Mpc-based copolymers and hydrogels, and uses thereof in regenerative medicine

Abstract

The present application relates to polymers, conjugates, hydrogels, and kits comprising 2- methacryloyloxyethyl phosphorylcholine (MPC) and / or copolymers comprising MPC, and collagen or a collagen-like peptide (CLP). The polymers, conjugates, hydrogels, and kits may be used as fillers and sealants for various applications, for example for treating a wound or tissue injury, such as corneal injury or perforation. Devices and systems for the administration of the polymers, conjugates, and hydrogels are also disclosed.

Description

[0001] MPC-BASED COPOLYMERS AND HYDROGELS, AND USES THEREOF IN REGENERATIVE MEDICINE

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the benefit of United States provisional patent application serial No. 63 / 507,795 filed on June 13, 2023, which is incorporated herein by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] A sequence listing is submitted herewith as an XML file named G12810-00862_Seq Listing. xml, created on June 13, 2024, and having a size of 83715 bytes. The content of the aforementioned file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] The present invention generally relates to the field of regenerative medicine, and more particularly to the regeneration of damaged corneas.

[0008] BACKGROUND ART

[0009] The cornea is the transparent front of the eye and main refractive surface, focusing light into the eye for vision. Any permanent loss of transparency due to injuries or diseases can result in blindness. Pathologies resulting in inflammation can progress to ulceration, neovascularisation, and perforation. Corneal perforations are emergencies and are treated by sealing with cyanoacrylate or surgical “Krazy glue”, which is toxic and kills surrounding cells. In general, ulcers that are non-responsive to medication and “Krazy glue”-sealed corneas require follow-on transplantation. While corneal transplantation can restore vision, severely inflamed and neovascularized corneas are often contraindicated as they have a high risk for graft failure. Where the supply of donor tissue is in a severe shortfall, they are preferentially reserved for patients’ corneas that are more likely to benefit from the surgery and not rejected. It is desirable to develop an alternative to transplantation that can be applied easily to seal wounds and promote functional regeneration of the damaged tissue.

[0010] An early proof-of-concept “liquid cornea” (LiQD Cornea) comprising self-assembling collagen-like peptides (CLP) delivered as an injectable liquid that gelled spontaneously within wounds, stimulating regeneration in mini-pig and rabbit corneas, was described in McTiernan et al. (Sci Adv. 2020 Jun 17;6(25):eaba2187. doi: 10.1126 / sciadv.aba2187. Print 2020 Jun). However, using fibrin glue for sealing necessitates the application of a second syringe and an extra step that can be problematic in emergency situations. In addition, extra amounts of PEG that is used in order to improve the mechanical properties of the formulation can cause severe adverse allergic reaction, as reported in a number of COVID- 19 vaccination recipients (Cox et al. J Clin Pharmacol. 2021 Jun;61 (6):832-835. doi: 10.1002 / jcph.1824. Epub 2021 Feb 28). Also, the inflammation and severe pathologies put patients at high risk (50-75%) of rejecting transplants even with immune suppression, and there was nothing in the hydrogel to reduce possible inflammation.

[0011] There is thus a need for novel approaches for sealing and filling injured corneal tissues.

[0012] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

[0013] SUMMARY

[0014] In various aspects and embodiments, the present disclosure provides the following items 1 to **:

[0015] 1 . A conjugate comprising:

[0016] (a) 2-methacryloyloxyethyl phosphorylcholine (MPC) and collagen or a collagen-like peptide (CLP) crosslinked to the MPC;

[0017] (b) (i) a copolymer comprising monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) and polymerizable monomers comprising one or more functional groups; and (ii) collagen or a CLP crosslinked to the copolymer; or

[0018] (c)

[0019] (i) a copolymer comprising a core comprising a multi-arm polymer; monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) attached to one or more arms of the multi-arm polymer; optionally polymerizable monomers or polymers comprising one or more functional groups and

[0020] (ii) collagen or a CLP attached to one or more arms of the multi-arm polymer and / or to the MPC.

[0021] 2. The conjugate of item 1 , wherein the one or more functional groups comprise carboxylic acid groups, hydroxyl groups, thiol groups, and / or amine groups.

[0022] 3. The conjugate of item 1 or 2, wherein the polymerizable monomers comprising one or more functional groups are acrylic acid (AA) or 2-hydroxyethyl acrylate (HEA).

[0023] 4. The conjugate of any one of items 1 to 3, wherein the copolymer of (b) has one of the following structures:

[0024] PMPC-PAA PMPC-PHEA wherein PC is phosphorylcholine, x represents the Mw of polymeric MPC (PMPC), and y represents the Mw of polyacrylic acid (PAA) or poly 2-hydroxyethyl acrylate (PHEA), preferably wherein the Mw of PMPC-PAA is about 387,000 kDa, and the Mw of PMPC-PHEA is about 532,000 kDa.

[0025] 5. The conjugate of item 4, wherein the Mw of PMPC-PAA is about 350,000 to about 400,000 kDa.

[0026] 6. The conjugate of item 5, wherein the Mw of PMPC-PAA is about 387,000 kDa.

[0027] 7. The conjugate of item 4, wherein the Mw of PMPC-PHEA is about 500,000 to about 550,000 kDa.

[0028] 8. The conjugate of item 4, wherein the Mw of PMPC-PHEA is about 532,000 kDa.

[0029] 9. The conjugate of any one of items 1 to 8, wherein the conjugate comprises (i) a first copolymer comprising monomers of MPC and polymerizable monomers comprising hydroxyl groups; (ii) a second copolymer comprising monomers of MPC and polymerizable monomers comprising carboxylic acid groups.

[0030] 10. The conjugate of any one of items 1 to 9, wherein the multi-arm polymer comprises 2 to 8 arms.

[0031] 11 . The conjugate of any one of items 1 to 10, wherein the multi-arm polymer comprises a multiarm polyethyleneglycol (PEG).

[0032] 12. The conjugate of item 11 , wherein the PEG has a molecular weight of 2000 to 5000 kDa.

[0033] 13. The conjugate of any one of items 1 to 12, wherein the copolymer of (c) has the following structure: wherein PC is phosphorylcholine, n represents the Mw of the polymer (300,000 to 500,000 kDa) and m is an integer of 2 to 8.

[0034] 14. The conjugate of any one items 1 to 13, wherein the MPC is directly linked to the collagen or CLP.

[0035] 15. The conjugate of any one items 1 to 13, further comprising a linker between the MPC and the collagen or CLP.

[0036] 16. The conjugate of item 15, where the linker is / V, / V'-methylenebisacrylamide (BIS).

[0037] 17. The conjugate of any one of items 1 to 16, wherein the conjugate comprises a CLP.

[0038] 18. The conjugate of item 17, wherein the CLP has a length of 27 to 36 amino acids.

[0039] 19. The conjugate of item 17 or 18, wherein the CLP comprises or consist of the following sequence:

[0040] (PKG)3or4(POG)3or4(DOG)2or3(DOX) (SEQ ID NO: 13), wherein X is G or C.

[0041] 20. The conjugate of item 19, wherein the CLP comprises or consist of the following sequence: (PKG)3(POG)3(DOG)2(DOX), wherein X is G or C.

[0042] 21. The conjugate of item 17, wherein the CLP comprises or consist of the following sequence: X1X2X3(PKG)3or4(POG)3or4(DOG)2 or3(DOC), wherein X1 , X2 and X3 are independently any amino acid, and wherein at least one of X1 , X2 and X3 is C.

[0043] 22. The conjugate of item 21 , wherein X1X2X3is GCG, CGG, SCG, or SCS

[0044] 23. A hydrogel comprising the conjugate of any one of items 1 to 22.

[0045] 24. The hydrogel of item 23, further comprising additional proteins, peptides, cells, and / or drugs.

[0046] 25. A collagen-like peptide (CLP) having a length of 27 amino acids.

[0047] 26. The CLP of item 25, wherein the CLP has the following structure: (PKG)3(POG)3(DOG)2(DOX) (SEQ ID NO: 13), wherein X is G or C.

[0048] 27. The CLP of item 26, wherein X is G.

[0049] 28. The CLP of item 26, wherein X is C.

[0050] 29. A collagen-like peptide (CLP) of one of the following structures:

[0051] X1X2X3(PKG)3(POG)3(DOG)2X4X5X6; or

[0052] X1X2X3(PKG)3(POG)5X4X5X6wherein X1, X2, X3, X4, X5and X6are independently any amino acid, and wherein at least one of X1, X2, and X3is C, and at least one of X4, X5, and X6is C.

[0053] 30. The CLP of item 29, wherein X4X5X6is DOC, SCS, GCG, SCG, or CGG.

[0054] 31 . The CLP of item 29 or 30, wherein X1X2X3is SCS, GCG, SCG, or CGG.

[0055] 32. An injection device comprising the conjugate of any one of items 1 to 22 or the hydrogel of item 23 or 24.

[0056] 33. The injection device of item 32, wherein the device comprises a plurality of barrels or compartments and a mixer connected to the plurality of barrels or compartments 34. An injection device comprising a plurality of barrels or compartments and a mixer connected to the plurality of barrels or compartments.

[0057] 35. The device of item 33 or 34, comprising two barrels or compartments.

[0058] 36. The device of any one of items 33 to 35, wherein the mixer is a static mixer.

[0059] 37. The device of any one of items 33 to 36, wherein a first barrel or compartment comprises 2-methacryloyloxyethyl phosphorylcholine (MPC), copolymers comprising MPC, and / or collagen or a collagen-like peptide, and a second barrel or compartment comprises a cross-linking agent.

[0060] 38. The device of item 37, wherein the second barrel or compartment further comprises polymerizable monomers or a polymer comprising one or more functional groups.

[0061] 39. The device of any one of items 33 to 38, wherein the barrels or compartments have a maximum capacity of 500 pL each.

[0062] 40. The device of any one of items 33 to 39, wherein the mixer has a dead volume of less than 40 pL.

[0063] 41 . The device of any one of items 33 to 40, which comprises a third barrel or compartment.

[0064] 42. A system comprising the injection device of any one of items 33 to 41 , and a heater adapted to heat one or more of the barrels or compartments of the device.

[0065] 43. A kit comprising:

[0066] (I)

[0067] (a) 2-methacryloyloxyethyl phosphorylcholine (MPC), and collagen or a collagen-like peptide (CLP);

[0068] (b) (i) monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) and polymerizable monomers comprising one or more functional groups; and (ii) collagen or a CLP; or

[0069] (c)

[0070] (i) a copolymer comprising a core comprising a multi-arm polymer; monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) attached to one or more arms of the multi-arm polymer; optionally polymerizable monomers or polymers comprising one or more functional groups and

[0071] (ii) collagen or a CLP attached to one or more arms of the multi-arm polymer and / or to the MPC; and

[0072] (II) optionally a cross-linking agent.

[0073] 44. The kit of item 43, wherein the cross-linking agent is 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4- methylmorpholinium chloride (DMTMM).

[0074] 45. The kit of item 43 or 44, further comprising additional proteins, peptides, cells, nanoparticles and / or drugs.

[0075] 46. The kit of item 45, further comprising an inhibitor of angiogenesis. 47. The kit of any one of items 43 to 46, wherein the kit further comprises the device of any one of items 32 to 41 or the system of item 42.

[0076] 48. Use of the conjugate of any one of items 1 to 22 or the hydrogel of item 23 or 24 for treating a wound or tissue injury.

[0077] 49. The use of item 25, wherein the wound or tissue injury is corneal wound or injury.

[0078] 50. The use of item 25 or 26, wherein the wound or injury is burn or perforation.

[0079] 51 . The use of any one of item 25 to 27, wherein the conjugate or hydrogel is for administration by injection into or in the vicinity of said wound or injury.

[0080] 52. The use of any one of item 25 to 27, wherein the conjugate or hydrogel is for administration using the device of of any one of items 32 to 41 .

[0081] 53. A method for treating a wound or tissue injury in a subject comprising contacting the wound or tissue with the conjugate of any one of items 1 to 22 or the hydrogel of item 23 or 24.

[0082] 54. The method of item 53, wherein the wound or tissue injury is corneal wound or injury.

[0083] 55. The method of item 53 or 54, wherein the wound or injury is burn or perforation.

[0084] 56. The method of any one of items 53 to 55, wherein the conjugate or hydrogel is injected into or in the vicinity of said wound or injury.

[0085] 57. The method of any one of items 53 to 56, wherein the conjugate or hydrogel is injected using the device of of any one of items 32 to 41 .

[0086] Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0087] BRIEF DESCRIPTION OF DRAWINGS

[0088] In the appended drawings:

[0089] FIGs. 1A-C depict the preparation of pendant carboxylated enriched phosphorylcholine I (PEPC I) (FIG. 1A), PEPC II (FIG. 1 B) and CLP-PEPCs hydrogel (FIG. 1 C).

[0090] FIGs. 2A-D depict the preparation of CLP-MPC copolymer (FIG. 2A), CLP-MPC bio copolymer (FIG. 2B), poly(acrylic acid) (PAA) (FIG. 2C) and the CLP-MPC bio copolymer hydrogel (FIG. 2D).

[0091] FIG. 3 depicts the preparation of a multi-arm or star MPC polymer (with 4 arms).

[0092] FIG. 4 depicts the precursors of click chemistry in 8 arm PEG-maleimide and star MPC- maleimide (dots) can be crosslinked with sulfur (S)-containing cysteine (represented by the dots at both ends of CLP) via click chemistry.

[0093] FIG. 5 depicts a dual-barreled syringe-like device with a static mixer.

[0094] FIG. 6A depicts a 3D-designed and printed head with joined static mixer. The design of the microfluidic delivery system can be altered based on the viscosity of the liquid being delivered. FIG. 6B: Assembled syringe delivery system with syringe barrels and biomaterials as shown in FIG. 4. However, the void volume was decreased from 150 pL to about 36 pL.

[0095] FIGs. 6C-H show handling and injection of the hydrogel (LiQD Cornea) into the 6 mm wound bed in a rabbit for an anterior lamellar keratoplasty filling and sealing.

[0096] FIG. 7 depicts the detailed view of a 2-barreled design of a static mixer according to the present disclosure with a dead volume equal to 36 pL. The mixing channel is composed of some narrowing and expansions which induce micro-vortexes along the path of the flow, enhancing the mixing of the two components. Also, the simplistic design of the channel would allow the printability of the construct with smaller feature size.

[0097] FIG. 8 depicts the detailed view of a 3-barreled design of a static mixer according to the present disclosure with a dead volume equal to 46 pL. The mixing channel is composed of some narrowing and expansions which induce micro-vortexes along the path of the flow, enhancing the mixing of the two components. Also, the simplistic design of the channel would allow the printability of the construct with smaller feature size. The third barrel could be used for the encapsulation of cells and / or nanoparticles, for example.

[0098] FIG. 9 depicts proposed designs for a powder-based formulation with the ability to separate the dry and wet components by 1) using a thin and impermeable membrane and 2) a bypass, two-plunger syringe system.

[0099] FIG. 10 shows a prototype syringe heater used to differentially thaw the frozen LiQD Cornea contents for application to the cornea.

[0100] FIG. 11 A shows the synthesis method of PEPC and FIG. 11 B shows the preparation of the hydrogel and the method of application.

[0101] FIGs. 12A-I show the characterization of PEPC. FIG. 12A: Fourier Transform Infrared (FTIR) spectrum of PEPC. FIG. 12B:1H-NMR of PEPC, FIG. 12C:31P-NMR of PEPC, FIG. 12D: differential scanning calorimetry (DSC) curve of PEPC, FIG. 12E: Transparency of CLP-PEPC hydrogel, FIG. 12F: collagenase degradation of CLP-PEPC hydrogel, FIG. 12G: bursting pressure of CLP-PEPC hydrogel, FIG. 12H: gelation time of CLP-PEPC hydrogel (average is reported), FIG. 121: compressive modules of CLP-PEPC hydrogel after one hour (average is reported).

[0102] FIGs. 13A-D show images of alkali burn-corneal perforations in mice filled with 36aa CLP- PEPC I hydrogel. Fluorescein exclusion images showing slower healing in 1 / 3 mice with problem of over-filling of the cornea. FIGs. 13A-B: CLP-PEPCI hydrogel, FIGs. 13C-D: heathy eye control.

[0103] FIG. 14 depicts images of mice corneas 2 weeks post-injection of 27aa CLP-PEPC l / ll hydrogel encapsulating lipid nanoparticles releasing mRNA encoding GFP (+ LNP-GFP mRNA), cDNA encoding GFP (+ LNP-GFP cDNA) or empty nanoparticles (+ LNP only).

[0104] FIGs. 15A-F show the characterization of CLP-MPC. FIG. 15A:1H-NMR of CLP-BIS. FIG. 15B:1H-NMR of CLP-MPC. FIG. 15C:31P-NMR of CLP-BIS. FIG. 15D:31P-NMR of CLP-MPC. FIG. 15E:1H-NMR of PAA. FIG. 15F: FTIR of CLP-MPC, CLP-BIS, and PAA. FIG. 16A shows the application of a thin layer of self gelling CLP-MPC containing rhodamine-labelled lipid nanoparticles containing siRNA against HSV-1 ICPO. The labeling indicates adherence of the hydrogel with its nanoparticle-siRNA cargo.

[0105] FIG. 16B is a graph showing the transparency of CLP-MPC hydrogel.

[0106] FIG. 16C is a graph showing the results of a collagenase test for CLP-MPC hydrogel.

[0107] FIGs. 17A-J show the temperature of the CLP-MPC hydrogel at various times. FIG. 17A: frozen form, FIG. 17B: 2 mins after heating by the designed heater, FIG. 17C: 4 mins after heating by the designed heater, FIG. 17D: 5 mins after heating by the designed heater, FIG. 17E: 6 mins after heating by the designed heater. FIG. 17F: right after it was removed from the heater. FIG. 17F: the hydrogel after it was applied by the delivery system, FIG. 17H: 2 mins after application, FIG. 171: 8 mins after application, FIG. 171: 20 mins after application. The triangles show the maximum and minimum temperature in each image.

[0108] FIG. 18 shows the corneal healing in both unvascularized and neovascularized corneas with bleeding following filling of corneal perforations with CLP-MPC.

[0109] FIG. 19A shows the epithelial coverage over time of all 8 pigs. Solid black shapes indicate 2 re-grafted animals. All pigs showed 100% wound coverage.by 2 months post-operation. Pig 1 , first animal operated showed slower wound coverage as this animal had an “underfilled” wound bed. Subsequent animals received sufficient CLP-MPC to overfill to obtain the convex curvature of the cornea.

[0110] FIG. 19B shows the intraocular pressure (IOP) in the treated eyes (OD) and untreated contralateral eyes (OS) as measured using tonometry. The IOP in the treated eyes vary from pig to pig but overall are fairly equivalent over the 6 months and within the normal range for pigs around 15 mm Hg.

[0111] FIG. 19C shows the corneal thickness in the operated eye (OD) and control unoperated contralateral eyes (OS). The operated and regenerated corneas at 39 weeks post-operation are slightly thinner than those of the unoperated eyes but nevertheless have regained thickness by week 26 and have stabilized.

[0112] FIG. 19D shows the tear production as measured by Schirmer's tear test (mm). Measurements deviate due mainly to the state of anaesthesia in pigs, with lower amounts produced in both eyes when the animals are deeply anaesthetized when sampled. Nevertheless, tear production is fairly equivalent in both eyes.

[0113] FIG. 20 shows control untreated cornea and CLP-MPC filled cornea after wounding with a light alkali burn and surgical perforation at 6-months post-operation. The epithelium has fully regenerated. Parallel bundles of corneal nerves are seen in both control and regenerated neo- corneal tissue, as indicated by the arrows. Larger stromal nerves (arrowed) are also seen in both the control and regenerated stromal tissue.

[0114] SUBSTITUTE SHEET (RULE 26) FIGs. 21A-D show the characterization of CLP-starMPC. FIG. 21A:1H-NMR of PEG- CTCTP. FIG. 20B:1H-NMR of PEG-CTCTP-MPC-AA. FIG. 21C:31P-NMR of PEG-CTCTP. FIG. 21D:31P-NMR of PEG-CTCTP-MPC-AA.

[0115] FIG. 22A shows an image of a rabbit cornea with 6 mm diameter, 250 m deep anterior lamellar keratectomy wound bed, filled with CLP-starMPC based hydrogel (LiQD Cornea), at 3 weeks post-operation. FIG. 22B: Corresponding OCT images showing curved surface. FIG. 22C: H&E image through the cornea showing the implant (*) and in-growing epithelium (arrow).

[0116] FIGs. 23A-B show the characterization of starMPC with chemically cross-linked core (polyMPC). FIG. 23A:1H-NMR of polyMPC. FIG. 23B:31P-NMR of polyMPC.

[0117] DETAILED DISCLOSURE

[0118] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0119] The terms "comprising", "having", "including", and "containing" are to be construed as open- ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0120] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0121] The use of any and all examples, or exemplary language (“e.g.”, "such as") provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.

[0122] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.

[0123] Herein, the term "about" has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).

[0124] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

[0125] Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives. Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0126] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1- 4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0127] The present inventors have developed hydrogels based on 2-(methacryloyloxy) ethyl 2- (trimethylammonio)ethyl phosphate (MPC) and CLP that may be used as fillers or sealants for various applications. These hydrogels can be applied easily by a one-step method, in contrast to existing hydrogels needed more than one step to be applied, e.g., requiring photoactivation or fibrin glue. Photoactivated hydrogels ignore the fact that patients with severe pathologies have photophobia and therefore a lot of pain when bright light is shone into their eyes. They will need significant anaesthesia to block the pain. The hydrogels described herein do not require light for polymerization. Most other hydrogels that contain biological content use gelatin or methacrylated gelatin. Gelatin is derived from bovine, porcine, or fish skin by denaturation of collagen. The final product typically contains a mixture of single fibrils, and portions of triple or double helices of unknown and varying lengths. Also, another important issue is that photoactivation or any other in situ crosslinking methods, generally leave a lot of unsaturated and unreacted bonds in the eye, which may be toxic.

[0128] In as aspect, the present disclosure provides a conjugate or hydrogel comprising:

[0129] (a) 2-methacryloyloxyethyl phosphorylcholine (MPC) and collagen or a collagen-like peptide (CLP) linked directly or indirectly (through a linker), or crosslinked, to the MPC;

[0130] (b) (i) a copolymer comprising monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) and polymerizable monomers comprising one or more functional groups; and (ii) collagen or a CLP crosslinked to the copolymer; or

[0131] (c)

[0132] (i) a copolymer comprising a core comprising a multi-arm polymer; monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) attached or cross-linked to one or more arms of the multi-arm polymer; optionally polymerizable monomers or polymers comprising one or more functional groups and collagen or a CLP attached or cross-linked to one or more arms of the multi-arm polymer and / or to the MPC.

[0133] In another aspect, the present disclosure provides a kit comprising:

[0134] (I)

[0135] (a) 2-methacryloyloxyethyl phosphorylcholine (MPC), and collagen or a collagen-like peptide (CLP);

[0136] (b) (i) monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) and polymerizable monomers comprising one or more functional groups; and (ii) collagen or a CLP; or

[0137] (c) (i) a copolymer comprising a core comprising a multi-arm polymer; monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) attached to one or more arms of the multi-arm polymer; optionally polymerizable monomers or polymers comprising one or more functional groups and collagen or a CLP attached to one or more arms of the multi-arm polymer and / or to the MPC; and

[0138] (II) optionally a cross-linking agent.

[0139] In another aspect, the present disclosure provides a method for preparing a conjugate or hydrogel as described herein, the method comprising mixing (i) the ingredients defined in (a), (b) or (c) above; and (ii) optionally a cross-linking agent.

[0140] The term "hydrogel" as used herein, refers to a crosslinked network of hydrophilic biopolymers. The term "cross-linked" refers to a structural configuration where individual monomer / polymer chains, peptides, proteins, are interconnected through covalent bonds. These crosslinks can occur between different monomer / polymer chains, peptides or proteins, or within the same monomer / polymer chain, peptide or protein, creating a three-dimensional network.

[0141] The term "collagen-like peptide" or "CLP" (also known as collagen-mimetic peptide [CMP] or collagen-related peptide [CRP]) refers to any peptide which is a structural or functional equivalent of collagen and that contains three peptide chains, each having a repeating structure whose basic unit is -(Xaa-Yaa-Gly)- to self-assemble in a solvent and form a helical structure. Each of Xaa and Yaa is independently selected from a proline (Pro or P) residue, a hydroxyproline (Hyp or O) residue, an arginine (Arg or R) residue, a lysine (Lys or K) residue, a valine (Vai or V) residue, a leucine (Leu or L) residue, an isoleucine (He or I) residue, a serine (Ser or S) residue, a threonine (Thr or T) residue, an alanine (Ala or A) residue, a glycine (Gly or G) residue, a phenylalanine (Phe or F) residue, a methionine (Met or M) residue, a glutamate (Glu or E) residue, an aspartate (Asp or D) residue, an asparagine (Asn or N) residue, a glutamine (Gin or Q) residue, a histidine (His or H) residue, a tryptophan (T rp or W) residue or a tyrosine (Tyr or Y) residue. In an embodiment, the basic units, which may be the same or different, are repeated at least 7 times. In an embodiment, the basic units, which may be the same or different, are repeated at least 8 times. In an embodiment, the basic units, which may be the same or different, are repeated at least 10 times. In an embodiment, the basic units, which may be the same or different, are repeated at least 12 times.

[0142] Examples of CLP include peptides of the sequences Ac-(Gly-Pro-Hyp)3-Gly-X-Hyp-(Gly- Pro-Hyp)4-Gly-Gly-CONH2(SEQ ID NO:1), Ac-(Gly-Pro-Hyp)3-Gly-Pro-Y-(Gly-Pro-Hyp)4-Gly-Gly- CONH2(SEQ ID NO:2), Cys-Gly-(Pro-Lys-Gly)4(Pro-Hyp-Gly)4(Asp-Hyp-Gly)4(SEQ ID NO:3), Cys-Gly-(Pro-Lys-Gly)4(Pro-Hyp-Gly)4(Glu-Hyp-Gly)4(SEQ ID NO:4), Cys-Gly-(Pro-Lys- Gly)4(Pro-Hyp-Gly)4(Asp-Hyp-Gly)4-Gly-Gly-(Pro-Lys-Gly)4(Pro-Hyp-Gly)4(Asp-Hyp-Gly)4(SEQ ID NO:5), Cys-Gly-Gly-Gly-Gly-(Pro-Lys-Gly)4(Pro-Hyp-Gly)4(Asp-Hyp-Gly)4(SEQ ID NO:6), Cys-Gly-(Pro-Lys-Gly)4(Pro-Hyp-Gly)4(Asp-Hyp-Gly)4-Arg-Gly-Asp-Ser-Pro-Gly (SEQ ID NO:7); Cys-Gly-(Pro-Lys-Gly)4(Pro-Hyp-Gly)4(Asp-Hyp-Gly)4-lle-Lys-Val-Ala-Val-Gly (SEQ ID NO:8), ((GPO)3GXY(GPO)4GG) (SEQ ID NO:9), or (PRG)4(POG)4(EOG)4(SEQ ID NO: 10).

[0143] In various embodiment, the CLP further comprises a thiol containing amino acid, preferably cysteine, at its N- and / or C-terminal end.

[0144] In an embodiment, the CLP comprises or consists of the following sequence:

[0145] (PKG)3or4(POG)3or4(DOG)2or3(DOX), wherein X is G or C. In an embodiment, the CLP comprises or consists of the following sequence: (PKG)3 or 4(POG)3 or 4(DOG)2 or 3(DOG), for example (PKG)4(POG)4(DOG)3(DOG) (SEQ ID NO:11). In another embodiment, the CLP comprises or consists of the following sequence: (PKG)3 or 4(POG)3 or 4(DOG)2 or 3(DOC), for example (PKG)4(POG)4(DOG)3(DOC) (SEQ ID NO: 12).

[0146] In an embodiment, the CLP has a length of less than 36 amino acids, for example a length of 33, 30, or 27 amino acids. In a further embodiment, the CLP has a length of 27 amino acids. It has been observed that the hydrogels formed from shorter sequences (27aa) CLP were generally superior to those with longer sequences (36aa), in terms of controlling the gelation time in CLP- MPC hydrogels.

[0147] In a further embodiment, the CLP comprises or consists of the following sequence: (PKG)3(POG)3(DOG)2(DOX), wherein X is G or C (SEQ ID NO: 13).

[0148] In an embodiment, the CLP comprises or consists of the following sequence: (PKG)3(POG)3(DOG)2(DOG) (SEQ ID NO:14).

[0149] In an embodiment, the CLP comprises or consists of the following sequence: (PKG)3(POG)3(DOG)2(DOC) (SEQ ID NO: 15). The replacement of the C-terminal Glycine (G) of the 27aa CLP is allows for bio-orthogonal chemical reaction for developing new biosynthetic polymers. A terminal cysteine allows for additional crosslinking options in addition to the K groups.

[0150] Other CLPs that may be used in the conjugate or hydrogel described herein include medium length CLP such as VB which is based upon a modified subdomain of the collagen-like Sc12 protein from Streptococcus pyogenes (Merrett K, Wan F, Lee CJ, Harden JL. Enhanced Collagen- like Protein for Facile Biomaterial Fabrication. ACS Biomater Sci Eng. 2021 Apr 12;7(4): 1414- 1427. doi: 10.1021 / acsbiomaterials.1c00069) as well as other prokaryotic collagen-like proteins (see, e.g., Jonathan Picker, Ziyang Lan, Srishtee Arora, Mykel Green, Mariah Hahn, Elizabeth Cosgriff-Hernandez, and Magnus Hook. Front Bioeng Biotechnol. 2022 Mar 17:10:840939. doi: 10.3389 / fbioe.2022.840939. eCollection 2022).

[0151] In other embodiments, a second cysteine (C) may be added to the N-terminus to allow a Michael addition between thiol- and maleimide-functionalized molecules to allow hydrogel crosslinking without an extra crosslinker. In an embodiment, the CLP comprises or consists of the following sequence: GCG-(PKG)3(POG)3(DOG)2DOC (SEQ ID NO: 16) or CGG- (PKG)3(POG)3(DOG)2DOC (SEQ ID NO:17), where O is hydroxyproline (Hyp). In an embodiment, one or both glycine residues (G) can be substituted with S to increase solubility to give a CLP that comprises or consists of the following sequence: SCS-(PKG)3(POG)3(DOG)2DOC (SEQ ID NO: 18) or SCG-(PKG)3(POG)3(DOG)2DOC (SEQ ID NO: 19). Other CLPs that may be used according to the present disclosure comprises or consists of the following sequence: SCS(PKG)3(POG)5DOC (SEQ ID NO:20), SCS(PKG)3(POG)5-SCS (SEQ ID NO:21), or GCG(PKG)3(POG)5-GCG (SEQ ID NO:22).

[0152] Polymerizable monomers comprising one or more functional groups such as carboxylic acid groups, hydroxyl groups, and / or amine groups are well known in the art, and include, for example, acrylic acid (AA), 2-hydroxyethyl acrylate (HEA), and analogs thereof. These monomers have the ability to form copolymers with MPC and comprise functional groups suitable to react with groups present on peptides and proteins, such as carboxylic acid groups (e.g., present on the side chain of acidic amino acids such as glutamic acid and aspartic acid) and / or amine groups (e.g., present on the side chain of certain amino acids such as lysine and arginine), as illustrated in FIGs. 1A- 1C.

[0153] In an embodiment, the MPC is copolymerized with acrylic acid. As shown in FIG. 1A, this copolymer (PEPC I) contains two different pendent groups, carboxylic acids and phosphorylcholine. The copolymer can be crosslinked with the CLP in hydrogel or collagen inside the eye by chemical conjugation method to activate the carboxylic acids (e.g., using EDC or DMTMM) and then letting amine groups in peptide or protein conjugate them.

[0154] In another embodiment, the MPC is copolymerized with 2-hydroxyethyl acrylate. As shown in FIG. 1B, this copolymer (PEPC II) contains two different pendent groups, hydroxyl and phosphoryl choline (PC). It allows better flexibility of incorporating different desired amounts of MPC. PEPC II can be crosslinked with the PEPC I and CLP to form an in situ gelling construct within a cornea using a chemical crosslinker (such as a carbodiimide like EDC, or a triazine such as DMTMM). Both EDC or DMTMM will activate the carboxylic acids that in turn become conjugated to the hydroxyl groups in PEPC II.

[0155] PEPCI and ll-CLP may be used to encapsulate different types of solid lipid nanoparticles (SLN) nanoparticles for the delivery of nucleic acids, as exemplified below.

[0156] In an embodiment, the conjugate or hydrogel as described herein comprises: a first copolymer of MPC and acrylic acid; a second co-polymer of MPC and 2-hydroxyethyl acrylate; and collagen or CLP, wherein the first co-polymer, second co-polymer and collagen or CLP are crosslinked.

[0157] In an embodiment, the MPC is conjugated directly to the collagen or CLP without the need for any secondary copolymer as depicted in FIG. 2A.

[0158] In an embodiment, the MPC and the collagen or CLP are linked indirectly through a linker. The term “linker” as used herein means a chemical structure connecting the CLP or collagen to the MPC. The linker can be connected to the CLP or collagen at different functional groups on the CLP or collagen. For example, the linker can be connected to the CLP or collagen at the primary amines (amines (-NH2): this group exists at the N-terminus of each polypeptide chain (called the alpha-amine) and in the side chain of lysine (Lys, K) residues (called the epsilon- amine). For example, the linker can be connected to the CLP or collagen at the carboxyl groups (-COOH): this group exists at the C-terminus of each polypeptide chain and in the side chains of aspartic acid (Asp, D) and glutamic acid (Glu, E). For example, the linker can be connected to the CLP or collagen at the sulfhydryl groups (-SH): this group exists in the side chain of cysteine (Cys, C). Often, as part of a protein's secondary or tertiary structure, cysteines are joined together between their side chains via disulfide bonds (-S-S-). These must be reduced to sulfhydryls to make them available for crosslinking by most types of reactive groups. For example, the linker can be connected to the CLP or collagen at the carbonyls (-CHO): ketone or aldehyde groups can be created in glycoproteins by oxidizing the polysaccharide post-translational modifications (glycosylation) with sodium meta-periodate. An example of a linker is N,I\T- methylenebisacrylamide (BIS). As depicted in FIG. 2B, BIS may be attached to the CLP, which then may be used to polymerize MPC to CLP. The advantage of using a linker is that the amount of MPC can be controlled by changing the feed.

[0159] In an embodiment, the conjugate or hydrogel comprises a multi-arm or star polymeric core (multi-arm or star MPC polymers). “Multi-arm” in reference to the geometry or overall structure of a polymer refers to polymer having 3 or more polymer-containing “arms”. Thus, a multi-armed polymer may possess 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymer arms, 7 polymer arms, 8 polymer arms, or more, depending upon its configuration and core structure. Multi-arm or star MPC polymers can be produced by using a template such a small PEG or using an in situ chemically crosslinked core.

[0160] As shown in FIG. 3A, a multi-arm chemically cross-linked core may be obtained using a cross-linking agent such as BIS or pentaerythritol tetraacrylate during polymerization process to shape the core. More specifically, CLP-BIS and polyMPC are prepared separately as described herein, and then these two different types of arms can form a star MPC by the addition of a crosslinking agent such as BIS or pentaerythritol tetraacrylate during the final polymerization process. MPC and collagen or CLP are conjugated / attached to the arms of the multi-arm or star polymeric core. The multi-arm or star polymers comprising MPC and collagen or CLP may be cross-linked using a suitable cross-linker (e.g., DMTMM) in the presence of a polymer comprising functional groups, such as PAA to obtain the conjugate or hydrogel.

[0161] In an embodiment, the multi-arm or star polymeric core comprises a multi-arm PEG. Multiarm PEGs comprise multiple PEG chains branching out from a central core. The central core can be a small molecule or a polymer structure that serves as the anchor point for the PEG arms. Common cores include molecules like pentaerythritol (four-arm PEG), hexaglycerol or tripentaerythritol (eight-arm PEG) or other multi-functional cores that can accommodate more arms. The arms are typically linear PEG chains, generally of equal length, extending from the central core. Each arm can be functionalized with various end groups, such as hydroxyl (-OH), amine (-NH2), carboxyl (-COOH), or methoxy (-OCH3) to allow the grafting / crosslinking of molecules.

[0162] For example, 3, 4, 5, 6, and 8 arms PEGs used as the templating core will result in a corresponding 3, 4, 5, 6, or 8 arms MPC polymer as shown in FIG. 3B. Furthermore, since PC pendent groups do not contain a suitable functional group to be crosslinked with collagen or a CLP, monomers comprising functional groups such as HEA or AA are added to the arms to make star MPC copolymers crosslinkable. The multi-arm MPC with PEG core with functional group (e.g., AA or HEA) may then be conjugated to collagen or CLP using a suitable crosslinker (e.g., DMTMM) to obtain the conjugate or hydrogel.

[0163] In an embodiment, the PEG in the multi-arm PEG has a molecular weight between 1 ,000 and 10,000 kDa, preferably between 1 ,000 and 5,000 kDa, for example PEG 2000, PEG 3000, PEG 4000, or PEG 5000.

[0164] In an embodiment, the arms of the multi-arm or star polymeric core and / or the MPC further comprises a functional group or moiety that reacts with a thiol group (e.g., present in cysteine residues) to allow the crosslinking of collagen or CLP comprising cysteine residues (e.g., at the N and / or C-terminal ends), as described above. Example a functional group or moiety that reacts with a thiol group is maleimide. Advantageously, this crosslinking may be performed in the absence of chemical crosslinking activator such as DMTMM or EDC. In an embodiment, the conjugate described herein is crosslinked using any suitable protein / peptide crosslinking agent or crosslinking activator. The reactive function of the crosslinking agent / activator can be an NHS ester compound (that reacts with primary amine), a maleimide compound (that reacts with sulfhydryl containing molecule), a hydrazide compound (that reacts with aldehyde containing molecule), a carbodiimide based compound such as 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDC) (that reacts with carboxylate containing molecule) or a triazine derivative that reacts with carboxylate containing molecules. Agents to induce protein crosslinking are well known in the art and include, for example, glutaraldehyde, DSG, disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), bis(succinimidyl) penta(ethylene glycol (BS(PEG)5), TSAT, DSP, DTSSP, DST, BSOCOES, EGS, sulfo-EGS, DMA, DMP, DMS, DTBP, DFDNB, SIA, SMAP, SIAB, sulfo-SIAB, AMAS, BMPS, GMBS, sulfo- GMBS, MBS, sulfo-MBS, SMCC, sulfo-SMCC, SMBP, sulfo-SMBP, SMPH, LC-SMCC, sulfo- KMUS, SPDP, LC-SPDP, sulfo-LC-SPDP, SMPT, EDC / NHS, / V-cyclohexyl- / V'-(2- morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC) or 4-(4,6-dimethoxy-1 ,3,5- triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). In an embodiment, the crosslinking agent is DMTMM.

[0165] The hydrogel may further comprise other components such as proteins (e.g., ECM proteins, growth factors, inhibitors of angiogenesis such as COCO / DAND5, etc.), peptides (e.g., cell adhesion motifs such as RGD motifs, YIGSR motifs), sugars (e.g., polysaccharides, glycosaminoglycans), drugs / therapeutic agents, cells (e.g., corneal stem cells, corneal epithelial cells), adhesives, nanoparticles, etc. In an embodiment, the hydrogel is free of peptide with cell adhesion motifs such as RGD motifs. In another embodiment, the hydrogel is free of adhesive. Examples of therapeutic agents that may be included into the hydrogel include an antibacterial agent, an antifungal agent, an antiviral agent, an anti-acanthamoebal agent, an anti-inflammatory agent, an immunosuppressive agent, an anti-glaucoma agent, an anti-angiogenesis agent, an anti-VEGF agent, a growth factor, or any combination thereof.

[0166] Certain exemplary embodiments of the present disclosure comprise a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation and is compatible with administration to a subject, for example a human. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits. Examples of pharmaceutically acceptable carriers include, but are not limited to, a solvent or dispersing medium containing, for example, water, pH buffered solutions (e.g., phosphate buffered saline [PBS], HEPES, TES, MOPS, etc.), isotonic saline, Ringer's solution, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), alginic acid, ethyl alcohol, and suitable mixtures thereof. In some embodiments, the pharmaceutically acceptable carrier can be a pH buffered solution (e.g., PBS) or water.

[0167] In another aspect, the present disclosure provides a device, for example an injection device such as a syringe or microfluidic system, comprising a plurality of barrels or compartments and a mixer, e.g., a passive (or static) mixer or an active mixer connected to the plurality of barrels or compartments. The static mixer is adapted to be connected to the plurality of barrels or compartments of the injection device (e.g., dual-barrel or triple-barrel syringe) to facilitate the thorough mixing of the different materials that are stored separately in the barrels of the device. This type of mixer ensures that the components are evenly blended upon dispensing, which permits to achieve the intended chemical or physical properties of the mixed product (hydrogel). The static mixer includes two or more inlet ports that align with the outlets of the barrels or compartments of the device (e.g., syringe). These ports are designed to securely attach to the device (e.g., syringe), ensuring a leak-proof connection. The static mixer also includes mixing elements, typically a series of stationary, helical or other geometrically configured elements that create turbulence and divide the flow of the materials into smaller streams. These elements are strategically arranged to continuously split and recombine the streams, promoting thorough mixing as the materials pass through. The mixed material exits through a single outlet nozzle, which can be designed to accommodate different application needs, such as dispensing directly onto a wound. The outer casing or housing of the static mixer is typically made of plastic or other suitable material that ensures chemical compatibility and durability. The housing is often transparent, allowing visual confirmation of the mixing process. The mixer is equipped with an attachment mechanism, such as a threaded connection or bayonet fitting, to facilitate easy and secure attachment to the barrels or compartments of the device (e.g., syringe). In operation, when the plunger of the multi-barrel syringe is depressed, the two or more separate components are pushed simultaneously into the inlet ports of the static mixer. As the components pass through the internal mixing elements, they are subjected to multiple divisions and recombinations, resulting in a homogeneously mixed product (hydrogel) by the time they reach the outlet nozzle. The mixed material is then ready for immediate application.

[0168] In an embodiment, the device comprises two barrels or compartments. In an embodiment, the device comprises three barrels or compartments. In an embodiment, the barrels or compartments of the device comprise the different components of the hydrogel defined herein. In an embodiment, a first channel or compartment comprises one or more of the monomers defined herein, e.g., monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC), multi-arm polymer, monomers comprising one or more functional groups (or polymers made from the monomers), and / or collagen or CLP. In an embodiment, a second channel or compartment comprises a crosslinking agent. In an embodiment, the barrels or compartments may be adapted to have a maximum capacity of 500 pL, for example a maximum capacity of 400 pL or 300 pL. In an embodiment, the second channel may also comprise one of the monomers defined herein, and / or collagen or CLP.

[0169] To produce a one-step delivery of the hydrogel described herein, copolymers with suitable functional groups which can be premixed were produced as shown in FIG. 5 and its enhanced design (FIGs. 6A-H) to enable more efficient mixing, easier delivery due to the slimer design and significant reduction of void volume from about 150 pL to about 36 pL. This may be achieved by the design depicted in FIG. 7. The static mixer allows for the components which are separated to prevent premature chemical reactions during storage, to be mixed immediately prior to application into the patient wound bed. Drawings of the enhanced printed head and static mixer are shown in FIG. 7. A static mixer for a 3-barrel / compartment delivery device is depicted at FIG. 8. The third barrel may be used to inject other ingredients in combination with the hydrogel such as drugs, bioactive molecules, growth factors, cells, etc. It may be preferable in certain embodiments that one of the components in the delivery device may in solid form (e.g., powder). For example, DMTMM has been shown to be unstable in solution, and thus should be preferably frozen before use. In order to be able to store the delivery device described herein in the refrigerator (at 4-8°C), it may be desirable to keep DMTMM in powder form.

[0170] The device with the various components may be frozen for storage and future use. For thawing prior to use, the device may be contacted with a heater. Preferably, the temperature may be adjusted independently in each of the barrel or compartment. For example, crosslinkers are often heat-sensitive and thus should not be heated at high temperatures. In contrast, the polymers need to be at a higher temperature (e.g., up to 65°C) to thaw. A prototype of a syringe heater is depicted in FIG. 10. In this prototype, the overall temperature may be set to between 35-60°C (e.g. , 45-47°C), but the configuration of a heating block on one side while open on the other allows the materials within both syringe barrels to thaw at the proper temperatures. However, any other configuration or system allowing the control of the temperature in the different barrels or compartments may be used. In another aspect, the present disclosure provides a system comprising the device and heater described herein.

[0171] The mixing of the contents of the first and second channels or compartments in the static mixer (e.g., by pushing on the plunger or piston of the syringe) induces the crosslinking of the collagen or CLP with the polymers to form the hydrogel, which may be directly injected to a patient in need of treatment.

[0172] In another aspect, the present disclosure provides a method for treating a soft tissue injury or wound in a subject comprising injecting the hydrogel described herein into the soft tissue of the subject, in the injury or wound, or in the vicinity of the injury or wound. The present disclosure also provides the use of the hydrogel described herein for treating a soft tissue injury or wound in a subject. The present disclosure also provides the use of the hydrogel described herein for the manufacture of a medicament for treating a soft tissue injury or wound. The present disclosure also provides the hydrogel described herein for use in treating a soft tissue injury or wound in a subject.

[0173] Generally, soft tissue includes all tissues of the body except bone. Examples of soft tissues include, but are not limited to, muscles, tendons, fibrous tissues, fat, blood vessels, nerves, and synovial tissues. Examples of injuries or wounds include punctures, incisions, excisions, lacerations, abrasions, necrotic wounds, or burns. In some embodiments, the hydrogel and methods of the disclosure are useful for enhancing the healing of wounds or injuries of the skin, cornea, heart, liver, cartilage, bones, vascular system, spleen, kidney, stomach, and intestine. In a further embodiment, the hydrogel and methods of the disclosure are useful for enhancing the healing of corneal wounds or injuries.

[0174] In another aspect, the present disclosure provides a method for treating an eye condition characterized by a corneal defect in a subject comprising injecting the hydrogel described herein into the eye of the subject. The present disclosure also provides the use of the hydrogel described herein for treating an eye condition characterized by a corneal defect in a subject. The present disclosure also provides the use of the hydrogel described herein for the manufacture of a medicament for treating an eye condition characterized by a corneal defect in a subject. The present disclosure also provides the hydrogel described herein for use in treating an eye condition characterized by a corneal defect in a subject.

[0175] The eye condition may be Fuchs' dystrophy, iridocorneal endothelial syndrome, keratoconus, lattice dystrophy, ocular herpes infections, trachoma, or physical damage to the eye (e.g., corneal burns, injury). The eye condition may be a corneal dysfunction such as corneal endothelial dysfunction, acute or chronic corneal endothelial cell (CEO) loss, bullous keratopathy (PBK), Fuchs’ endothelial dystrophy, corneal transplant failure, corneal transplant rejection, corneal inflammation, corneal edema, corneal degeneration, corneal melting, or corneal ectasia. The hydrogel disclosed herein may be used in combination with other therapeutic agents for the treatment of the eye condition and / or associated symptoms, such as antimicrobial agents (antibacterial, antiviral, antifungal, and antiprotozoal agents), antifibrotic agents, antiinflammatory agents (e.g., steroids and non-steroidal agents), anti-angiogenic agents, antithrombotic agents, etc. The other therapeutic agent(s) may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form. Coadministration in the context of the present disclosure refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time. For example, a first agent (the hydrogel disclosed herein) may be administered to a patient before, concomitantly, before and after, or after a second active agent is administered. The agents may in an embodiment be combined / formulated in a single composition and thus administered at the same time.

[0176] The term "subject" or "mammalian subject" refers to any mammalian subject for whom treatment or therapy is desired, particularly humans. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as non-human primates, dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is a human.

[0177] EXAMPLES

[0178] The present disclosure is illustrated in further details by the following non-limiting examples.

[0179] Example 1: Preparation of CLP-PEPC l / ll hydrogels

[0180] The aim of the work described in this example was to develop an injectable “liquid cornea” (LiQD Cornea) alternative to transplantation that can be applied easily and will promote both wound closure and functional regeneration of the damaged tissue.

[0181] For all the following experiment, the number of amino acids of CLP does not affect the gelation time so that all the CLPs (27 and 36) or (Vb) or (27C and 36C) can be used interchangeably.

[0182] Methods

[0183] Preparation ofPEPC I (FIG. 1A). Pendant carboxylated enriched phosphorylcholine (PEPC I) was prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization. In this method, 1.6 g of acrylic acid (AA), 400 mg of MPC, and 13 mg of CTCTP were dissolved in 15 mL of DW in a three-neck round bottomed flask. Then, a flow of nitrogen and a stopcock were used to evacuate all oxygen from the flask. After 15 mins, 0.1 mmol each of APS and SHS were dissolved in 0.5 mL of DW separately. SHS and then APS were added to the flask while it was still being purged by N2gas. The solution was stirred for 24 hrs. The resulting PEPC content was transferred into a dialysis tube with a 6.5 KD cut-off and dialyzed against DW for three days. The PEPC dialysate was then lyophilized at -15°C.

[0184] Preparation of PEPC II. PEPC II was prepared to allow increase of the proportion of MPC within the final hydrogels. PEPC II was prepared using a similar method as for PEPC I (FIG. 1 B). First, proper ratios of HEA:MPC (500:1500 to 250:1750 mg) were added to 30 mL of deionized water (DW) in a three neck flask; then, 0.03 mmol 3-((((1- carboxyethyl)thio)carbonothioyl)thio)propanoic acid (CTCTP) was added to the solution and nitrogen gas was purged into the flask for 30 mins to make the environment inert and suitable for the polymerization, Then, 0.1 mmol of each APS and SHS were dissolved in 500 pL separately, and added to the solution while it was being stirred at inert environment. The solution was stirred for 24 hrs at ambient temperature. After 24 hrs, the solution (often viscous) was transferred to a dialysis bag (6.5 kDa cut-off), and after three days of dialyzing, the final solution was lyophilized at -15°C.

[0185] Preparation of CLP-PEPC hydrogels using a thermomixer. Hydrogels were prepared using the combination of CLP, PEPC (AA), and EDC. Therefore, suitable amounts of CLP (between 10 to 30 mg) were dissolved in PBS (0.25 M), and a thermomixer was used to heat it up to 65°C until it was melted and dissolved completely. Then, PEPC (20 to 40 pL; 5 % W / W in 0.2 M PBS, pH 7 adjusted by NaOH) was added to the CLP solution at the same temperature. After these two compounds formed a clear solution, they were removed to cool down around 37°C, and then 20 pL of 0.25 M PBS containing (0.6 to 2 mg) of EDC or equal of amounts of DMTMM was added to the CLP / PEPC solution. After mixing, the hydrogel was ready to be injected to fill the perforation.

[0186] As shown in FIG. 11 , the hydrogel can be easily applied by a syringe or pipette, it is not thermosensitive (like collagen) or humid sensitive (like cyanoacrylate glue), and it does not need any curing or activation or second adhesive glue either which makes it easy to be applied and user friendly. It is believed that the carboxylic groups of PEPC can be conjugated to the lysine groups of both CLP and collagen in wound bed, thereby allowing to cover and seal the wound within few minutes (the gelation time is about 190 seconds, as described below).

[0187] NMR characterization of materials and hydrogels. Phosphorous nuclear magnetic resonance spectroscopy (31P-NMR) and proton nuclear magnetic resonance spectroscopy (1H- NMR) in D2O at 600 MHz (Bruker, Avance III HD).

[0188] Collagenase degradation assay. Following the initial dissolution of 1 mg of collagenase in 1 mL of PBS (1X), 240 pL of the resulting collagenase / PBS solution was once again dispersed in 20 mL of PBS to create a concentration of roughly 0.75 U / mL. After blotting off the surface water and soaking the samples in the prepared solution at 37°C, they were weighed to determine the rate of mass loss. The following equation was used to determine the residual weight: Residual mass is calculated as Residual mass =^- x 100, where Wi is the weight ofthe hydrogel at a specific time point and Wois the hydrogel's original weight.

[0189] Water content of hydrogels. The water content (Wwc) was determine using a simple (W-W 01 formulation as follows: Wwc= — x 100 where Wois wet weight and the W is the final weight that the hydrogel can reach after remaining in PBS overnight.

[0190] Light transmission of hydrogels. The hydrogels were placed into a cuvette filled with PBS (1X), and the absorption was measured in the range of 200 to 1000 nm by SpectraMax M2e series plate / cuvette spectrophotometer (Molecular Devices, San Jose, CA, USA). Then, the absorbance was converted to transmittance. Ex vivo model of corneal perforation and bursting pressure. Excised pig eyes were obtained from a local butcher shop. The corneoscleral buttons were dissected out and placed into artificial anterior chambers (Barron Artificial Anterior Chamber, Katena, NJ) for bursting pressure testing. A 5-mm trephine was used to partially dissect each cornea to about a 50% depth (200-300 pm), after which a 1 mm biopsy punch was used to make a central, full-thickness perforation. The liquid cornea formulation was applied and allowed to set.

[0191] After three hours, the bursting pressure was measured to confirm that the hydrogel inside the perforation had fully crosslinked. An artificial anterior chamber was attached to an intra-arterial blood pressure monitor (TruWave, Edwards Lifesciences) to a saline infusion bag using a pressure cuff at a rate of 1 mL / min in order to standardize the procedure for determining the bursting pressure. The maximum pressure that the hydrogel can withstand was determined by plotting the burst pressure (mmHg) with time and using the peak on the curve as a reference point. N=3 repeats.

[0192] In vivo testing in mice. With ethical permission, the right cornea of 12 mice were each given a light alkali burn sufficient to cause lymphatic and vascular changes, by placing a 0.25 N NaOH soaked 2 mm diameter filter paper on each cornea for 15 sec, and then flushing the eye. A surgical perforation was created and filled with the liquid cornea formulation. Animals were given Tobradex (tobramycin and dexamethasone solution) 3 times daily post-operatively for 1 week. The corneas were followed with fluorescein staining to determine the rate of wound closure using the Micron IV system (Phoenix-Micron, Inc, Bend, OR). Images of the ocular surface were also captured.

[0193] Results of / n vitro experiments

[0194] FTIR spectrum of PEPC copolymer is shown in FIG. 12A. As it can be seen the curve has the functional groups of AA and MPC were appeared at 1057 cm-1(C-O-C), 1159 cm-1(P-O), 1230 cm1(P=O), 1712 cm1(C=O), and 2944 cm1(CH2), and a broad at 3200-3700 cm1(- COOH). FIG. 12B presents1HNMR spectrum of PEPC, peaks at 1.8 and 2.28 can be assigned to the backbone of AA, and the sharp peak at 3.11 ppm is due to the methyl group of MPC which are attached to the nitrogen. The other characteristic peaks are marked in FIG. 12G. In the Phosphorous NMR (FIG. 12C) a single peak at 0.69 ppm can prove the presence of MPC polymer in PEPC copolymer. Given that information, the results of FTIR and NMR could prove the successful synthesis of PEPC. DSC curve of PEPC is shown in FIG. 12D. PEPC showed an endothermic peak around 90°C which can confirm the presence of MPC in copolymer. The second endothermic peak in PEPC can be assigned to the disintegration of the copolymer which occurs at around 213°C.

[0195] The Mn and Mw of PEPC was determined to be 377 (kg / mol) and 387 (kg / mol), respectively. Therefore, the polydispersity index (DP) was 1.026 which is satisfactory and proves the consistency of the synthesized copolymer. The wavelength that the transparency passes 85% (FIG. 12E) was 386 nm for CLP-PEPC hydrogel. Also, the percentage of water content of CLP-PEPC was 90.2 ± 2.5 %, which is in the 84 to 90% range that is an acceptable swelling and similar to the human cornea water content. This shows that the hydrogel can strike a balance to avoid eye dryness while the integration of it with the cornea does not increase the thickness of cornea to anything above 500 pm that could cause problems.

[0196] Collagenase test results are shown in FIG. 12F. The weight remained almost steady for one day so that the collagenase rate of CLP-PEPC hydrogel was 0.52 ± 0.08 mg / day. The results proved that the hydrogel biodegradability is in a suitable range which provide a great condition for cornea to regenerate (it needs three weeks) itself, which is the main goal, before being degraded completely, and excreted from the eye. The results confirmed that the hydrogel is stable even in a accelerate test with a high concentration of collagenase.

[0197] The results of ex vivo bursting pressure (FIG. 12G) proved that CLP-PEPC did not burst up to 46.7 ± 1 .5 mmHg. FIG. 12G presents the condition of hydrogel after applying on pig dissected cornea as well.

[0198] FIG. 12H shows the change in storage modules, loss modules, and tan delta by time, since loss modules represents the viscous portion of the viscoelastic, a loss modulus larger than the storage modulus, indicating a more liquid behaviour. Therefore, the cross point of curves of loss and storage modules can be considered as the gelation time; and as it is seen in FIG. 12H, the gelation time was about 190 seconds. Also, tan delta is the other parameter that can further prove that the gelation time is around 190 seconds (tan delta is 0 when the materials is completely solid but above 0 when it is liquid). The gelation time frame is great because it gives enough time to a surgeon to apply the hydrogel carefully, the hydrogel is not moisture-sensitive or thermo-sensitive, which is one its great advantage relative to cyanoacrylate glue and collagen or fibrin glue. FIG. 121 shows the gradual increase of storage modulus, and decrease of tan delta after the gel was formed, according to the figure, in the beginning and up to 1500 sec, in each 500 seconds, their was an increase of 500 Pas, but after 1500 sec the rate became almost steady and plateaued. There was no significant increase in storage modulus up 3600 sec (one hour). The high rate of gelation in the beginning is convenient for the patients as they can received the treatment in the emergency room and leave in about 20 to 30 mins.

[0199] Overall, these findings demonstrate that the hydrogel constitutes a promising therapy for cornea alkali burn perforations that is easy to use, can decrease the time of treatment in emergency situation, and that is convenient for both patients and surgeons.

[0200] Results of / n vivo experiments

[0201] Mouse corneas with alkali burns and surgical perforations were filled with CLP-PEPC I made from 36aa CLP with the sequence (PKG)4(POG)4(DOG)4as published by O’Leary et al. (O'Leary LE, Fallas JA, Bakota EL, Kang MK, Hartgerink JD. Multi-hierarchical self-assembly of a collagen mimetic peptide from triple helix to nanofiber and hydrogel. Nat Chem. 2011 Aug 28;3(10):821-8. doi: 10.1038 / nchem.1123. PMID: 21941256). The solution from the syringe-mixer was viscous and it was noted that the cornea was overfilled for one mouse. This resulted in slower wound healing as seen in FIGs. 13A-B. Wound closure in the other two mice was rapid. Neovascularization seen in Mouse 2 during week 1 was mostly resolved by 1 -month post-surgery.

[0202] 27CLP-PEPC l / ll can be used to encapsulate lipid nanoparticles to deliver bioactive factors such as nucleic acids as shown. FIG. 14 shows the delivery of mRNA vs DNA for GFP at 2 weeks after implantation.

[0203] Example 2: Preparation of CLP-MPC hydrogels

[0204] CLP-MPC was synthesized with two different methods, in the first method, MPC was added to the CLP directly, and the conjugation was done using Michael reaction. But, in the second method, a linker (BIS) was attached to the CLP by Michael addition, and then MPC was attached to the linker using radical polymerization method.

[0205] Preparation of CLP-MPC by direct conjugation

[0206] In this method (FIG. 2A), 500 mg CLP first was dissolved in 30 mL of distilled water (DW); then, 500 mg MPC was added and dissolved in the CLP solution. Afterwards, the pH of the solution was adjusted at 6.5 using TEA. The reaction became milky at this step and it was stirred at room temperature (RT) for 96 hours (hrs). After 96 hrs, HCI was used to adjust the pH to 4 to make the solution transparent again. Then, a dialysis bag (cut off, 1 K Dalton) was used to purify the CLP-MPC, and after 3 days of dialysis, the dialysate was lyophilized at -15°C.

[0207] Preparation of CLP-BIS-MPC

[0208] CLP-BIS-MPC was synthesized using a 2-step method, in the first step, a linker (BIS) was attached to the CLP by Michael addition. 500 mg of the CLP was dissolved in 50 mL of DW in a tree neck flask equipped with stopcock. Then, 60 mg of BIS (excess amounts 2) was added to the solution. Nitrogen cylinder and pipette Pasteur was used to purge nitrogen to the liquid for 5 mins. Then a balloon full of nitrogen was attached to the top of one of the septa. The pH was adjusted to 6 using TEA, and the solution remained to be stirred for 96 hrs at RT under N2environment. After 96 hrs, HCI was used to decrease the pH to about 4 to make the solution transparent again. Then, a dialysis bag (cut off, 1K Dalton) was used to purify the CLP-BIS, and after three days of dialyzing (5 L beaker full of DW was changed once per day), the solution was lyophilized at -40°C and 100 mTorr for 30 min, then, the polymerization was started after the addition of APS (0.1 mmol) and SHS (0.1 mmol). After 24 hrs of stirring at RT, the solution transferred to a dialysis bag, (cut off, 3.5K Dalton) to be purified. The CLP-BIS was freeze-dried at -15°C and 10 mTorr.

[0209] 500 mg of the CLP-BIS was dissolved in 30 mL of DW in a tree neck flask equipped with stopcock. Then, 100 mg of MPC (ratio CLP: MPC 5:1) was dissolved in the solution. Nitrogen cylinder and pipette Pasteur was used to purge nitrogen to the liquid for 5 mins. Then a balloon full of nitrogen was penetrated to the top of one of the septa. Then, the polymerization was started after the addition of APS (0.1 mmol) and SHS (0.1 mmol). The solution remained to be stirred for 24 hrs at RT under N2environment. Then, a dialysis bag (cut off, 1 K Dalton) was used to purify the CLP-BIS-MPC, and after three days of dialysis (5 L beaker full of DW was changed once per day), the CLP-BIS-MPC was lyophilized at -40°C and 100 mTorr.

[0210] Preparation of acrylic acid homopolymer (PAA)

[0211] 2 mL of AA was dissolved in 30 mL of DW in a tree neck flask equipped with stopcock. Nitrogen cylinder and pipette Pasteur was used to purge nitrogen to the liquid for 5 mins. Then, a balloon full of nitrogen was penetrated to the top of one of the septa. Then, APS (0.1 mmol) and SHS (0.1 mmol) were added to the flask to start polymerization. The solution remained to be stirred for 24 hrs at RT under N2environment. Then, a dialysis bag (cut off, 14K Dalton) was used to purify the PAA, and after 3 days of dialysis (5 L beaker full of DW was changed once per day), the dialysate was lyophilized at -40°C and 100 mTorr.

[0212] Then, 50 mg of the lyophilized PAA was dissolved in 1 mL of 0.25 M PBS in a 5-mL conical tube to prepare 5% (W / V) of PAA solution (stirred overnight). Then, the pH of the solution was adjusted to between 7 and 7.4 using NaOH (5 M).

[0213] Preparation of double-barrel static mixer

[0214] A double-barrel static mixer as illustrated in FIG. 5) was used to simultaneously mix different components of the hydrogel. This method permits to apply the hydrogel on the eye easily and carefully, and it also avoid any premixing step. The delivery system contains two syringes are filled separately before assembling them.

[0215] First syringe: 120 mg of CLP-MPC was dissolved in 100 pL of PBS (0.25 M) in a 1.5 mL microtube at 65°C using thermomixer, after two mins, 100 pL of PAA (5%) was added to the tube, (vortex and spin can be used to help dissolving the materials). After two more mins of heating at 65°C, the content of the tube (~200 pL) was aspirated into one of the syringes, and the syringe was connected to the static mixer.

[0216] Second syringe: 20 mg of DMTMM was dissolved in 50 pL of PBS (0.25 M) in a 1.5 mL microtube at room temperature. Then, 150 pL of PAA (5%) was added to the tube. Vortexing and spinning can be used to help mixing the materials. 200 pL of solution was aspirated into the syringe, which was then connected to the static mixer. The filled double-barrel static mixer may be used right away or frozen at -20°C for future use.

[0217] Characterization of CLP-MPC

[0218] FIG. 15A presents1H-NMR spectrum of CLP-BIS, peaks at 1.2, 1.4, 4.8, and 6.2 can be assigned to the BIS groups before polymerization. Moreover, different peaks of amino acids in CLP were appeared at their corresponding chemical shifts. However, after incorporation of MPC to the CLP (FIG. 15B), the peak at 6.2 ppm was significantly suppressed and moved to 2.5 ppm, which confirms the polymerization of BIS with MPC. The1H-NMR of CLP-MPC (FIG. 15B) also contains characteristic peaks of methylene groups of MPC which appeared at 3.2 ppm. In the31P- NMR of CLP-MPC (FIG. 15D), a single peak confirms the presence of MPC polymer in CLP-MPC while the31PNMR of CLP-BIS (FIG. 15C) did not show any peak. Also,1H-NMR peak of PAA (FIG. 15E) showed two characteristic peaks of PAA belong to methylene groups at 1.7 and 2.35 ppm.

[0219] FIG. 16A shows that the CLP-MPC formulation is able to incorporate nanoparticles for delivery of drugs or bioactive molecules, notably an siRNA as an anti-viral against the Herpes Simplex Virus serotype I (HSV-1).

[0220] FIG. 16B shows that the wavelength that the transparency passes 85% was 293 nm for CLP-MPC hydrogel; the transparency of the hydrogel in visible region is above 95% which is desirable. Also, the percentage of water content of CLP-MPC was 90.7 ± 1.5 % which is an acceptable swelling and water content very similar to the human cornea water content which showed that the hydrogel can strike a balance to avoid eye dryness while the integration of it with the cornea does not increase the thickness of cornea to anything above 500 pm to cause problems.

[0221] Collagenase test results are shown in FIG. 16C. The hydrogel experienced a gradual weight loss during one month and the weight decreased to 55.2 % of its initial weight after 8 weeks, the collagenase rate of CLP-MPC hydrogel was 0.67 ± 0.05. The results proved that the hydrogel biodegradability is in a suitable range which provide a great condition for cornea to epithelialize completely (it was observed to be between 5 to 8 weeks in pigs), which is the main objective before the hydrogel becomes degraded completely and excreted from the eye.

[0222] The change in the temperature of the gel upon heating is shown in FIG. 17A-**. At time 0, the temperature of both syringes is around -17°C, and after 5 min in the heater, and because only one of the syringes was heated, the temperature of the first and second syringe increased to 38.4 and 18.5 °C. After applying the hydrogel, two liquids were mixed together and the final temperature of the gel reached about 21 °C, which is close to room temperature and comfortable for patients. Treatment of Yucatan mini-pigs

[0223] The right eyes of each pig received an alkali burn centrally, made from placing a 3 mm filter paper soaked in 0.5N NaOH for 30 seconds after which excess alkali was rinsed off thoroughly. The light burns were design to attract immune and inflammatory cells. Following the burn, perforations were made by trephining the central cornea with a 6 mm trephine down to 60% depth. The corneal tissue was removed. The perforation was completed by using a 1 mm biopsy punch centrally down through most of the cornea to create a cylindrical wound which was completed by using a scalpel blade to penetrate the inner or endothelial surface.

[0224] The perforation was filled with CLP-MPC after thawing with heater as illustrated in FIGs. 10 and 17A-J. The max temperature measured using a thermal camera after heating at 47°C was 29°C for the CLP hydrogel syringe barrel. The unheated barrel left to thaw at ambient temperature remained at ambient temperature.

[0225] It was observed that CLP-MPC sealed 100% of all wounds in all pigs tested (FIG. 19A). Two animals reinjured their eyes and were regrafted. Unlike cyanoacrylate glue which does not work with moisture, CLP-MPC sealed the wound after removal of previous application using a scalpel, resulting in some bleeding. 2 / 2 regrafts were sealed. One animal was regrafted twice. FIG. 18 shows healing in unvascularized but alkali-burned and surgically perforated corneas. The two pigs that had to be regrafted due to their scratching their eyes also healed despite the neovascularisation and bleeding into the wound (FIG. 18). It was found that the wounds had to be filled to restore a smooth surface to allow rapid re-epithelialization

[0226] Intraocular pressure (FIG. 19B) and tear production (FIG. 19D) were comparable between operated and unoperated eyes. The operated and regenerated corneas at 39 weeks postoperation are slightly thinner than those of the unoperated eyes but nevertheless have regained thickness by week 26 and have stabilized (FIG. 19C). Cell and nerve regeneration observed, with nerves seen by 6 months post-operation (FIG. 20).

[0227] Example 3: Preparation of Star MPC hydrogels

[0228] Preparation of multi arms PEG-CTCTP

[0229] In this method (FIG. 3A), 100 mg of the prepared multi arms PEG (mw 5000) was dissolved in 100 mL of DW at 4°C. Then, 102 mg of CTCTP (excess amounts) was added to the solution. The pH was adjusted at 6 using NaOH (5M). Then, simultaneously, 0.8 mmol (excess amounts) of EDC and NHS was added to the solution, and the solution was stirred for 96 hrs at 4°C. After 96 hrs, HCI was used to decrease the pH to about 4 to make the solution transparent again. Then, a dialysis bag (cut off, 1 K Dalton) was used to purify the multi arms PEG-CTCTP, and after 3 days of dialyzing, the dialysate was lyophilized at -40°C and 100 mTorr.

[0230] Preparation of multi arms PEG-CTCTP-MPC-AA In this method (FIG. 3B), 100 mg of multi arms PEG-CTCTP was dissolved in the 30 mL of DW. Then, the weight ratio of AA:MPC (75:25) were dissolved in the solution. N2gas was purged into the solution for 30 min, then, the polymerization was started after the addition of APS (0.1 mmol) and SHS (0.1 mmol). After 24 hrs of stirred at RT, the solution transferred to a dialysis bag, (cut off, 3.5K Dalton) to be purified. The CLP-BIS-MPC was freeze-dried at -40°C and 100 mTorr.

[0231] Preparation of hydrogels

[0232] In this step, a double-barreled syringe with a new printed tip and static mixer to reduce the void volume and allow more efficient application was designed (FIG. 7), was used to simultaneously mix different components of the hydrogel. The delivery system contains two syringes which are filled separately before assembling them:

[0233] First syringe: 60 mg of CLP-MPC was dissolved in 55 L of PBS (0.25 M) in a 1.5 mL microtube at 65°C using thermomixer, after two mins, 50 L of STAR-MPC with PEG core (10%) was added to the tube. Vortexing and spinning may be used to help dissolving the materials. After two more mins of heating at 65°C, 100 pL of the content of the tube was aspirated into one of the syringes, which was connected to the mixer.

[0234] Second syringe: 7.5 mg of DMTMM was dissolved in 75 pL of PBS (0.25 M) in a 1.5 mL microtube at room temperature. Then, 75 pL of STAR-MPC with PEG core (10%) was added to the tube. Vortexing and spinning may be used to help mixing the materials. 100 pL of the context was aspirated into one of the syringes, which was connected to the mixer.

[0235] The filled double-barrel static mixer may be used right away or frozen at -20°C for future use.

[0236] Characterization of Star MPC with PEG core

[0237] FIG. 21A presents1H-NMR spectrum of PEG-CTCTP. Peaks of CTCTP and PEG are identified in FIG. 21A. The1H-NMR of STAR-MPC with PEG core (PEG-CTCTP-MPC-AA) (FIG. 21 B), also contains characteristic peaks of methylene groups of MPC and PEG which appeared at 3.2 ppm. In the31P-NMR (FIG. 21 D) of PEG-CTCTP-MPC-AA a single peak confirms the presence of MPC polymer while the31PNMR of PEG-CTCTP (FIG. 21C) did not show any peak.

[0238] In vivo study of Star MPC with PEG core

[0239] By 3-weeks post-operation, H&E sections of corneas harvested from a treated rabbit showed that the hydrogel was stably incorporated and that there was already over-growth of the injected hydrogel by corneal epithelial cells from the host (FIG. 22).

[0240] Preparation of STAR MPC with chemically crosslinked core In this method first CLP-BIS and polyMPC (RAFT Method see 4.1.1) are prepared separately and then these two different types of arms can form a star MPC by the addition of a crosslinking agent such as BIS or pentaerythritol tetraacrylate during final polymerization process.

[0241] Characterization of STAR MPC with chemically crosslinked core

[0242] FIG. 23 presents1H-NMR and31P-NMR spectra of PolyMPC. Since the interpretation of the similar compounds was explained previously, here only the characteristic peaks are identified on the curve.

[0243] Example 4: Synthesis of bioorthogonal reaction hydrogel

[0244] Polyacrylic acid (PAA) was prepared as follows. Two grams of AA was dissolved in 30 mL of DW in a three-neck round bottom flask. Then, nitrogen gas was purged into the flask for 30 min. to make the environment inert and suitable for the polymerization, Then, 0.1 mmol of each APS and SHS were dissolved in 500 pL separately, and added to the solution under stirring in an inert environment. The solution was stirred for 24 hrs at RT. After 24 hrs, the solution (often viscous) was transferred to a dialysis bag (6.5 kDa cut-off), and after three days of dialyzing, the final solution was lyophilized at -15°C.

[0245] Propargylamine and cystamine were added to PAA separately by the same method; therefore, only the addition of cystamine is described herein. In order to prepare PAA-S (PAA- cystamine), 500 mg of PAA was dissolved in DW water, then 0.2 mmol cystamine were added to the flask. The solution became a little viscous after dissolving cystamine and adjusting of the pH to around 7 by addition of HCI decreased the viscosity. Then, an excess amount of DMTMM (0.4 mmol) was added to the flask, and the solution was stirred for 96 hrs before transferring to a dialysis bag (14 kDa cut-off). After three days of dialyzing, the final solution was lyophilized at - 15°C. The PAA-propargylamine (PAA-E) was prepared with the same method.

[0246] To prepare a hydrogel, CLP-MPC was weighed and added to a 1 .5 mL microcentrifuge tube (T1), PAA-S (5% in 0.25 M PBS, pH adjusted to 7) and PAA-E (5% in 0.25 M PBS, pH adjusted to 7) added to the same tube. A sufficient amount of 0.25 M PBS was added to the T 1 to make a total volume of 80 pL. Then, the tube was put in the thermomixer and heated up to 65°C, and simultaneously DMTMM was dissolved in 20 pL of PBS (0.25 M) (T2). Then, T1 was removed from thermomixer and cooled down to almost 37°C, and T2 was added to T1 to obtain the hydrogel ready to be applied.

[0247] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise.

[0248] REFERENCES

[0249] Liu W, Deng C, McLaughlin CR, Fagerholm P, Lagali NS, Heyne B, Scaiano JC, Watsky MA, Kato Y, Munger R, Shinozaki N, Li F, Griffith M. Collagen-phosphorylcholine interpenetrating network hydrogels as corneal substitutes. Biomaterials. 2009 30(8): 1551-1559. doi: 10.1016 / j. biomaterials.2008.11 .022.

[0250] Islam MM, Buznyk O, Reddy JC, Pasyechnikova N, Alarcon El, Hayes S, Lewis P, Fagerholm P, He C, lakymenko S, Liu W, Meek KM, Sangwan VS, Griffith M. Biomaterials-enabled cornea regeneration in patients at high risk for rejection of donor tissue transplantation. NPJ Regen Med. 2018 3: 2. doi: 10.1038 / S41536-017-0038-8.

[0251] Simpson FC, McTiernan CD, Islam MM, Buznyk O, Lewis PN, Meek KM, Haagdorens M, Audiger C, Lesage S, Gueriot FX, Brunette I, Robert MC, Olsen D, Koivusalo L, Liszka A, Fagerholm P, Gonzalez-Andrades M, Griffith M. Collagen analogs with phosphorylcholine are inflammationsuppressing scaffolds for corneal regeneration from alkali burns in mini-pigs. Commun Biol. 2021 4(1): 608. doi: 10.1038 / s42003-021-02108-y.

[0252] Popovic N, Hooker E, Barabino A, Flamier A, Provost F, Buscarlet M, Bernier G, Larrivee B. COCO / DAND5 inhibits developmental and pathological ocular angiogenesis. EMBO Mol Med. 2021 13(3): e12005.

[0253] McTiernan CD, Simpson FC, Haagdorens M, Samarawickrama C, Hunter D, Buznyk O, Fagerholm P, Ljunggren MK, Lewis P, Pintelon I, Olsen D, Edin E, Groleau M, Allan BD, Griffith M. LiQD Cornea: Pro-regeneration collagen mimetics as patches and alternatives to corneal transplantation. Sci Adv. 2020 6(25): eaba2187. DOI: 10.1126 / sciadv.aba2187.

[0254] Cox F, Khalib K, Conlon N. PEG that reaction: a case series of allergy to polyethylene glycol. J Clin Pharmacol. 2021 61(6): 832-835. doi: 10.1002 / jcph.1824. Epub 2021 Feb 28.

Claims

WHAT IS CLAIMED IS:1 . A conjugate comprising:(a) 2-methacryloyloxyethyl phosphorylcholine (MPC) and collagen or a collagen-like peptide (CLP) crosslinked to the MPC;(b) (i) a copolymer comprising monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) and polymerizable monomers comprising one or more functional groups; and (ii) collagen or a CLP crosslinked to the copolymer; or(c)(i) a copolymer comprising a core comprising a multi-arm polymer; monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) attached to one or more arms of the multi-arm polymer; optionally polymerizable monomers or polymers comprising one or more functional groups and(ii) collagen or a CLP attached to one or more arms of the multi-arm polymer and / or to the MPC.

2. The conjugate of claim 1 , wherein the one or more functional groups comprise carboxylic acid groups, hydroxyl groups, thiol groups, and / or amine groups.

3. The conjugate of claim 1 or 2, wherein the polymerizable monomers comprising one or more functional groups are acrylic acid (AA) or 2-hydroxyethyl acrylate (HEA).

4. The conjugate of any one of claims 1 to 3, wherein the copolymer of (b) has one of the following structures:PMPC-PAA PMPC-PHEA wherein PC is phosphorylcholine, x represents the Mw of polymeric MPC (PMPC), and y represents the Mw of polyacrylic acid (PAA) or poly 2-hydroxyethyl acrylate (PHEA), preferably wherein the Mw of PMPC-PAA is about 387,000 kDa, and the Mw of PMPC-PHEA is about 532,000 kDa.

5. The conjugate of claim 4, wherein the Mw of PMPC-PAA is about 350,000 to about 400,000 kDa.

6. The conjugate of claim 5, wherein the Mw of PMPC-PAA is about 387,000 kDa.

7. The conjugate of claim 4, wherein the Mw of PMPC-PHEA is about 500,000 to about 550,000 kDa.

8. The conjugate of claim 4, wherein the Mw of PMPC-PHEA is about 532,000 kDa.

9. The conjugate of any one of claims 1 to 8, wherein the conjugate comprises (i) a first copolymer comprising monomers of MPC and polymerizable monomers comprising hydroxyl groups; (ii) a second copolymer comprising monomers of MPC and polymerizable monomers comprising carboxylic acid groups.

10. The conjugate of any one of claims 1 to 9, wherein the multi-arm polymer comprises 2 to 8 arms.

11. The conjugate of any one of claims 1 to 10, wherein the multi-arm polymer comprises a multi-arm polyethyleneglycol (PEG).

12. The conjugate of claim 11 , wherein the PEG has a molecular weight of 2000 to 5000 kDa.

13. The conjugate of any one of claims 1 to 12, wherein the copolymer of (c) has the following structure:wherein PC is phosphorylcholine, n represents the Mw of the polymer (300,000 to 500,000 kDa) and m is an integer of 2 to 8.

14. The conjugate of any one claims 1 to 13, wherein the MPC is directly linked to the collagen or CLP.

15. The conjugate of any one claims 1 to 13, further comprising a linker between the MPC and the collagen or CLP.

16. The conjugate of claim 15, where the linker is / V, / V'-methylenebisacrylamide (BIS).

17. The conjugate of any one of claims 1 to 16, wherein the conjugate comprises a CLP.

18. The conjugate of claim 17, wherein the CLP has a length of 27 to 36 amino acids.

19. The conjugate of claim 17 or 18, wherein the CLP comprises or consist of the following sequence:(PKG)3 or4(POG)3 or4(DOG)2 or3(DOX), wherein X is G or C.

20. The conjugate of claim 19, wherein the CLP comprises or consist of the following sequence: (PKG)3(POG)3(DOG)2(DOX) (SEQ ID NO: 13), wherein X is G or C.21 . The conjugate of claim 17, wherein the CLP comprises or consist of the following sequence: X1X2X3(PKG)3or4(POG)3or4(DOG)2or3(DOC), wherein X1 , X2 and X3 are independently any amino acid, and wherein at least one of X1 , X2 and X3 is C.

22. The conjugate of claim 21 , wherein X1X2X3is GCG, CGG, SCG, or SCS23. A hydrogel comprising the conjugate of any one of claims 1 to 22.

24. The hydrogel of claim 23, further comprising additional proteins, peptides, cells, and / or drugs.

25. A collagen-like peptide (CLP) having a length of 27 amino acids.

26. The CLP of claim 25, wherein the CLP has the following structure: (PKG)3(POG)3(DOG)2(DOX) (SEQ ID NO: 13), wherein X is G or C.

27. The CLP of claim 26, wherein X is G.

28. The CLP of claim 26, wherein X is C.

29. A collagen-like peptide (CLP) of one of the following structures: X1X2X3(PKG)3(POG)3(DOG)2X4X5X6; or X1X2X3(PKG)3(POG)5X4X5X6wherein X1, X2, X3, X4, X5and X6are independently any amino acid, and wherein at least one of X1, X2, and X3is C, and at least one of X4, X5, and X6is C.

30. The CLP of claim 29, wherein X4X5X6is DOC, SCS, GCG, SCG, or CGG.31 . The CLP of claim 29 or 30, wherein X1X2X3is SCS, GCG, SCG, or CGG.

32. An injection device comprising the conjugate of any one of claims 1 to 22 or the hydrogel of claim 23 or 24.

33. The injection device of claim 32, wherein the device comprises a plurality of barrels or compartments and a mixer connected to the plurality of barrels or compartments34. An injection device comprising a plurality of barrels or compartments and a mixer connected to the plurality of barrels or compartments.

35. The device of claim 33 or 34, comprising two barrels or compartments.

36. The device of any one of claims 33 to 35, wherein the mixer is a static mixer.

37. The device of any one of claims 33 to 36, wherein a first barrel or compartment comprises 2-methacryloyloxyethyl phosphorylcholine (MPC), copolymers comprising MPC, and / or collagen or a collagen-like peptide, and a second barrel or compartment comprises a cross-linking agent.

38. The device of claim 37, wherein the second barrel or compartment further comprises polymerizable monomers or a polymer comprising one or more functional groups.

39. The device of any one of claims 33 to 38, wherein the barrels or compartments have a maximum capacity of 500 pL each.

40. The device of any one of claims 33 to 39, wherein the mixer has a dead volume of less than 40 pL.41 . The device of any one of claims 33 to 40, which comprises a third barrel or compartment.

42. A system comprising the injection device of any one of claims 33 to 41 , and a heater adapted to heat one or more of the barrels or compartments of the device.

43. A kit comprising:(I)(a) 2-methacryloyloxyethyl phosphorylcholine (MPC), and collagen or a collagen-like peptide (CLP);(b) (i) monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) and polymerizable monomers comprising one or more functional groups; and (ii) collagen or a CLP; or(C)(i) a copolymer comprising a core comprising a multi-arm polymer; monomers of 2-methacryloyloxyethyl phosphorylcholine (MPC) attached to one or more arms of the multi-arm polymer; optionally polymerizable monomers or polymers comprising one or more functional groups and(ii) collagen or a CLP attached to one or more arms of the multi-arm polymer and / or to the MPC; and(II) optionally a cross-linking agent.

44. The kit of claim 43, wherein the cross-linking agent is 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)- 4-methylmorpholinium chloride (DMTMM).

45. The kit of claim 43 or 44, further comprising additional proteins, peptides, cells, nanoparticles and / or drugs.

46. The kit of claim 45, further comprising an inhibitor of angiogenesis.

47. The kit of any one of claims 43 to 46, wherein the kit further comprises the device of any one of claims 32 to 41 or the system of claim 42.

48. Use of the conjugate of any one of claims 1 to 22 or the hydrogel of claim 23 or 24 for treating a wound or tissue injury.

49. The use of claim 25, wherein the wound or tissue injury is corneal wound or injury.

50. The use of claim 25 or 26, wherein the wound or injury is burn or perforation.51 . The use of any one of claim 25 to 27, wherein the conjugate or hydrogel is for administration by injection into or in the vicinity of said wound or injury.

52. The use of any one of claim 25 to 27, wherein the conjugate or hydrogel is for administration using the device of of any one of claims 32 to 41 .

53. A method for treating a wound or tissue injury in a subject comprising contacting the wound or tissue with the conjugate of any one of claims 1 to 22 or the hydrogel of claim 23 or 24.

54. The method of claim 53, wherein the wound or tissue injury is corneal wound or injury.

55. The method of claim 53 or 54, wherein the wound or injury is burn or perforation.

56. The method of any one of claims 53 to 55, wherein the conjugate or hydrogel is injected into or in the vicinity of said wound or injury.

57. The method of any one of claims 53 to 56, wherein the conjugate or hydrogel is injected using the device of of any one of claims 32 to 41 .

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