4 POG repetitive units Pep-1 NH2-GCG(POG)4GCG-OHcontaining two glycine flanking 0.032 ± 44.1 ± cysteines at each end 0.005 0.8 5 POG repetitive units Pep-2 NH2-GCG(POG)5GCG-OHcontaining two glycine flanking 0.034 ± 49.0 ± cysteines at each end 0.001 1.0 6 POG repetitive units Pep-3 NH2-GCG(POG)6GCG-OHcontaining two glycine flanking 0.072 ± 52.0 ± cysteines at each end 0.008 2.0 7 POG repetitive units Pep-4 NH2-GCG(POG)7GCG-OHcontaining two glycine flanking 0.084 ± 54.1 ± cysteines at each end 0.003 0.4 8 POG repetitive units Pep-5 NH2-GCG(POG)8GCG-OHcontaining two glycine flanking 0.087 ± 59.0 ± cysteines at each end 0.002 3.09 POG repetitive unitsPep-6 NH2-GCG(POG)9GCG-OHcontaining two glycine flanking 0.094 ± 64.0 ± cysteines at each end 0.002 4.0 10 POG repetitive unitsPep-7 NH2-GCG(POG)10GCG-OHcontaining two glycine flanking 0.094 ± 65.0 ± cysteines at each end 0.001 6.0 Replacing amino terminalPep-8 NH2-GGG(POG)8GCG-OHcysteine with glycine in the 8 0.113 ± 61.9 ± POG repetitive containing 0.001 0.4 sequence Replacing carboxy terminalPep-9 NH2-GCG(POG)8GGG-OHcysteine with glycine in the 8 0.118 ± 49.4 ± POG repetitive containing 0.002 0.2 sequence Replacing both cysteines withPep-10 NH2-GGG(POG)8GGG-OHglycine in the 8 POG repetitive 0.120 ± 55.9 ± containing sequence 0.002 0.2 Cysteine shift to middle ofPep-11 NH2-GGG(POG)3(PCG)2(POG)3GGG-OHsequence in the 8 POG 0.054 ± 43.1 ± repetitive containing sequence 0.002 0.7 Amino terminal cysteine shift toPep-12 NH2-GGG(POG)2(PCG)(POG)5GCG-OHcentre of sequence in the 8 0.078 ± 50.7 ± POG repetitive containing 0.001 0.3 sequence Carboxy terminal cysteine shiftPep-13 NH2-GCG(POG)5(PCG)(POG)2GGG-OHto centre of the 8 POG 0.091 ± 50.5 ± repetitive containing sequence 0.001 0.3 NH2- Both cysteine shift to centre of Pep-14 GGG(POG)2(PCG)(POG)2(PCG)(POG)2GGG- sequence in the 8 POG 0.074 ± 39.0 ± OH repetitive containing sequence 0.003 0.3 Disrupting folding by replacingPep-15 NH2-GCG(POG)4POV(POG)3GCG-OHone POG by POV at the centre 0.028 ± 45.0 ± of the sequence in the 8 POG 0.001 2.0 repetitive containing sequence Disrupting folding by replacingPep-16 NH2-GCG(PPG)8GCG-OHhydroxyproline with proline in 0.007 ± 43.0 ± the 8 POG repetitive containing 0.001 2.0 sequence Removing glycine spacer in thePep-17 NH2-GC(POG)8CG-OH8 POG repetitive containing 0.080 ± 58.0 ± sequence 0.002 2.0Replacing glycine with valine asPep-18 NH2-GCV(POG)8VCG-OHspacer in the 8 POG repetitive 0.055 ± 54.0 ± containing sequence 0.001 2.0 Replacing glycine with argininePep-19 NH2-GCR(POG)8RCG-OHas spacer in the 8 POG 0.081 ± 60.0 ± repetitive containing sequence 0.001 2.0 Multiple cysteine residues atPep-20 NH2-GCC(POG)8CCG-OHboth ends in the 8 POG 0.062 ± 51.4 ± repetitive containing sequence 0.001 0.6 NH2- Multiple cysteine residues at Pep-21 GCG(POG)2(PCG)(POG)2(PCG)(POG)2GCG- ends and middle in the 8 POG 0.055 ± 47.9 ± OH repetitive containing sequence 0.003 0.3 CLP NH2-CG(PKG)4(POG)4(DOG)4-OHControl peptide0.071 ± 42.0 ± 0.009 1.0 Mass spectrometry was then performed for peptides 1-21 and the control CLP. Figures 1-22 shows the spectra from the mass spectrometry data for peptides 1-21 and the control CLP whereas the data for peptides 1-21 and the control CLP is summarized described in Table 2.Table 2. List of peptide codes, their sequences, molecular weights, and crude purity from mass spectraillustrating detected ions are presented below. MolecularSample SequenceResidue Crude WeightM+2H Ion M+3H IonNumber purity (%) (g / mol) Pep-1 NH2-GCG(POG)4GCG-OH 1522 18 761.8 508.2 73Pep-2 NH2-GCG(POG)5GCG-OH 1789 21 895.5 597.3 64Pep-3 NH2-GCG(POG)6GCG-OH 2056 24 1029.1 686.4 79Pep-4 NH2-GCG(POG)7GCG-OH 2323 27 1162.7 775.5 74Pep-5 NH2-GCG(POG)8GCG-OH 2591 30 1296.4 864.6 75Pep-6 NH2-GCG(POG)9GCG-OH 2858 33 1430.0 953.7 54Pep-7 NH2-GCG(POG)10GCG-OH 3125 36 1563.7 1042.8 43Pep-8 NH2-GGG(POG)8GCG-OH 2545 30 1273.3 849.2 54Pep-9 NH2-GCG(POG)8GGG-OH 2545 30 1273.3 849.2 61Pep-10 NH2-GGG(POG)8GGG-OH 2499 30 1250.3 833.9 69Pep-11 NH2-GGG(POG)3(PCG)2(POG)3GGG-OH 2479 30 1240.3 827.2 75Pep-12 NH2-GGG(POG)2(PCG)(POG)5GCG-OH 2535 30 1268.3 845.9 67Pep-13 NH2-GCG(POG)5(PCG)(POG)2GGG-OH 2535 30 1268.3 845.9 74NH2- Pep-14 GGG(POG)2(PCG)(POG)2(PCG)(POG)2GG 2479 30 1240.3 827.2 G-OH 69Pep-15 NH2-GCG(POG)4POV(POG)3GCG-OH 2633 30 1317.5 878.7 64Pep-16 NH2-GCG(PPG)8GCG-OH 2463 30 1232.4 821.9 64Pep-17 NH2-GC(POG)8CG-OH 2477 30 1239.3 826.6 73Pep-18 NH2-GCV(POG)8VCG-OH 2675 30 1338.5 892.6 68Pep-19 NH2-GCR(POG)8RCG-OH 2789 30 1395.5 930.7 56Pep-20 NH2-GCC(POG)8CCG-OH 2683 30 1342.5 895.3 72NH2- Pep-21 GCG(POG)2(PCG)(POG)2(PCG)(POG)2GC 2571 30 1286.4 857.9 G-OH 57 CLP NH2-CG(PKG)4(POG)4(DOG)4-OH 3518 38 1759.9 1173.6 NA

[0357] To minimize steric repulsion and increase reactivity in peptides containing cysteineresidues, each cysteine residue was flanked by two flexible glycine residues that allowed the sulfhydryl group flexibility to move in space.27Further, in designing the peptide sequences, terminal cysteines were avoided as they could make synthesis challenging and increase the cost.28Thus, the GCG motif was added to each end of the peptide sequences with the repetitive POG unit, from (POG)4Pep-1 (18 amino acids) to (POG)10 Pep-7 (36 amino acids; see Table 1). Notably, the addition of two cysteine residues did not result in significant formation of dimers in solution (Figure 23). Further, available number of cysteines in phosphate buffer (pH 7.4) was found to be similar between our peptides and free Cys (>90%) (see Figure 24 for a representative example).

[0358] The effect of peptide length on the CLPs’ ability to form triple helices stable at humanphysiological temperature was then assessed, the results of which are summarized in Table 1. Analysis of the melting curves indicated that as expected, increasing sequence length resulted in cooperative melting transitions (Figure 25). Further, to assess the functional impact of changes in the peptide sequence on 3D structure assembly when using nucleophilic Michael-addition to PEG-maleimide, aseries of peptide sequences were designed and synthesized using Pep-5 as a building block as it is cost- effective, has good crude yield (Table 2), and displayed a cooperative melting behaviour (Figure 25). Table 1 summarizes the main modifications for this group of peptide sequences (Pep-8 to Pep-21).

[0359] The Rpn parameter (Rpn = 225nm / 200nm circular dichroism signal) was used todifferentiate between the triple-helix conformation and poly(Pro)-II helix.29-30Considering poly(Pro)-II helix conformations show very low Rpnvalues (<0.005),29and collagen displays Rpnvalues ≈0.12,29Pep- 1 to 8 presented Rpn ≥ 0.035±0.004, which corresponds to ≈50% of the Rpn value measured for the CLP control sequence. Further, the structural G-X-G(POG)nG-X-G motif, with X being G or C, and n>7 results in Rpnvalues larger (0.087-0.084) than those measured for the control CLP (≈0.071). Upon screening of different lengths, sequence Pep-5 and Pep-6 showed the best ellipticity and higher denaturation temperature. The need for having the structural POG repetitive unit to yield highly elliptical peptides is illustrated when the POG repetitive unit is replaced with POV (Pep-15) or PPG (Pep-16), for example. In peptides whose peptide ellipticity is disrupted, such as Pep-15 that bears a single G to V substitution, there is no cooperative denaturation behavior, which aligns well with the low Rpn value of 0.028. Similar results were observed for Pep-16, which has the lowest Rpn value (0.007) from the peptides synthesized. It was also observed that peptides, such as Pep-5, increase their Rpnas a function of the peptide concentration, similar to the control peptide CLP (Figure 26). Further, the presence of cysteine decreases the peptide packing as showed by comparing the ellipticity of Pep-5 vs. Pep-10.

[0360] The foundational peptide sequences, in particular Pep-1 to Pep-8, present suitable self-assembling properties for testing their ability to form on-the-spot hydrogels for soft tissue and organ applications. In the next section, we will present the rationale and main findings for the preparation and testing of hydrogels using the peptide library developed herein.

[0361] The main modifications in the peptide sequences can be summarized as following:Cysteine position in peptide sequence 1Replacing amino terminal cysteine by glycine (Pep-8)2 Replacing carboxy terminal cysteine by glycine (Pep-9)3 Replacing both cysteines with glycine (Pep-10)4 Cysteine shift to middle of sequence (Pep-11)5 Amino terminal cysteine shifts to centre of sequence (Pep-12)6 Carboxy terminal cysteine shifts to centre of sequence (Pep-13)7 Both cysteine shift to centre of sequence (Pep-14)Disruption of peptide folding 1. Disrupting folding by replacing one POG by POV at the centre of the sequence (Pep- 15)2. Disrupting folding by replacing hydroxyproline with proline (Pep-16)Effect of terminal glycine spacer 1. Removing glycine spacer (Pep-17)2. Replacing glycine with valine as spacer (Pep-18)3. Replacing glycine with arginine as spacer (Pep-19)Impact of multiple cysteine residues 1. Multiple cysteine residues at both ends (Pep-20)2. Multiple cysteine residues at ends and middle (Pep-21)Example 2: 3D-biomimetic hydrogel structures for soft tissue repair

[0362] The basis for the hydrogel design is a multi-arm polyethylene glycol (PEG) polymercrosslinked by CLPs. PEG is a hydrophilic, cytocompatible polymer approved by the FDA for human use and thus represents an excellent base material for clinically translatable biomaterials.31-33As the PEG molecule itself possesses little chemical reactivity, we functionalized its ends with the chemically active group maleimide (generating PEG-maleimide) to facilitate the CLP crosslinking process. Among functional groups, maleimide was chosen as it is an electrophilic Michael-addition acceptor with a superior kinetic profile compared to other commonly used groups such as acrylamides.21Rapid kinetics are important to create a gel within a clinically relevant timeframe and ensure complete reactivity to minimize cross-reactions upon application to the tissue.

[0363] Considering the different nucleophilic Michael-addition donors that can react with ourPEG-maleimide, sulfhydryl groups possess rapid reaction kinetics and selectively form a stable Michaeladduct21allowing for the creation of a stable hydrogel that is quickly formed in a clinically relevant timeframe. The natural amino acid cysteine contains a reactive sulfhydryl group34and is thus chosen for incorporation into the peptide design as outlined in Table 1. Considering the peptide sequences used in this work, which include two cysteines in each strand, the click reaction with maleimides can yield up to two PEG-maleimides per strand. If the assembly of three CLP monomers alone can bring together three strands, the reaction of this triple helix-like structure could yield up to six different PEG-maleimides per helix. This creates unique network dynamics that we show to result in the rapid formation of a durable, cytocompatible gel for tissue engineering. Note that these large trimer structures can be sterically hindered, and thus removal of the glycine spacer residues results in less efficient tissue bonding (Figure 27).

[0364] The possibility to tune the mechanical properties of our peptide-based material opens thedoor for us to assess the suitability of this material to be used in three applications that normally would require materials prepared using different chemistries / compositions. In the following sections, we present proof of concept experimental data for assessing the feasibility of using our material for skin wound closure, for treating cornea perforations and in situ cornea reshaping, and as injectable intramyocardial material.

[0365] Figures 28-29 shows a simplified schematic of the peptide-PEG assembly using our bi-functional peptides in combination with the multi-armed PEG maleimide. This figure also contains an overview of the low volume and rapid strategy we used for identifying the most promising peptide candidates. First, we monitored the stability of the PEG-maleimide in carbonate buffers (Figure 30). As expected, the maleimide has moderate stability at slightly basic pH 7.4. However, stability at low temperatures (≈4.0ºC) is quite good for up to 90 min. Thus, our experimental design keeps the reagent solution on ice until it is applied and warmed up to physiological temperatures. Furthermore, having the hydrogels formed within 1 / 10thof the half-life of the maleimide in solution (i.e., < 5 min) is desirable to reduce side reactions. We next screened the peptides’ capacities to form hydrogels (see Figure 31 for a representative example). Our endpoint for selecting the peptide(s) for moving forward included materials with a gelation time between 15 and 120s; see Figure 32 whereby heatmaps are used to illustrate the findings. This figure also depicts representative images of small drops (50 µL) of the formulations prepared at CMratio4.0 on a 45º inclined surface taken at different timepoints (0, 15, and 30 s). The images illustrate that the material prepared using Pep-10 remains viscous for +30 s compared to the Pep-5 thatrapidly becomes a gel (≈30s). Target values for other evaluated parameters (also illustrated using heat maps) include transmittance > 85% (Figure 33A), refractive index < 1.380 (Figure 33B), denaturation temperature > 42ºC (Figure 33D), water content >95% (Figure 33C), and stability in type I collagenase solution, i.e., degradation of <5.0 mg / h which is the degradation rate for a 1.0% collagen hydrogel in solution (Figure 33E). We have also examined the relative abundance of the maleimide FITR signal vs. the C=C after the material has been formed (Figure 34A-B). Table 3 displays the tabulated data for the heatmap plot results shown in Figures 32-33C. Table 3. Heatmap Data Denaturation Gelation time Water Content * Transmittance (%)**Refractive Index***†Temperature (s) (%) (°C)††4 N / A N / A 66 ± 12. 6 59 ± 5.3 54 ± 5.655.38 1 1.3507 1.3585 6- 59 ±53.4 ± .34120 4 ± 9 ± 16 ± ± 96.7 96.6 96.0 p ± 66 ± 95.938 2 N / N / N / e P 10 11 10 ± 3.21 ± 5 ± 0 ± 030 00049 020 005 A A A 2834 00 030 N / A N / A N / A N / A N / A N / A N / A N / A N / A99.009 97.42 1.33948 1.3565 1.3516 6 1- 24 ± 33 ± 118 ± 98.19 ± ± ± 5 ± 0 ± N / N / N 1 ± 73 N / A N / A / pe41 2 N / A A A A P 233 00001 00010 N / A N / A N / A N / A N / A N / A

[0366] In summary, the low volume high throughput process allowed us to reduce the number ofpotential peptide-based formulations from ≈200 (each formulation repeated at least in triplicate) to only 9 top formulations with peptides 3, 4, and 5 as primary candidates for further testing.

[0367] As mentioned, this initial screening process allowed us to narrow down three top peptidesas primary candidates for hydrogel preparation (Pep-3, -4, and -5) at three different CMratio. Since the aim was to apply / deliver the materials directly to the tissue, we chose buffers that allow for fast reaction in a pH range safe for cells such as bicarbonate or Hanks buffer (pH 7.0).35 The materials showed hydrogel formation using all the buffers tested (see representative example in Figure 31). Further, since our goal is to develop a clinically translatable material, we designed the physical properties of the material prior to hydrogel formation to make them deliverable via a ≈400 µm cannula (G27 needle) orin the form of a topical / superficial application. To precisely deliver the intended volumes of peptide formulation, we developed a handheld device that can accurately deliver volumes ranging from 10 µL to 1.0 mL (Figure 35). This handheld device is equipped with interchangeable nozzle adapters, which were designed for the different applications reported here. Further specifications on the device are found throughout, for example, Figures 36-38, including details for the engineering of the nozzle adapters (Figures 40-41).

[0368] The skin bonding properties of the different formulations was first assessed by performingex vivo wound closure testing using murine skin (Figure 42). The results indicated that only the Pep-5 formulation possessed skin bonding properties comparable to those obtained for BioGlue®, a clinically used glutaraldehyde-protein based tissue adhesive whose mechanical properties remain constant within our screened timeframe (Figure 42). The Pep-5 formulation bonding strength was superior to the rest of the screened peptides except for peptides 19, 20 and 21, which are variants of Pep-5 (Figure 43). However, the gelation for those peptides was too rapid, making them unpractical for on-the-spot tissue repair application (Figure 32). Further, our findings indicate that the structural properties and amino acid sequence of Pep-5 are critical for functional tissue bonding, as the effectiveness of these gels is affected by the self-assembly of the peptide chains. Thus, for example, removal of a cysteine from either side of the peptide (Pep-8 and -9) results in a bonding strength of only a few kPa while removing both cysteines (Pep-10) results in gelation failure. Disruption of folding with a Gly-Val substitution in the middle of the sequence (Pep-15) results in a significant loss of ~50% in bonding strength. Using a lower CMratioof Pep-5 (1.0) or diluting the CMratio(4.0) results in weaker bonding (Figure 44). Further testing of the CMratio 4.0 non-diluted formulation indicated that bonding strength gradually increases after application, from ≈50 kPa at 30 min to ≈ 100 kPa at 60 min (Figure 44). While bonding is one of the key parameters, shear adhesion strength is also a useful parameter to evaluate. Tests for adhesivity of the Pep-5 CMratio4.0 show our peptide-based material has comparable adhesivity as that from BioGlue® (Figure 44). Having identified Pep-5 as the top peptide candidate, the material porosity was further assessed. An average pore size of ≈10 µm was observed after swelling for the Pep-5 materials (Figure 45). In the next section, the focus was on presenting data on the Pep-5 CMratio 4.0 formulation as a tissue sealant for skin and cornea applications.

[0369] To test the material’s potential cytotoxicity, in vitro tests assessing the viability of humanskin cells was carried out. The data indicated no adverse effect on the viability of cells exposed to thepeptide-based materials (Figure 46). the wound closure ability of the peptide-based material in a full- thickness murine wound model was then assessed (Figure 47). The animal model used a 7-day endpoint to evaluate inflammatory response and tissue remodelling as previously reported by our team.36Pictures of the wounds at 0, 1, 3, and 7 days clearly show that our peptide-based material is cosmetically superior to sutures or BioGlue® (Figure 48). Note that wound dehiscence after 1 day was observed in ≈25% of the animals that received BioGlue®. The data presented here corresponds to those wounds that did not dehisce. Mechanical testing of wound skin freshly harvested after euthanasia indicate that the peptide- based material is superior to sutures and BioGlue®, and comparable to unwounded skin (Figure 49). Histological analyses of the tissues show that the peptide-based material presents a more functional wound healing, including increased collagen deposition and reduced epithelial thickness (Figure 50). Further histological assays carried out to evaluate the number of pro-healing macrophages indicate the animals treated with the peptide-based material have no statistically significant differences in the number of CD206+ macrophages compared to the sutures or BioGlue® groups (Figure 51).

[0370] CLP-based materials for cornea repair

[0371] Considering the bonding capacity of our peptide-based material, it was assessed if it couldbe used for rapid closure of perforated soft tissues. As an ex vivo model, a cornea perforation system was chosen, which is readily used to test the suitability of a material for sealing a perforated tissue.37Figure 55 depicts the data and schematic representation for the ex vivo model we used. Pig corneas were used as they are physiologically similar to humans.38The data indicated that the peptide-based material is considerably stronger and more consistent than cyanoacrylate as a corneal sealant (Figure 52). While cyanoacrylate remains one of the gold standards for rapid corneal sealing in emergency situations, its observed toxicity towards corneal tissues presents the main drawback for its safe use.39In contrast, the peptide-based material did not exert significant toxicity towards human corneal cells (Figure 53), which positions our technology ahead in the space of corneal repair. Further, another potential application of the peptide-based material could be as an in situ corneal filler. The data indicates that rapid injection of CMratio4.0 into the pig cornea renders implants that are considerably thicker and more stable than those produced using Viscoat (Figure 54). Further, we assessed the ability of the peptide-based material to reshape cornea curvature in our ex vivo model. Figure 55 includes a schematic representation for our experiments, showing the creation of an intrastromal pocket in the pig cornea, which can be filled with a viscous material, such as our peptide hydrogel. After injection of our peptide-based material, weobserved an increase in cornea curvature compared to the control (Figure 55). Upon application of the rigid contact lens, a change in the cornea shape is observed for the peptide-based material when compared to the injected and no lens. Curvature changes for corneas treated with Viscoat and lens shows that injection of Viscoat does not achieve a significative change in curvature when compared to the pocket alone. In fact, there is an overall decline in cornea curvature for the corneas that received the centracone lens and Viscoat. Thus, in summary, only the CMratio 4.0 group achieved While preliminary, our data suggest the peptide-based material developed in this work offers a cost-effective alternative to cyanoacrylate, including the possibility to in situ reshape the cornea. While preliminary, the data suggest the peptide-based material developed in this work offers a cost-effective alternative to cyanoacrylate, including the possibility to in situ reshape the cornea

[0372] CLP-based materials for treating the infarcted heart

[0373] The data presented in Figures 35 to 55 indicate that the CMratio 4.0 hydrogel has highpotential as a tissue adhesive, sealant, and filler material; however, for intramyocardial injection, such materials are too viscous to be delivered using a G27 needle. Thus, for intramyocardial applications, the suitability of the CMratio 1.0 material was tested, which is approximately an order of magnitude less viscous at high shear rates (20, 3.0, and 7.5 Pa•s for CMratio4.0 and 1.0 and Viscoat at 100 s-1). For in vivo testing, a clinically relevant murine myocardial infarction model was used that recapitulates patients who have not responded to revascularization therapeutics or who have delayed seeking treatment.43The model involves the permanent ligation of the artery descendent blood flow supply for 7 days prior to treatment delivery (Figure 56). The CMratio1.0 material did not present toxicity in human cardiac endothelial cell viability in vitro. Results showed that the percentage of viable cells was not different for the peptide-based material compared to a collagen-based hydrogel after 2 days of culture (Figure 56). Assessment of the left ventricular ejection fraction 4 weeks after treatment delivery showed that the intramyocardial injection of the CMratio 1.0 material preserved cardiac function of the MI heart (i.e., no fold-change vs. baseline, p>0.05) compared to the control group (Figure 57). However, no differences in scar size (Figure 4D / 61) or the number of pro-healing macrophages (Figure 59) were observed. Use of fluorescent labelling to track the CMratio 1.0 material in vivo, showed that it could still be visualized in the cardiac muscle for up to 7 days after injection (Figure 60). This finding presents an interesting opportunity for use of the CMratio1.0 material for the in situ delivery of therapeutic products, including stem cells, small molecules, or other therapeutic agents that typically have low retention rates whendelivered to the myocardium.

[0374] The technological development of biomaterials remains costly and time consuming.44Further, material testing often necessitates the use of relatively large sample volumes, which hinders biomaterial discoveries using peptide libraries. In Examples 1 and 2, a strategy that considerably and time-cost effectively reduced the number of potential peptide candidates and formulations to one order of magnitude was used. The proposed screening approach uses small sample volumes of the peptide candidates, which considerably reduces screening costs and time burden of their synthesis. The number of amino acids in each peptide was limited as much as possible as each extra residue increases synthetic cost and difficulty, thus the ideal candidate will possess the desired properties while minimizing residue number. The choice of screening tests used in our strategy combined with the facile material assembly process enabled by the selection of chemistries allows us to rapidly screen different formulations in a high-throughput manner.

[0375] The peptide-based material utilizes the structural backbone of the peptide as both thecrosslinker and a structural component. This approach uses a synergistic dual mechanism, inspired by how structural collagen organizes in natural tissue, where orthogonal chemistry and supramolecular assembly are key. Peptide supramolecular assembly is usually thought of in terms of alpha helix and beta sheet folding, but we show that collagen triple helical folding can be utilized to create and reinforce materials. Through our screen of a library of peptide structures we illustrate the key molecular characteristics of successful CLPs. The resulting hybrid peptide-copolymer displays desirable and tunable physical properties that can be adjusted simply by altering reagent stoichiometry, which allows for use in different tissues. Further, the material preparation is straightforward requiring only reagent solvation in minutes, without the need for external crosslinkers or solvents and is sufficiently robust to be performed in situ.

[0376] Some key advantages of the material include simple, rapid in situ gelation, tunablehydrogel strength and adhesivity, and a well-defined chemical composition. Further, the synthetic nature of the peptides will allow for additional post-refinement of the matrix composition. For example, this could include adding motifs that recognize specific cellular proteins for promoting cell interaction and function. The tunable gelation of the material allows for its use as a topically applied material or in the form of an injectable material by simply using a syringe-mixing system. Typically, peptide gels that form solely from supramolecular assembly are weak and applied only as a topical dressing.45Instead, our gelscan achieve a wound closure strength of ~50 kPa, whereas a pure peptide gel such as a recently reported tryptophan zipper system had a yield strength of 0.075 kPa.46Another LDLK12 peptide gel reported a failure stress of 0.019 kPa that could be increased to 0.035 kPa with EDC / NHS crosslinking.47Compared to other PEG-peptide systems,48our peptide-based material renders stronger gels more quickly at physiological pHs. Peptide hydrogels are often used as carriers for drug delivery,49which is another promising application for which our materials would be highly suited.

[0377] Biomedical systems should be cost-effective and practical to find adaptation. The systemis composed of only two components that can be stored as solids. These components are cost-effective as peptide synthesis is a scalable industrial process that can produce kilograms50or even tons51of finished product as the number of peptide drugs in clinical trials continues to increase52and novel peptide drugs such as Semaglutide53receive media attention. Peptides of ~30 amino acid residues, such as the 31 residue Semaglutide, are considered relatively short and can readily be scaled for industrial production.51,54The cost to access peptides is reasonable for biomedical standards, with retail Semaglutide selling for approximately 150 USD / g in the least expensive markets.55

[0378] Due to their rapid gelation and adhesivity, peptide hydrogels could be applied to thecornea surface to seal perforation defects or injected into the corneal stroma to alter its shape. Furthermore, treatment with topical application of our peptide hydrogels resulted in visibly accelerated healing along with histological markers of improved healing including reduced epithelial thickness and increased collagen content. While our data illustrated low macrophage polarization, it has been described that macrophage expression varies across time during the process of wound healing as initially M1 macrophages are seen, then replaced with M2 macrophages, and lastly both are gone from the wound as it heals and matures.56Further, a moist wound environment, such as one provided by hydrogels, is beneficial towards wound healing57-58and thus our material is suitable as a wound sealant.

[0379] Peptides and other biological molecules can be rapidly degraded in living organisms. Thepeptide-based material can also beinjected into the ischemic area of the heart after mixing two components, rapidly forming a gel. In a mouse model of myocardial infarction, injecting it in situ for 28 days-maintained heart function in contrast to the loss of function observed for the control group. Imaging experiments show that the hydrogel remained in the myocardium for at least seven days, offering the potential for in situ drug or cell delivery. Previous peptide-based injectable materials include an elastin- mimetic peptide hydrogel with great mechanical strength under the action of upregulated trans-glutaminase (TGase) for treating heart tissue post-MI.59However, a limitation is the complex and time- consuming preparation methods that are needed60in contrast, our peptide-based material is prepared rapidly, which is more convenient and simpler to operate with, thus making it more suitable to clinical application. Further, our findings open new avenues for enabling modifications of the materials use the peptide structures and orthogonal click chemistry, such as chemical functionalization of the PEG core,61-62which would allow for the prolonged delivery of small drugs and molecules.

[0380] In summary, the cumulative data illustrate that engineering the chemical structure ofcollagen-like peptides allows for the development of functional crosslinking backbones suitable for the formation of hydrogels. Our data indicates that those peptides must contain at least two cysteine thiol- reactive groups for Michael addition click chemistry with adequate steric spacing, and supramolecular assembly of these peptides contributes to hydrogel strength. This unique combination of engineered click-CLP allows for the rapid in situ production of adhesive, multifunctional, and tunable hydrogels. These materials were assessed in three separate soft tissue repair sites: The cornea, skin and heart as an injectable formulation. The simple yet robust chemistry, combined with the peptide engineering used to produce these gels leaves ample room for future biofunctionalization and / or loading of drugs or cells. EXAMPLE 3 – Ex vivo and in vitro testing Materials and Methods for Example 3 Materials and Hydrogel preparation

[0381] The various hydrogel formulations were created by combining varying concentrations ofPoly-L-Lysine (PLL) (Sigma-Aldrich, St. Louis, USA), HEPES (Fisher Scientific, Pittsburg, USA), 8- Arm PEG-MAL MW 40k (PEG-MAL) (Creative PEGworks, Durham, USA), 8-Arm – PEG20K- Acrylate Hexaglycerol core (PEG-Ac) (Sigma-Aldrich, St. Louis, USA), Poly (ethylene Glycol) diacrylate average Mn 700 (PEGDA-700) (Sigma-Aldrich, St. Louis, USA), NaOH, C3-peptide (BEaTs, Ottawa, Canada) , Rose Bengal (RB) (Sigma-Aldrich, St. Louis, USA) and Eosin Y (Will review,USA). All materials were resuspended in MilliQ water for the purpose of making stock solutions. The stock solutions were the following: PLL (10 wt%), HEPES (200 mM, pH 8), PEG-MAL (50 wt%), PEG-Ac (50 wt%), NaOH (1M), C3 (30 wt%), Eosin Y (0.20 wt%), and RB (0.20 wt%).

[0382] The addition of chemicals was done at room temperature where two solutions were made.Solution 1 was composed of nanopure water, PLL, PEG-MAL, PEG-Ac, and PEGDA-700. Solution 2was composed of C3-peptide, HEPES, Eosin Y or Rose Bengal and sodium hydroxide.

[0383] The concentration of all elements previously described varied depending on theexperiment being performed. Moreover, various PLL molecular weights (MW) were tested such as Poly- L-lysine hydrobromide (MW 4,000 – 15,000) (Sigma-Aldrich, St. Louis, USA), Poly-L-lysine hydrobromide (MW 30,000 – 70,000) (Sigma-Aldrich, St. Louis, USA), Poly-L-lysine hydrobromide (MW 150,000 – 300,000) (Sigma-Aldrich, St. Louis, USA) and Poly-L-lysine hydrobromide (MW 300,000+) (Sigma-Aldrich, St. Louis, USA). To assess the wound-closure strength the samples were compared to BioGlue® (CryoLife, Kennesaw, USA). Ex-vivo wound simulation on mice skin

[0384] The wound closure strength of tissue adhesives and sealants was determined using amodified standard test technique (ASTM F2458-05)35. The University of Ottawa Heart Hospital donated skin samples from a cohort of male and female C57BL / 6 mice (Charles Rivers Laboratories, Canada). The mouse skin was partitioned into strips of 30 ± 5 mm length, 5 ± 0.5 mm width, and 0.7 ± 0.2 mm thickness. A surgical blade was then used to completely sever the samples at mid-length and simulate a wound (Figure 66). The simulated wound was treated with 30 µL of hydrogel sample. A few drops of PBS were applied to each end of the skin strips every 5 minutes until testing to prevent the mouse skin from drying out. Hydrogel application and crosslinking

[0385] Prior to partitioning, the width, thickness, and length of the mouse skin strips weremeasured and modified as necessary. The mouse skin samples were fully sliced at the mid-section of their total length using a surgical blade. The bisected mouse skin strips were positioned in contact with each other at the cut section and treated with hydrogel. The treatment involved the topical administration of 30 µL of hydrogel to the simulated wound region. Unless otherwise specified, the samples were irradiated with a single color (Green) Engin High Power LED (Mouser Electronics, USA) at a wavelength of 532 nm (irradiance 120-150 mW / cm2) for a period of two minutes at room temperature (25° C). The Green LED was powered by YIHUA PS 3010DC power supply (YIHUA, Guangzhou, China). Quantifying adhesive properties of hydrogel in ex vivo murine wound model

[0386] A modified ASTM F2458-05 test measured tissue adhesive and sealant wound closurestrength35. The bisected mouse skin with the irradiated hydrogel at the desired formulation was clamped on an Instron 3342 universal testing machine (Instron, USA) at 5 ± 2 mm distance from the simulated wound on both ends. The Instron transductor sensor Model 2519 – 101 (Instron, USA) was used to record the formulation’s resistance force during the tensile test at a cross head speed of 1 mm / min until material failure. The software used was the INSTRON Series IX / S (Instron, Norwood, USA). Testing the mechanical properties of hydrogels

[0387] Rectangular pieces of hydrogel were prepared with dimensions 2.5 mm length x 5 mmwidth and clamped on an Instron 3342. The Young’s modulus reported here was calculated as the slope of the stress-strain curve at 10% strain. The maximum percent strain was taken as the strain at which the maximum tensile stress was reached. Rheology

[0388] Shear thinning and viscosity measurements were collected with a Brookfield Rheometerand the software Rheo3000 (AMETEK, USA). For measuring the sample, 100 µL of material was tested with an RCT-25-1 head where the shear rate was measured at a constant rate of 1 / s for 60 seconds at room temperature (25 °C). A total of 270 points for each sample was recorded at a rate of 1 point / sec. Water content

[0389] Water retention was measured by swelling the hydrogels in PBS and measuring the massof the hydrogels at different time points (W(measured)). The degree of swelling was relative to the initial hydrogel mass (W(initial)). Swelling percentage was calculated by using the following equation: swelling (%) = W(measured) / W(initial). The dry mass (W) of each sample was measured by lyophilizing the hydrogel samples over a period of 48h and then measuring the weight of sample. Water content was calculated according to the equation: Wt % = (W-Winitial) / W % Collagen degradation

[0390] The hydrogels were placed in phosphate-buffered saline and incubated for 48 hours at 37°C. After allowing the hydrogel to swell, the stability of the hydrogels was evaluated using collagenase from Clostridium histolyticum (Sigma-Aldrich, USA) at 5 Unit / mL in 0.1 M Tris-HCL (pH 7.4) Buffer containing 5 mM CaCl2 was used to evaluate the stability of the hydrogels33. To determine the degradation rate, the samples had their masses measured at multiple time points. Percentage of residual weight was calculated using the following equation: residual mass (%) = W(total) / W(initial).Statistical Analysis

[0391] Statistical analysis was performed in GraphPad Prism 10, one-way ANOVA. Outlierswere processed by ROUT statistical analysis, with a Q of 1%. The simple axial design Heat map model was made with Origin 2021b software. Results Adhesion optimization of light-activated material

[0392] Figure 67 is a schematic of the research methodology for the development and testing ofour tissue adhesives. Step 1: Optimization of light-activated formulation with respect to adhesion to skin tissue. Step 2: Selection of most effective material delivery method to ensure good adhesion strength and material stability over time. Steps 3, 4 and 5: Application of tissue adhesive to skin incision and material activation in situ using green light to establish strong and stable wound closure.

[0393] An axial design was performed to determine the most favourable proportion of polymersand peptide for achieving the maximum adhesion strength, where the concentrations of PEG- Ac, PEG- Mal and CLP was manipulated and their effect on wound closure strength (WCS) was observed (Figure 68-69). The concentration of PEG-Mal, PEG-Ac and CLP were maintained below 10 wt%, 5 wt% and 5 wt% respectably and the wound closure strength (WCS) was reported as this equates to the adhesion strength of the material at the point where the material holds the two pieces of skin together (Figure 69). The data pertaining to the axial design was sorted on a two-dimensional triangle heatmap plot, enabling the identification of key areas for further analysis and optimization (Figure 69). Overall, a trend that was identified is that adhesive candidates with a high concentration of PEG-Ac, with relative lower amounts of peptide and PEG-Mal demonstrated the highest wound closure strength. Specifically, the three studied formulations, dubbed as L, P and H had an average wound closure strength of 6.9 kPa, 7.4 kPa, and 7.2 kPa respectively (Figure 72). The composition of the top three tissue adhesive candidates, labeled H, L and P, are presented in Figures 68-69 and 72.

[0394] To enhance the adhesive properties of the material presented here, the concentration ofthe primary active components was augmented by a factor of 2.5, while maintaining a constant ratio of these components (Figure 69A). The higher concentration of PEG-Mal, PEG-Ac, and peptide in the precursor formulation also resulted in an increased viscosity. The observed rise in viscosity thataccompanied the higher wound closure strength of the materials was an added benefit as it would allow the developed materials to stay predominantly on the desired wound site. Formulation H still had a low viscosity even after applying the concentration multiplication factor of 2.5 and was deemed unsatisfactory for wound closure applications due to high risk of runoff from wound bed and was thus discarded from further consideration. (Figure 68-69). Importance of the photoinitiator and green light exposure effect

[0395] Prior research had established a correlation between green light exposure and productionof free radicals from photoinitiators36–38. To this end, experiments were performed to investigate whether the photoinitiator used would have a noticeable effect on the adhesion properties and if modulating the exposure time to green light could result in tunability of adhesion and mechanical properties of the hydrogel. Regarding the first section, it was found that despite using the same concentration of photoinitators, Eosin Y resulted in superior wound closure strength (Figure 69C). Additionally, as previously theorized it was found that light exposure had a direct effect on the wound closure strength of the material (Figure 69D). Although there was a strong relationship between light exposure time and tensile strength, there was no statistical difference between 4 and 2 minutes potentially indicating that network formation plateau is reached after a specific period of exposure. To test this hypothesis, the hydrogel absorption profiles were monitored over time and the peak signifying Eosin Y at 516 nm was shown to continue decreasing even after 4 minutes of green light exposure (Figure 69D). The reduction in absorption peak demonstrates that Eosin Y is still available for free radical generation. As a result, negligible improvement in tensile strength after 2 minutes of light exposure could be due to near- complete consumption of reactive groups instead of a lack of available free radicals. Future testing will measure the number of acrylate groups after varying light irradiation using solid state NMR. As for hydrogel mechanical properties, the results indicate that green light exposure could be used as a tunable mechanism for the material as less green light resulted in weaker materials compared to the stiffer hydrogels exposed to 4 minutes of green light (Figure 69F). The optimized formulation has comparable mechanical qualities of commercially available options

[0396] The optimized formulations L and P, which were deemed to be the best hydrogelformulations, were compared to commercially available surgical glues for their wound closure strengthas well as flexibility. Regarding wound closure strength, when tested L presented a large range of values that in some instances was superior to BSA- Glutaraldehyde, while P demonstrated more clustered values (Figure 69E). Percent strain refers to the displacement of the material compared on a single axis prior to breakage, and thus this was used as a measure of material elasticity. Regarding percent strain, L demonstrated significant higher elasticity than the control and while P did not demonstrate statistical significance compared to the control, some individual values were superior to the control perhaps indicating sample size needs to be increased (Figure 69F). From the results of this section, overall formulation P demonstrated to be a suitable material that could provide to be superior to currently used methods (Figure 69E & 69F). Water content and collagenase degradation

[0397] The assessment of biocompatibility in materials, particularly hydrogels, necessitates theconsideration of several crucial factors. The aforementioned factors encompass quantification of water content, evaluation of swelling capabilities, analysis of thermal profiles, and examination of degradation properties24,33,39–41. To this end, series of diverse assays were conducted. Starting with the water content and retention of the material, the material presented here exhibits hydrophilic properties, as evidenced by its water content and retention characteristics. The water content of the hydrogels was determined to be about ~80-85%, indicating a significant proportion of water within the material (Figure 71B). In addition, the hydrogel swells in phosphate buffered solution, reaching equilibrium within approximately one hour and capable of retaining a swelled state of 60- 80% its initial weight even after 48 hours (Figure 71A & 71B).

[0398] To determine if the material can be degraded by commonly found enzymes in the body asimple in vitro collagenase assay was performed (Figure 71C). Regarding the results, this assay demonstrated that the collagenase enzyme from this specific strain used was unable to degrade neither L nor P (Figure 71C).

[0399] The principal findings in this example suggest that the tensile strength of our bioadhesiveis comparable to commercially available alternatives with the added benefit of having tunable adhesion. The present example focused on the development of a tissue adhesive, wherein the concentration of individual components within the hydrogel was systematically optimized to enhance the wound closurestrength of the material.

[0400] In particular, the mechanical tunability of the developed materials is an advantage overcommercial adhesives that are unable to be finely tune for different target tissues. Considering the type of reaction and the three primary functional groups (thiol, maleimide, and acrylate), an ideal mixture ratio of reagents could yield a material with desirable adhesive and physical properties which ultimately lead to finding three potential formulations labeled L, H and P (Figure 68-69 and Figure 72). These formulations were further optimized regarding their tensile strength by increasing the concentration of the various key elements which, as expected, resulted in a steady increase in tensile strength (Figure 69A). Subsequent analysis of the material mechanical properties, particularly their viscosity, resulted in the exclusion of formulation H from further consideration as this material presented a liquid consistency like water prior to photocross-linkage, which could potentially give rise to complication during in vivo testing as it could shift from the targeted area (Figure 69B).

[0401] The significance of the photosensitizing molecule became a subject of interest in theadvancement of the material, as the utilisation of Eosin Y in comparison to Rose Bengal resulted in a substantial enhancement in the wound closure strength of the material (Figure 69C). Rose Bengal and Eosin Y are both classified as xanthene dyes, a group characterized by presence of three aromatic ring arranged linearly and possess the property of generating free radicals and promoting photopolymerization of acrylate and other functional groups upon exposure wavelengths of 500 – 580 nm44. One possible explanation for the observed discrepancies could be attributed to the efficiency of free-radical release in the materials, which may result in an increased formation of polymer networks. This observation appears to align with the findings reported in the existing literature, as it has been documented that Eosin Y exhibits higher polymerization rates compared to Rose Bengal44. With the more efficient polymerization rates, subjecting Eosin Y to extended durations of green light exposure was tested to enable the fine- tuning of the mechanical characteristics of the hydrogel, particularly its tensile strength and wound closure capabilities. A notable and consistent positive trend in tensile strength with light duration increased from between the 0-, 1- and 2-minute dosage. However, the tensile strength values from 4- minute dosage did not exhibit a discernible disparity when compared to the 2-minute dosage (Figure 69D). Intriguingly, a subsequent set of experiments conducted that looked at the absorption profile of the hydrogel at different green light dosages demonstrated that the well characterized peak 520-510 nm is reduced over time (Figure 71D).

[0402] Degradation studies in biomaterials hold significant importance as they provide valuableinsights into the interactions between the material and the body when applied. These studies enable a precise understanding of whether the material requires replacement and whether it can be safely eliminated from the area. Poly (ethylene glycol) (PEG) hydrogels have been extensively studied and it is well established that they exhibit a tendency to undergo gradual degradation in vivo, primarily through oxidative degradation and ester hydrolysis. The material developed herein demonstrated very limited degradation (Figure 71C). The results observed could be attributed to several factors, such as the absence of identifiable sequences within the CLP where the enzyme could potentially exert its activity47. Alternatively, the minimal degradation could also be attributed to a higher abundance of acrylate bonds compared to peptide bonds. This observation appears to be supported by tensile tests that look at the effect of CLP in the formulation where removal of the CLP from the formulation presented a product with considerable tensile strength, albeit noticeably weaker than those containing CLP (Figure 72).

[0403] This example describes the development of a tunable novel synthetic material for woundclosure. The optimized hydrogel formulations produce a tunable material capable of archiving superior mechanical qualities to conventional commercially available options depending on light irradiation. The material described here offers great promise as a wound-sealant and as a delivery method of nutrients or pharmaceuticals to the targeted area.

[0404] One or more illustrative embodiments have been described by way of example. It will beunderstood to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

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Claims

WHAT IS CLAIMED IS:

1. A self-assembling collagen-like peptide (CLP) comprising a plurality of amino acid trimer repeatshaving the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

2. The self-assembling collagen-like peptide (CLP) of claim 1, wherein the plurality of amino acidtrimer repeats is flanked at the N-terminal and the C-terminal by at least two amino acid residues.

3. The self-assembling collagen-like peptide (CLP) of claim 3, wherein the at least two amino acidresidues are cysteine, glycine, valine or a combination thereof.

4. The self-assembling collagen-like peptide (CLP) of claim 3, wherein the at least two amino acidresidues is defined by amino acid sequence GCG.

5. The self-assembling collagen-like peptide (CLP) of any one of claims 1-4, wherein the collagen-like peptide (CLP) is defined by amino acid sequence G-X-G(POG)nG-X-G, wherein X is G or C, and 3 ≤ n ≤ 20.

6. The self-assembling collagen-like peptide (CLP) of claim 5, wherein X is C and 6 ≤ n ≤ 10.

7. The self-assembling collagen-like peptide (CLP) of claim 6, wherein n = 8.

8. A hydrogel comprising a self-assembling collagen-like peptide (CLP) and a multi-armpolyethylene glycol (PEG) polymer, wherein the CLP comprises a plurality of amino acid trimer repeats having the amino acid sequence POG, wherein P is a proline, O is a hydroxyproline or a cysteine, and G is a glycine or a valine.

9. The hydrogel of claim 8, wherein the multi-arm PEG polymer is crosslinked to the CLP.

10. The hydrogel of claim 9, wherein the multi-arm PEG polymer is a 2‐Arms‐PEG acrylate, 4‐Arms‐PEG acrylate, 8‐Arms‐PEG acrylate, a 2‐Arms‐PEG maleimide, 4‐Arms‐PEG maleimide, 8‐ Arms‐PEG maleimide or any combination thereof.

11. The hydrogel of claim 10, wherein the PEG is an 8‐Arms‐PEG acrylate, an 8‐Arms‐PEGmaleimide or any combination thereof.

12. The hydrogel of any one of claims 8-11, wherein the plurality of amino acid trimer repeats isflanked at the N-terminal and the C-terminal by at least two amino acid residues.

13. The hydrogel of claim 12, wherein the at least two amino acid residues are cysteine, glycine,valine or a combination thereof.

14. The hydrogel of claim 12 or 13, wherein the at least two amino acid residues is defined by aminoacid sequence GCG.

15. The hydrogel of any one of claims 8-14, wherein the collagen-like peptide (CLP) is defined byamino acid sequence G-X-G(POG)nG-X-G, wherein X is G or C, and 3 ≤ n ≤ 20.

16. The hydrogel of claim 15, wherein X is C and 6 ≤ n ≤ 10.

17. The hydrogel of claim 16, wherein n = 8.

18. The hydrogel of any one of claims 8-17, wherein the water content in the hydrogel is betweenabout 60% to about 90%.

19. The hydrogel of any one of claims 8-18, wherein the self-assembling CLP has a CMratio ofbetween about 1.0 to about 5.0.

20. A photoactivatable hydrogel precursor (PHP) comprising:a self-assembling collagen-like peptide (CLP), wherein the CLP comprises a plurality of amino acid trimer repeats having the amino acid sequence POG, wherein P is a proline, O is a hydroxyproline or a cysteine, and G is a glycine or a valine; and a multi-arm polyethylene glycol (PEG) polymer comprising at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator.

21. The photoactivatable hydrogel of claim 20, wherein a hydrogel is obtained by photoactivatingthe hydrogel precursor with visible light.

22. The photoactivatable hydrogel of claim 21, wherein the visible light has a wavelength of betweenabout 500 nm to about 580 nm.

23. The photoactivatable hydrogel of claim 21 or 22, wherein the visible light is green light.

24. The photoactivatable hydrogel of any one of claims 20-23, wherein the photoinitiator is RoseBengal, Eosin Y, or any other photoactive molecule.

25. The photoactivatable hydrogel of claim 24, wherein the photoinitiator is Eosin Y.

26. The photoactivatable hydrogel of any one of claims 20-25, wherein the concentration of PEG-maleimide and / or PEG-acrylate is between about 1% w / v and about 25% w / v.

27. A bioadhesive comprising the self-assembling CLP of any one of claims 1-7, the hydrogel of anyone of claim 8-19 or the photoactivatable hydrogel of any one of claims 20-26.

28. A scaffold comprising the self-assembling CLP of any one of claims 1-7, the hydrogel of any oneof claim 8-19 or the photoactivatable hydrogel of any one of claims 19-26.

29. A composition comprising the self-assembling CLP of any one of claims 1-7 and a multi-armpolyethylene glycol (PEG) polymer.

30. The composition of claim 29, wherein the PEG polymer comprises at least one acrylate groupand / or at least one maleimide group, and at least one photoinitiator.

31. A method of preparing a hydrogel comprising :mixing a self-assembling collagen-like peptide (CLP) with a multi-arm polyethylene glycol (PEG) polymer, wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

32. The method of claim 31, wherein a hydrogel precursor is first obtained by mixing the self-assembling CLP with the multi-arm PEG polymer, wherein the hydrogel is obtained after the hydrogel precursor is incubated for a period of time to allow crosslinking of the self-assembling CLP to the multi-arm PEG.

33. The method of claim 32, wherein the period of time is less than 5 minutes.

34. The method of any one of claims 31-33, wherein the self-assembling CLP has a CMratio ofbetween about 1.0 to about 5.0.

35. A method of preparing a photoactivatable hydrogel precursor (PHP), the method comprising :mixing a self-assembling collagen-like peptide (CLP) with a multi-arm polyethylene glycol (PEG) polymer, wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, andwherein the multi-arm PEG polymer comprises at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator.

36. A method of preparing a photoactivated hydrogel, the method comprising photoactivating thePHP of claim 35.

37. The method of claim 36, wherein photoactivating the PHP comprises irradiating the PHP withlight.

38. The method of claim 37, wherein the light is visible light.

39. The method of claim 38, wherein the visible light has a wavelength of between about 500 nm toabout 580 nm.

40. The method of claim 38 or 39, wherein the visible light is green light.

41. The method of any one of claims 35-40, wherein the photoinitiator is Rose Bengal or Eosin Y.

42. The method of claim 41, wherein the photoinitiator is Eosin Y.

43. The method of any one of claims 35-42, wherein the concentration of PEG-maleimide and / orPEG-acrylate is between about 1% w / v and about 25% w / v.

44. The method any one of claims 35-43, wherein the CLP has a CMratio of between about 1.0 toabout 5.0.

45. A method of treating a wound, the method comprising:exposing a wound to a therapeutically effective amount of a hydrogel precursor, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) with a multi-arm polyethylene glycol (PEG) polymer, wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

46. The method of claim 45, further comprising waiting for a period of time after exposing the woundto allow crosslinking of the self-assembling CLP to the multi-arm PEG.

47. The method of claim 45 or 46, wherein exposing the wound comprises covering the wound withthe hydrogel precursor.

48. The method of any one of claims 45-47, wherein exposing the wound comprises injecting thetherapeutically effective amount of the hydrogel precursor in a subject in need thereof.

49. The method of claim 48, wherein injecting is performed at or around a wound site.

50. The method of any one of claims 45-49, wherein the wound is in a soft tissue.

51. The method of claim 50, wherein the soft tissue is muscle.

52. The method of claim 50, wherein the wound is in a cornea.

53. The method of claim 50, wherein the wound is in skin.

54. A method of treating a corneal disease in a subject in need thereof, the method comprising:injecting a therapeutically effective amount of a hydrogel precursor in the subject, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

55. The method of claim 54, further comprising waiting for a period of time after injecting to allowcrosslinking of the self-assembling CLP to the multi-arm PEG polymer.

56. The method of claim 54 or 55, wherein the corneal disease comprises corneal thinning and / or acorneal wound.

57. A method of treating a myocardial infarct in a subject in need thereof, the method comprising:injecting a therapeutically effective amount of a hydrogel precursor in the subject, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

58. The method of claim 57, further comprising waiting for a period of time after injecting to allowcrosslinking of the self-assembling CLP to the multi-arm PEG polymer.

59. A method of promoting cell growth, the method comprising:treating a cell with a therapeutically effective amount of a hydrogel precursor, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

60. The method of claim 59, further comprising waiting for a period of time after injecting to allowcrosslinking of the self-assembling CLP to the multi-arm PEG polymer.

61. A method of treating cellular degeneration in a subject in need thereof, the method comprising:injecting a therapeutically effective amount of a hydrogel precursor in the subject, wherein the hydrogel precursor comprises a self-assembling collagen-like peptide (CLP) and a multi-arm polyethylene glycol (PEG) polymer, and wherein the CLP comprises a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine.

62. The method of claim 61, further comprising waiting for a period of time after injecting to allowcrosslinking of the self-assembling CLP to the multi-arm PEG polymer.

63. A method of treating a wound, the method comprising:exposing a wound to a therapeutically effective amount of a photoactivatable hydrogel precursor (PHP), wherein the PHP comprises: a self-assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, anda multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator; and photoactivating the PHP.

64. The method of claim 63, further comprising waiting for a period of time after photoactivating toallow crosslinking of the PHP to the PEG polymer.

65. The method of claim 63 or 64, wherein photoactivating the PHP comprises irradiating the woundwith light.

66. The method of claim 65, wherein the light is visible light.

67. The method of claim 66, wherein the visible light has a wavelength of between about 500 nm toabout 580 nm.

68. The method of claim 66 or 67, wherein the visible light is green light.

69. The method of any one of claims 63-68, wherein the photoinitiator is Rose Bengal or Eosin Y.

70. The method of claim 69, wherein the photoinitiator is Eosin Y.

71. The method of any one of claims 63-70, wherein the concentration of PEG-maleimide and / orPEG-acrylate is between about 1% w / v and about 25% w / v.

72. The method any one of claims 63-71, wherein the CLP has a CMratio of between about 1.0 toabout 5.0.

73. The method of any one of claims 63-72, wherein exposing the wound comprises covering thewound with the PHP.

74. The method of claim 63-72, wherein exposing the wound comprises injecting the therapeuticallyeffective amount of the PHP in a subject in need thereof.

75. The method of any one of claims 63-74, wherein the wound is in a soft tissue.

76. The method of claim 75, wherein the soft tissue is muscle.

77. The method of claim 75, wherein the wound is in a cornea.

78. The method of claim 75, wherein the wound is in skin.

79. A method of treating a corneal disease in a subject in need thereof, the method comprising:injecting a therapeutically effective amount of a photoactivatable hydrogel precursor (PHP) in the subject, wherein the PHP comprises: a self-assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, and a multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator; and photoactivating the hydrogel precursor.

80. The method of claim 79, further comprising waiting for a period of time after photoactivating toallow crosslinking of the PHP to the PEG polymer.

81. The method of claim 79 or 80, wherein photoactivating the PHP comprises irradiating the subjectwith light.

82. The method of claim 81, wherein the light is visible light.

83. The method of claim 82, wherein the visible light has a wavelength of between about 500 nm toabout 580 nm.

84. The method of claim 82 or 83, wherein the visible light is green light.

85. The method of any one of claims 79-84, wherein the photoinitiator is Rose Bengal or Eosin Y.

86. The method of claim 85, wherein the photoinitiator is Eosin Y.

87. The method of any one of claims 79-86, wherein the concentration of PEG-maleimide and / orPEG-acrylate is between about 1% w / v and about 25% w / v.

88. The method any one of claims 79-87, wherein the CLP has a CMratio of between about 1.0 toabout 5.0.

89. The method of any one of claims 79-88, wherein the PHP is injected in the cornea.

90. The method of any one of claims 79-89, wherein the corneal disease comprises corneal thinningand / or a corneal wound.

91. A method of treating a myocardial infarct in a patient in need thereof, the method comprising:injecting a therapeutically effective amount of a photoactivatable hydrogel precursor (PHP) in the patient, wherein the PHP comprises: a self-assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, and a multi-arm polyethylene glycol (PEG) polymer having at least one acrylate group and / or at least one maleimide group, and at least one photoinitiator; and photoactivating the hydrogel precursor.

92. The method of claim 91, further comprising waiting for a period of time after photoactivating toallow crosslinking of the PHP to the PEG polymer.

93. The method of claim 91 or 92, wherein photoactivating the PHP comprises irradiating the subjectwith light.

94. The method of claim 93, wherein the light is visible light.

95. The method of claim 94, wherein the visible light has a wavelength of between about 500 nm toabout 580 nm.

96. The method of claim 94 or 95, wherein the visible light is green light.

97. The method of any one of claims 91-96, wherein the photoinitiator is Rose Bengal or Eosin Y.

98. The method of claim 97, wherein the photoinitiator is Eosin Y.

99. The method of any one of claims 91-98, wherein the concentration of PEG-maleimide and / orPEG-acrylate is between about 1% w / v and about 25% w / v.

100. The method any one of claims 91-99, wherein the CLP has a CMratio of between about 1.0to about 5.0.

101. The method of any one of claims 91-100, wherein the PHP is injected in the infract or ina region proximal to the infarct.

102. A method of promoting cell growth, the method comprising:treating a cell with a therapeutically effective amount of a photoactivatable hydrogel precursor (PHP), wherein the PHP comprises:a self-assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, and a multi-arm polyethylene glycol (PEG) polymer having at least one photoinitiator at least one acrylate group and / or at least one maleimide group; and photoactivating the PHP.

103. The method of claim 102, further comprising waiting for a period of time afterphotoactivating to allow crosslinking of the PHP to the PEG polymer.

104. The method of claim 102 or 103, wherein photoactivating the PHP comprises irradiatingthe cell with light.

105. The method of claim 104, wherein the light is visible light.

106. The method of claim 105, wherein the visible light has a wavelength of between about500 nm to about 580 nm.

107. The method of claim 105 or 106, wherein the visible light is green light.

108. The method of any one of claims 102-107, wherein the photoinitiator is Rose Bengal orEosin Y.

109. The method of claim 108, wherein the photoinitiator is Eosin Y.

110. The method of any one of claims 102-109, wherein the concentration of PEG-maleimideand / or PEG-acrylate is between about 1% w / v and about 25% w / v.

111. The method any one of claims 102-110, wherein the CLP has a CMratio of between about1.0 to about 5.0.

112. A method of treating cellular degeneration in a subject in need thereof, the methodcomprising: treating the subject with a therapeutically effective amount of a photoactivatable hydrogel precursor (PHP), wherein the PHP comprises:a self-assembling collagen-like peptide (CLP) having a plurality of amino acid trimer repeats defined by the amino acid sequence POG, wherein P is a proline, O is an hydroxyproline or a cysteine, and G is a glycine or a valine, and a multi-arm polyethylene glycol (PEG) polymer having at least one photoinitiator at least one acrylate group and / or at least one maleimide group; and photoactivating the hydrogel precursor.

113. The method of claim 112, further comprising waiting for a period of time afterphotoactivating to allow crosslinking of the PHP to the PEG polymer.

114. The method of claim 112 or 113, wherein photoactivating the PHP comprises irradiatingthe cell with light.

115. The method of claim 114, wherein the light is visible light.

116. The method of claim 115, wherein the visible light has a wavelength of between about500 nm to about 580 nm.

117. The method of claim 115 or 116, wherein the visible light is green light.

118. The method of any one of claims 112-117, wherein the photoinitiator is Rose Bengal orEosin Y.

119. The method of claim 118, wherein the photoinitiator is Eosin Y.

120. The method of any one of claims 112-119, wherein the concentration of PEG-maleimideand / or PEG-acrylate is between about 1% w / v and about 25% w / v.

121. The method any one of claims 112-120, wherein the CLP has a CMratio of between about1.0 to about 5.0.

122. Use of the hydrogel of any one of claims 8-19, the PHP of any one of claims 20-26, thebioadhesive of claim 27, the scaffold of claim 28 or the composition of claim 29 or 30, as a wound sealant, for improving wound closure, for treating a corneal disease, for treating a myocardial infarct, for treating a corneal disease, for promoting cell growth and / or for treating cellular degeneration.

123. A device for delivering a peptide-based material in a subject, the device comprising:- an on / off button or switch for initiating and stopping delivery of the peptide-based material in the subject; - a chamber for containing the peptide-based material; - an aperture through which the peptide-based material exits the chamber, wherein the aperture is adapted for receiving a nozzle adapter; - a camera port for securing a camera thereto; and - a digital control display.

124. The device of claim 123, wherein the chamber is adapted for receiving one or morepeptide cartridge containing the peptide-based material.

125. The device of claim 123, wherein the peptide cartridge is a preloaded syringe.

126. The device of claim 123, wherein the camera allows live monitoring when delivering thepeptide-based material in the subject.

127. The device of claim 123, wherein the digital control display allows adjusting at least onedelivery parameter.

128. The device of claim 127, wherein the at least one delivery parameter comprises volumeto be delivered and speed at which the volume is delivered in the subject.

129. The device of claim 123, wherein the nozzle adapter is adapted to receive a variety ofnozzles.

130. The device of claim 123, wherein the variety of nozzles comprise a nozzle for topicalapplication, a nozzle for cornea sealant delivery, and a nozzle for intratissue injection.

131. The device of claim 123, further comprising a handle.

132. The device of claim 131, wherein the device is a handheld device.

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