Polymer compositions and uses thereof

Abstract

The present invention relates to polymer compositions, in particular to biocompatible cross-linkable polymer compositions, methods of preparing the same and their use in the preparation of ocular implants.

Description

POLYMER COMPOSITIONS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from Australian Provisional Patent Application No. 2023902079 filed 30 June 2023, the entire contents of which are incorporated herein by cross-reference.TECHNICAL FIELD

[0002] The present invention relates in general to polymer compositions and uses thereof. In particular, the present invention relates to biocompatible cross-linkable polymer compositions, methods of preparing the same and their use in the preparation of laminate structures for use in tissue regeneration, such as ocular implants for the treatment of corneal endothelial dysfunction.BACKGROUND

[0003] Globally over 8 million people suffer from corneal diseases with rising indications in ageing populations. Corneal endothelial dysfunction, which is characterised by damage to and loss of corneal endothelial cells, leads to a loss of transparency of the cornea and eventual blindness. Currently, the only treatment for corneal endothelial dysfunction is corneal transplantation. However, the technical difficulty of performing such surgeries limits adoption and patient outcomes.

[0004] Surgical options for treating comeal endothelial dysfunction currently comprise two main techniques: Descemet’s stripping automated endothelial keratoplasty (DSAEK) and Descemet's membrane endothelial keratoplasty (DMEK). Both procedures involve removal of the damaged comeal endothelial cells and Descemet's membrane to which they are attached, and replacing them with a donor tissue graft. The grafts are typically inserted by a surgeon in the form of a scroll and, once positioned, are manually unscrolled. However, unlike the DSAEK graft, which contains stromal cells, the DMEK graft consists only of the thinDescemet's membrane and the attached endothelium layer (15 pm), making it difficult to unscroll and more susceptible tearing during surgery.

[0005] Thus, although the DMEK graft leads to better patient outcomes, including greater improvements in vision and lower rejection rates, many surgeons still opt for the simpler DSAEK procedure, despite associated complications such as poorer optical clarity, aberrations (ripples) in the graft due to differences in curvature, and hyperopic shifts due to curvature differences when cutting (e.g., with a microkeratome).

[0006] Accordingly, there is an ongoing need for improved or alternative methods for treating comeal endothelial dysfunction.SUMMARY

[0007] The present invention provides a biocompatible cross-linkable polymer composition comprising: a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group.

[0008] In another aspect, the present invention provides an aqueous bioglue composition comprising the biocompatible cross-linkable polymer composition of the invention, water and a biocompatible photoinitiator.

[0009] In another aspect, the present invention provides a kit when used for adhering a biocompatible hydrogel to a biological substrate, the kit comprising:(a) a first part comprising the biocompatible cross-linkable polymer composition of the invention; and(b) a second part comprising water and a biocompatible photoinitiator.

[0010] In another aspect, the present invention provides a kit when used for preparing a biological substrate having adhered thereto a cross-linked network polymer scaffold, the kit comprising:(a) a first part comprising the biocompatible cross-linkable polymer composition of the invention; and(b) a second part comprising water and a biocompatible photoinitiator.

[0011] In another aspect, the present invention provides use of the cross-linkable polymer composition according to the invention for adhering a biocompatible hydrogel to a biological substrate.

[0012] In another aspect, the present invention provides use of the cross-linkable polymer composition according to the invention for preparing a biological substrate having adhered thereto a cross-linked network polymer scaffold.

[0013] In another aspect, the present invention provides a process for adhering a biocompatible hydrogel to a biological substrate comprising:(i) combining the biocompatible cross-linkable polymer composition according to the invention with water and a biocompatible photoinitiator to form an aqueous bioglue composition of the invention;(ii) providing the aqueous bioglue composition between a biocompatible hydrogel layer and a biological substrate layer to form a laminate structure;(iii) irradiating the laminate structure at a wavelength suitable to activate the biocompatible photoinitiator and initiate cross-linking of the cross-linkable branched polyether compound, the process of which promotes adhesion between the biocompatible hydrogel and the biological substrate.

[0014] In another aspect, the present invention provides a process for preparing a biological substrate having adhered thereto a cross-linked network polymer scaffold comprising:(i) combining the biocompatible cross-linkable polymer composition according to the invention with water and a biocompatible photoinitiator to form an aqueous bioglue composition of the invention;(ii) providing a layer of the aqueous bioglue composition on a biological substrate layer to form a laminate structure;(iii) irradiating the laminate structure at a wavelength suitable to activate the biocompatible photoinitiator and initiate cross-linking of the cross-linkable branched polyether compound, the process of which promotes formation of a cross-linked network polymer scaffold adhered to the biological substrate.

[0015] In another aspect, the present invention provides a laminate structure comprising a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the cross-linked network polymer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three poly ether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group.

[0016] In another aspect, the present invention provides a laminate structure comprising a biocompatible cross-linked network polymer layer and a biological substrate layer, wherein the cross-linked network polymer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three poly ether arms each comprise a polymerisable ethylenically unsaturated group.

[0017] In another aspect, the present invention provides an ocular implant having a laminate structure comprising a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three poly ether arms, wherein the at least three poly ether arms each comprise a polymerisable ethylenically unsaturated group, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages, and wherein the biological substrate layer comprises Descemet's membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells.

[0018] In another aspect, the present invention provides an ocular implant having a laminate structure comprising a biocompatible cross-linked network polymer layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to whichis covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, and wherein the biological substrate layer comprises Descemet's membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells.

[0019] In another aspect, the present invention provides a method for treating corneal endothelial dysfunction in a subject, the method comprising a step of implanting an ocular implant into an eye of the subject, the ocular implant having a laminate structure comprising a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three poly ether arms, wherein the at least three poly ether arms each comprise a polymerisable ethylenically unsaturated group, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages, and wherein the biological substrate layer is Descemet’s membrane having attached thereto a layer of comeal cells comprising corneal endothelial cells.

[0020] In another aspect, the present invention provides a method for treating corneal endothelial dysfunction in a subject, the method comprising a step of implanting an ocular implant into an eye of the subject, the ocular implant having a laminate structure comprising a biocompatible cross-linked network polymer layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three poly ether arms, wherein the at least three poly ether arms each comprise a polymerisable ethylenically unsaturated group, and wherein the biological substrate layer is Descemet’s membrane having attached thereto a layer of comeal cells comprising corneal endothelial cells.

[0021] In yet another aspect, the present invention provides an ocular implant for use in treating corneal endothelial dysfunction, the ocular implant having a laminate structurecomprising a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three poly ether arms, wherein the at least three poly ether arms each comprise a polymerisable ethylenically unsaturated group, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages, and wherein the biological substrate layer is Descemet’s membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells.

[0022] In yet another aspect, the present invention provides an ocular implant for use in treating corneal endothelial dysfunction, the ocular implant having a laminate structure comprising a biocompatible cross-linked network polymer layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, and wherein the biological substrate layer is Descemet’s membrane having attached thereto a layer of comeal cells comprising corneal endothelial cells.

[0023] In yet another aspect, the present invention provides use of comeal endothelial cells in the manufacture of a medicament for treating corneal endothelial dysfunction, wherein the medicament comprises a laminate structure comprising a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages, andwherein the biological substrate layer is Descemet's membrane having attached thereto a layer of the corneal endothelial cells.

[0024] In yet another aspect, the present invention provides use of comeal endothelial cells in the manufacture of a medicament for treating corneal endothelial dysfunction, wherein the medicament is an ocular implant having a laminate structure comprising a biocompatible cross-linked network polymer layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, and wherein the biological substrate layer is Descemet's membrane having attached thereto a layer of the corneal endothelial cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Embodiments of the invention will now be described with reference to the following Figures, which are intended to be exemplary only, and in which:

[0026] Figure 1.]H NMR spectrum showing the chemical characterization of bioglue Part B in CDCh.

[0027] Figure 2. Sample preparation of bioglue formulations showing: a) the commercial LED white light plate; b) curing of samples under light; c) samples prepared in disposable syringes and; d) an example of the resulting bioglue sample for testing.

[0028] Figure 3. Dimensions of dog-bone samples for tensile mechanical testing using cutting blade C0024 - Type B (ASTM D412-C).

[0029] Figure 4. Lap shear test of photo-cross-linked bioglue (glass-bioglue-glass).

[0030] Figure 5. a) Illustration of overlapping test samples photo-cross-linked with bioglue (Part A and B, 5:1) for PHF-glue-PHF tests.

[0031] Figure 6. Glass slides coated with PHF and sheep cornea treated with bioglue (50 pL, 30%B).

[0032] Figure 7. Schematic representation of bioglue and PHF formation, a) GEM synthesis (Part B) and b) photo-cross-linking under light after combination with Part A solution and c) PHF synthesis as previously reported (Ozcelik et al., 2014).

[0033] Figure 8. ATR-FTIR analysis of a) glycerol ethoxylate, b) glycerol ethoxy trimethacrylate (GEM, Part B), c) aqueous Part A solution and d) bioglue formulation post- photo-cross-linking.

[0034] Figure 9. UV-Vis analysis of standardized photoinitiator Eosin Y (Xmax ~ 520 nm) absorbance was used to monitor the extent of the bioglue photo-cross-linking under exposure to white (o), green (♦) and blue (A) LED lights. White light (400-700 nm, 1022 mW / cm2), green light (kmax~ 520 nm, 2.3 mW / cm2) and blue light (z.max~ 455 nm, 3.8 mW / cm2) were shone on the samples for a given time at a distance of 2 cm from the sample. Gelation times are indicated by the dashed lines and red labels for each light source.

[0035] Figure 10. Effect of dilution on gelation time with increasing ratio of aqueous photoinitiator solution Part A, containing Eosin Y, triethanolamine and / V-vinylcaprolactam relative to Part B, containing trifunctional glycerol ethoxy methacrylate (GEM). Inset images show 0.75 mL samples prepared after white-light irradiation for 10 min.

[0036] Figure 11. Representative compressive stress-strain curves to break, for bioglue formulations of varying Part B concentrations from 10-50 v / v% Part B (denoted as X%B).

[0037] Figure 12. Mechanical compressive properties of bioglue formulations with varying amounts of Part B from 10-50 v / v%. The compressive testing of three samples (0.75 mL) were measured and the average presented for a) stress at break (o); b) strain at break (a); c) Young’s Modulus (E) at 10% strain; and d) toughness (UT).

[0038] Figure 13. Swellability ratio (%) of bioglue formulations (entries 1-5, Table 1) after photo-cross-linking and incubation in PBS at 37 °C for compositions containing Part B 10- 50 v / v%.

[0039] Figure 14. a) Transparency ratio (%), calculated by ImageJ analysis of photographs taken of bioglue cross-linked hydrogels (B-10, 20, 30, 40, and 50%) prepared as 0.75 mm3cylinders (10 mm thickness) and cast in 24-well plates (1.3 mm thickness) and b, c) shown photographed on top of a Koren lens chart respectively.

[0040] Figure 15. UV-Vis analysis of PHF membrane and PHF-bioglue samples at t = 0, 5, 10 min exposure to white light (1400 Lumen).

[0041] Figure 16. Lap shear stress of commercial ocular adhesive CoSEAL™ (polyethylene glycol based) com relative to (i) PHF-bioglue-glass; (ii) PHF-bioglue-PHF; and (iii) PHF- bioglue-tissue.

[0042] Figure 17. In vitro cytotoxicity analysis of two batches of Part B of bioglue: precrosslinked a) GEM-B1, b) GEM-B2; and c, d) cross-linked bioglue (Part A and B).

[0043] Figure 18. Human HBT in vitro trial performed at Eversight, USA showing a) bioglue addition to PHF membrane; b) photoinitiation of bioglue between PHF and human Descemet’s membrane under a surgical scope light; c) fourth passage of HBT graft through an Endoglide containing Life4C media; and d) intact HBT graft.

[0044] Figure 19. Ex vivo fabrication of HBT graft.

[0045] Figure 20. 'H NMR spectrum of bioglue formulation in D2O compared to Part A and B separately. Eosin Y is not evident in the NMR due to the low concentration.

[0046] Figure 21. 'H NMR spectrum of bioglue formulation (Part A in D2O: 80 v / v%, Part B: 20 vol%) after white light irradiation (700 Lumen, 1022 mW / cm2).

[0047] Figure 22.1H NMR spectrum of bioglue formulation (Part A in D2O: 80 v / v%, Part B: 20 vol%) after white light irradiation (1000 Lumen, 1460 mW / cm2).

[0048] Figure 23. The effect of Part B concentration in BSS on the gelation time of bioglue formulations.

[0049] Figure 24. Effect of bioglue direct casting on BT model in vitro. Mixed bioglue with Part A and Part B at different ratios was applied directly to a monolayer of B4G12 human corneal endothelial cells, followed by photo-crosslinking for 10 minutes.

[0050] Figure 25. Cell viability of bioglue when applied directly to a monolayer of B4G12 cells. The bioglue shows non-toxicity when the percentage of Part B is below 12.5% (A:B = 7:1). Green line marks the cell viability level at 80%, which is used as the benchmark for endothelial cell viability of an effective DMEK graft from a cadaveric donor cornea.DEFINITIONS

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0052] Unless otherwise specified, the indefinite articles “a”, “an” and “the” as used herein, include plural aspects. Thus, for example, reference to “a” substance includes a single substance, as well as two or more substances; likewise, reference to the “composition” or “formulation” includes a single composition or formulation, as well as two or more compositions or formulations; and so forth.

[0053] As used herein, the term “alkyl” or “alkyl group" refers to a monovalent (“alkyl”) and divalent (“alkylene”) straight chain or branched chain saturated aliphatic group and is intended to encompass monovalent and multivalent alkyl groups. The alkyl group may have from 1 to 12 carbon atoms, denoted Ci-nalkyl, or it may have from 1 to 6 carbon atoms, denoted Ci -ealkyl, and so forth. Examples of suitable alkyl moieties may include, but are not limited to, methyl, ethyl, 1-propyl, isopropyl, 1-butyl, 2-butyl, isobutyl, tert-butyl, amyl, 1.2- dimethylpropyl, 1,1 -dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl, 1- methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2- dimethylbutyl, 1.3-dimethylbutyl, 1 ,2,2-trimethylpropyl and 1,1,2-trimethylpropyl, heptyl, octyl, nonyl, decyl undecyl, dodecyl and the like.

[0054] As used herein, the term “heteroalkyl” or “heteroalkyl group” refers to a straight chain or branched chain saturated aliphatic group, wherein at least one (e.g., 1, 2, 3, 4 or 5) atom in the chain is a heteroatom independently selected from O, N, NH, or S. The alkyl group may have from 1 to 12 carbon atoms, denoted Ci- heteroalkyl, or it may have from 1 to 6 carbon atoms, denoted Ci-eheteroalkyl, and so forth. Examples of suitable heteroalkyl moieties may include, but are not limited to, ethers (e.g., R-O-R', wherein R and R' are independently an alkyl group as defined herein), tertiary amines (e.g., R-NR"-R', wherein R, R' and R" are independently an alkyl group as defined herein), secondary amines (e.g., R-NH-R', wherein R and R' are independently an alkyl group as defined herein), thioethers (e.g., R-S-R', wherein R and R' are independently an alkyl group as defined herein), and the like.

[0055] As used herein, the term “alkenyl” or “alkenyl group” refers to a monovalent (“alkenyl”) and divalent (“alkenylene”) straight or branched chain unsaturated aliphatic hydrocarbon groups having at least one double bond anywhere in the chain. Unless indicated otherwise, the stereochemistry about each double bond may be independently cis or trans, or E or Z, as appropriate. The alkenyl group may have from 2 to 12 carbon atoms, denoted C2 nalkenyl, or it may have from 2 to 6 carbon atoms, denoted Cz-ealkenyl, and so forth. Examples of suitable alkenyl groups may include, but are not limited to, ethenyl, vinyl, allyl,1-methylvinyl, 1 -propenyl, 2-propenyl, 2-methyl-l -propenyl, 2-methyl-l -propenyl, 1- butenyl, 2-butenyl, 3-butentyl, 1,3-butadienyl, 1 -pentenyl, 2-pententyl, 3-pentenyl, 4- pentenyl, 1,3 -pentadienyl, 2,4-pentadienyl, 1 ,4-pentadienyl, 3-methyl-2-butenyl, 1 -hexenyl,2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1 ,4-hexadienyl, 2-methylpentenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, and the like.

[0056] As used herein, the term “cycloalkyl” or “cycloalkyl group” refers to a saturated or partially saturated, monocyclic, fused or spiro polycyclic, carbocycle, and is intended to encompass monovalent and multivalent cycloalkyl groups. The cycloalkyl group may have from 3 to 12 carbon atoms per ring, denoted Cs ncycloalkyl. Examples of suitable cycoalkyl groups may include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, spiro[3.3]heptanyl, decalin and adamantyl.

[0057] As used herein, the term “heterocycloalkyl” or “heterocycloalkyl group” refers to a saturated or partially saturated, monocyclic, bicyclic, fused or spiro polycyclic carbocycles, wherein at least one (e.g., 1, 2, 3, 4 or 5) ring atom is a heteroatom independently selected from O, N, NH, or S. The heterocycloalkyl group may have from 2 to 6 carbon atoms per ring, denoted Cs-eheterocycloalkyl. Examples of suitable heterocycloalkyl groups may include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, quinuclidinyl, morpholinyl, diazaspiro[3.3]heptane (e.g., 2,6-diazaspiro[3.3]heptane), tetrahydrothiophenyl, tetrahydrofuranyl and tetrahydropyranyl. The heterocycloalkyl group may be a terminal group or a bridging group and may be attached through a heteroatom or any carbon ring atom.

[0058] As used herein, the term “aryl” or “aryl group” refers to a monocyclic, or fused polycyclic, aromatic carbocycle (i.e., a ring structure having ring atoms that are all carbon), and is intended to encompass monovalent and multivalent aryl groups. The aryl group may have from 6-12 atoms per ring, denoted C6-i2aryl. Examples of suitable aryl groups may include, but are not limited to, phenyl, naphthyl, phenanthryl As used herein, the term “aryl” is also intended to encompass optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a cycloalkyl or cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl.

[0059] As used herein, the term “heteroaryl” or “heteroaryl group” refers to an optionally substituted monocyclic, or fused polycyclic, aromatic heterocycle, wherein at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) ring atom is a heteroatom independently selected from O, N, NH, or S. The heteroaryl group may have from 1-12 carbon atoms per ring, denoted Ci-i2heteroaryl. Examples of suitable heteroaryl groups include, but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl (e.g., 1 ,3-oxazolyl, 1 ,2-oxazolyl), pyridinyl (e.g., 2-, 3-, 4-pyridinyl), pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl (e.g., 1 ,2,3-triazolyl, 1 ,2,4-triazolyl), triazinyl, tetrazinyl and carbazolyl. Representative examples of bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, ben zoxadi zol yl (e.g., 2,1 ,3- benzoxadiazolyl), cinnolinyl, dihydroquinolinyl, dihydroisoquinolinyl, furopyridinyl, indazolyl, indolyl (e.g., 2- or 3-indolyl), isoquinolinyl (e.g., 1-, 3-, 4-, or 5-isoquinolinyl), naphthyridinyl (e.g., 1 ,5-naphthyridinyl, 1 ,7-naphthyridinyl, 1,8-naphthyridinyl, etc),pyrrolopyridinyl (e.g., pyrrolo[2,3-b]pyridinyl), quinolinyl (e.g., 2-, 3-, 4-, 5-, or 8- quinolinyl), quinoxalinyl, tetrahydroquinolinyl, and thienopyridinyl. In one or more embodiments the heteroaryl group is an N-heteroaryl group having one or more nitrogen heteroatoms, e.g., 1, 2, 3 or 4 nitrogen heteroatoms depending on the particular structure. N- heteroaryl groups may also have heteroatoms other than nitrogen, but N-heteroaryl groups are characterized by having at least one nitrogen heteroatom. Exemplary N-heteroaryl groups include imidazolyl, indolyl, (e.g., 2- or 3- indolyl), naphthyridinyl, pyrazinyl, pyridyl (e.g., 2- , 3- or 4-pyridyl), pyrrolyl, pyrimidinyl, quinolinyl (e.g., 2-, 3-, 4-, 5-, or 8-quinolinyl), isoquinolinyl, quinazolinyl, quinoxalinyl and triazinyl, benzimidazolyl, triazolyl, tetrazinyl and carbazolyl. As used herein, the term “heteroaryl” or “heteroaryl group” is also intended to encompass optionally substituted partially saturated bicyclic aromatic heterocyclic moiety in which a heterocycle and a cycloalkyl or cycloalkenyl group are fused together to form a cyclic structure. The heteroaryl group may be a terminal group or a bridging group and may be attached through a heteroatom or any carbon ring atom. The present invention is also intended to encompass salts of the N-heteroaryl groups disclosed herein. For example, the salt of an N-heteroaryl may be an acid addition salt, such as an HC1 or HBr addition salt. Nonlimiting examples of N-heteroaryl salts include benzimidazolium, imidazolium, triazolium and pyridinium salts.

[0060] As used herein, the term “biocompatible” when used in relation to a substance means that the substance is not substantially harmful or toxic to living tissue. A substance may be inherently biocompatible, or it may be present in an amount that is not harmful or toxic to living tissue.

[0061] The terms “treat”, “treating” or “treatment” with regard to a condition (e.g., corneal endothelial dysfunction) refers to alleviating or abrogating the cause and / or the effects of the condition. As used herein, the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and / or duration of the condition, or the amelioration of one or more symptoms (e.g., one or more discernible symptoms) of the condition (i.e., “managing” without “curing” the condition), resulting from the administration of one or more therapies (e.g., an ocular implant as described herein). In specific embodiments, the terms “treat”, “treatment” and “treating” refer to the amelioration of at leastone measurable physical parameter of a condition described herein. In other embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a condition described herein, either physically by, e.g., stabilization of a discernible symptom or physiologically by, e.g., stabilization of a physical parameter, or both.

[0062] As used herein, and unless understood otherwise in the art, the term “about” typically means ±10% of the recited value.

[0063] The terms “v / v%” and “vol%” are used interchangeably herein and have the same meaning.

[0064] Throughout this specification and the claims that follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0065] The term “consisting of’ means “consisting only of’, that is, including and limited to the integer or step or group of integers or steps, and excluding any other integer or step or group of integers or steps.

[0066] The term “consisting essentially of’ means the inclusion of the stated integer or step or group of integers or steps, but other integer or step or group of integers or steps that do not materially alter or contribute to the working of the invention may also be included.

[0067] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge.

[0068] Other definitions are provided throughout the description.DETAILED DESCRIPTION

[0069] The present invention provides biocompatible cross-linkable polymer compositions that upon being cross-linked can be used as a biocompatible adhesive or “bioglue” for adhering a biocompatible hydrogel to a biological substrate, or which may be used in theabsence of a hydrogel to provide a suitable scaffold for a biological substrate. The present inventors have found that the biocompatible cross-linkable polymer compositions disclosed herein may be useful in preparing laminate structures suitable for use as ocular implants for the treatment of corneal endothelial dysfunction. Advantageously, the use of ocular implants in accordance with the present invention may lead to higher success rates in corneal endethelial layer tranasplants, reduced procedure times and / or lower rates of long term rejection.

[0070] A biocompatible cross-linkable polymer composition in accordance with the present invention comprises a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three poly ether arms each comprise a polymerisable ethylenically unsaturated group. In one or more embodiments, the cross-linkable branched polyether compound is not:SUBSTITUTE SHEET (RULE 26)wherein n is an integer independently ranging from about 2 to about 75.

[0071] The biocompatible cross-linkable polymer composition may be provided neat, or it may be provided in an aqueous solution or suspension. Thus, the biocompatible cross-linkable polymer composition may be soluble in, or miscible with, an aqueous liquid. In one or more embodiments, the biocompatible cross-linkable polymer is aqueous soluble. In that case, the biocompatible cross-linkable polymer composition may also be described as an aqueous soluble biocompatible cross-linkable polymer composition. In other embodiments, the biocompatible cross-linkable polymer is miscible with an aqueous liquid. For example, a biocompatible cross-linkable polymer in accordance with the present invention may be suspended in aqueous solution in the form of a self-assembled polymer, micelle, dispersion or emulsion.

[0072] The core moiety of the cross-linkable branched polyether compounds disclosed herein may be any suitable core moiety including, but not limited to, a hydrocarbon, carbohydrate, heteroalkyl, heterocycloalkyl or heteroaryl moiety. It will be appreciated by those skilled inSUBSTITUTE SHEET (RULE 26)the art that, in order to be covalently bonded to at least three polyether arms, the core moiety must be a 3- or more valent (e.g., a 3-valent, 4-valent, 5-valent, 6- valent, or more) core moiety.

[0073] In embodiments in which the core moiety is a hydrocarbon moiety, the hydrocarbon moiety may be a linear, branched, cyclic or aryl hydrocarbon moiety. It is to be understood that a hydrocarbon moiety includes any suitable moiety consisting of carbon and hydrogen atoms (e.g., an alkyl, cycloalkyl, or aryl group as defined elsewhere herein). In some embodiments, the hydrocarbon moiety comprises 1 carbon atom (i.e., a trivalent or tetravalent methyl group). In other embodiments, the hydrocarbon moiety is a linear or branched or cyclic hydrocarbon moiety comprising from 2 to 12 carbon atoms (e.g., Cz alkyl, Cs-izcycloalkyl), or from 3 to 8 carbon atoms (e.g., C3-8 alkyl, C3-8 cycloalkyl), or from 3 to 5 carbon atoms (e.g., Cs-salkyl, Ca-scycloalkyl). In other embodiments, the hydrocarbon moiety may be an aryl moiety comprising, for example, 6 to 12 carbon atoms (Ce ryl), or 6 to 10 carbon atoms (Ce-ioaryl). In one or more embodiments, the hydrocarbon moiety is a linear or branched hydrocarbon moiety comprising from 2 to 12 carbon atoms (e.g., C2-i2alkyl), or from 3 to 8 carbon atoms (e.g., Cs-salkyl), or from 3 to 5 carbon atoms (e.g., Cs-salkyl).

[0074] In embodiments in which the core moiety is a carbohydrate moiety, the carbohydrate moiety may be a linear, branched, cyclic or aryl carbohydrate moiety. It is to be understood that a carbohydrate moiety includes any suitable moiety consisting of carbon, hydrogen and oxygen atoms, including but not limited to, sugars, starches and cellulose. In some embodiments, the carbohydrate is a monosaccharide, examples of which may include, but are not limited to, glucose, galactose, mannose, fructose and ribose. In other embodiments, the carbohydrate moiety is a polysaccharide (i.e., comprising two or more saccharide units), examples of which may include, but are not limited to, dextran, pullulan, chitin, chitosan, inulin, cyclodextrin and hyaluronic acid.

[0075] The core moiety may also be selected from any other suitable core moiety known in the art, including but not limited to, a heteroalkyl, heterocycloalkyl or heteroaryl moiety as described elsewhere herein. The core moiety (e.g., the hydrocarbon, carbohydrate, heteroalkyl, heterocycloalkyl or heteroaryl moieties disclosed herein) may be optionally substituted with one or more substituents, such as hydroxyl (-OH), halo (-F, -Cl, -Br or -I),cyano (-CN), nitro (-NO2), alkoxy (-O-alkyl), carboxyl (-COOH), ester (-COO-alkyl), amino (-NR2, wherein each R is independently selected from H and alkyl), amido (-CONR, wherein R is H or alkyl), and the like. It is to be further understood that any optional substituents are to be selected such that the cross-linkable polymer remains biocompatible (i.e., not substantially harmful or toxic to living tissue). The selection of such optional substituents and processes for their introduction into the biocompatible cross-linkable polymer compositions as disclosed herein will be within the purview of a person skilled in the art.

[0076] The poly ether arms of the cross-linkable branched poly ether compounds disclosed herein comprise a polyether segment and a polymerisable ethylenically unsaturated group. The at least three polyether arms may be the same or they may be different. Further, each polyether segment may comprise the same repeat units, or they may be a random or block copolymer comprising different repeat units. The polymerisable ethylenically unsaturated group may independently present pendant from or at the terminus of each polyether arm. In one embodiment, the polymerisable ethylenically unsaturated group presents at the terminus of each polyether arm.

[0077] In one or more embodiments, the polyether segment is a polyoxyalkylene segment. In the context of a polyoxyalkylene, the term “oxyalkylene” used herein is intended to mean a divalent -O(CRxRY)i- group, where Rxand RYare each independently selected from hydrogen and optionally substituted alkyl, and i is an integer ranging from 1 to 10. Generally, Rxand RYare each independently selected from hydrogen and optionally substituted Ci -ealkyl, and i is an integer selected from 2, 3, and 4. When i > 1 , each (CRXRY) may be the same or different. For example, when the oxyalkylene unit is an oxyethylene unit, Rxand RYare both hydrogen and i=2 (i.e., -O(CH2)2-), and when the oxyalkylene unit is an oxypropylene unit, i=2 and Rxand RYof the first “i” are both hydrogen and Rxand RYof the second “i” can respectively be hydrogen and methyl (i.e., -OCH2CH(CH3)-). Each oxyalkylene group or unit within the polyoxyalkylene may be the same or different. In other words, the polyoxyalkylene may be a homopolymer or a copolymer (including a random or block copolymer). The oxy alkylene units may be derived from an alkylene oxide such as ethylene oxide, propylene oxide, or butylene oxide. The polyoxyalkylene segment may have a molecular weight in a range offrom about 100 to 10,000 Da, from about 150 to 5000 Da, and from about 200 to 1000 Da.

[0078] In one or more embodiments, the poly ether segment is selected from a poly (alkylene glycol), such as poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(isopropylene glycol) or a copolymer or terpolymer of ethylene glycol, propylene glycol and / or isopropylene glycol. In one or more embodiments, the polyether segment is a poly(ethylene glycol) segment or a polypropylene glycol) segment. The poly(ethylene glycol) segment or the polypropylene glycol) segment may contain between about 1 and about 100 repeat units, or between 4 and 40 repeat units, or between 6 and 12 repeat units. Other suitable polyether segments will be apparent to those skilled in the art and may include, for example, polyethers based on dioxanes, such as poly(l,4-dioxane), poly(4-methyl-l ,3- dioxane), and the like.

[0079] The poly ether arms may be covalently linked to the core moiety by a direct bond or by a linker moiety, e.g., a divalent linker moiety. The linker moiety may be any suitable linker moiety including, but not limited, to an alkyl (e.g., a Ci-ealkyl) or an alkoxy (e.g., a -O-Ci- ealkyl) linker. In some embodiments, other linkers such as carbonyl, ester or amide moieties may also be suitable.

[0080] The poly ether arms of the cross-linkable branched poly ether compounds disclosed herein each comprise a polymerisable ethylenically unsaturated group. Each polyether arm may comprise the same or different polymerisable ethylenically unsaturated group. Suitable polymerisable ethylenically unsaturated groups will be apparent to those skilled in the art and may include, but are not limited to, monovalent acrylates, monovalent methacrylates, monovalent vinyl carbonates, monovalent O-vinyl carbamates, monovalent N-vinyl carbamates, monovalent acrylamides and monovalent methacrylamides. In one more embodiments, the polymerisable ethylenically unsaturated groups forms part of a (meth)acryloyl, (meth) acryloyloxy, styrenyl, vinyl ether, vinyl ester or (meth)acrylamide group. In one or embodiments, the polymerisable ethylenically unsaturated groups form part of a (meth)acryloyl group.

[0081] The polymerisable ethylenically unsaturated groups may be covalently linked to the polyether arms by a direct bond or by a linker moiety, e.g., a divalent linker moiety. The linkermoiety may be any suitable linker moiety including, but not limited to, an alkyl (e.g., a Ci- ealkyl), alkenyl (e.g., Cz-ealkenyl) ether (e.g., -R-O-R', wherein each R and R' is independently Ci-ealkyl), ester (e.g., -R-C(O)O-R', wherein each R and R' is independently Ci-ealkyl), amide (e.g., -R-C(O)N(R")-R', wherein each R, R' and R" is independently Ci- ealkyl), carbamate (e.g., -R-OC(O)N(R")-R', wherein each R, R' and R" is independently Ci- ealkyl), isocyanate, succinimidyl, or succinmidyl carbonate linker moiety.

[0082] In some embodiments, the cross-linkable branched polyether compound has a structureA(BX)nwherein:A is an n-valent core moiety;BX is a poly ether arm, wherein B is a poly ether segment and X is a polymerisable ethylenically unsaturated group; and n is at least 3.

[0083] In one or more embodiments, A(BX)nis not:SUBSTITUTE SHEET (RULE 26)wherein n is an integer independently ranging from about 3 to about 75.

[0084] Suitable n-valent core moieties (A), polyether arms (BX), polyether segments (B) and polymerisable ethylenically unsaturated groups (X) are as described above.

[0085] In one or more embodiments, the cross-linkable branched polyether compound is:where each n is an integer independently ranging from about 4 to about 40, preferably from about 6 to about 12.SUBSTITUTE SHEET (RULE 26)

[0086] In one or more embodiments, the cross-linkable branched polyether compound is:where each n is an integer independently ranging from about 4 to about 40, or from about 6 to about 12.

[0087] The cross-linkable branched polyether compounds disclosed herein may be prepared by any suitable method known in the art. One such method, as exemplified in Scheme 1 below, involves esterification of a polyether polyol compound, such as commercially available glycerol ethoxylate, with a suitable electrophile (e.g., a carboxylic acid, acid chloride or anhydride) comprising an ethylenically unsaturated compound, such as (meth)acryloyl chloride or (meth)acrylic anhydride, to form ester bonds. Other suitable methods will be apparent to those skilled in the art and may include, for example, thiol to alkene click reactions, isocyanate to amine reactions; azide (N3) to alkene click reactions, N- hydroxysuccinimide to alcohol or amine reactions, and the like.Scheme 1SUBSTITUTE SHEET (RULE 26)

[0088] When cross-linked, the biocompatible cross-linkable polymer compositions disclosed herein form a cross-linked network polymer. Such cross-linked network polymers may be suitable for use as biocompatible polymer adhesives (or “bioglues”), for example, for adhering a biocompatible hydrogel to a biological substrate. Such cross-linked network polymers may also be suitable for use as a scaffold for a biological substrate in the absence of a hydrogel. Thus, the present invention also provides a cross-linked network polymer comprising polymerised residues of a cross-linkable branched polyether compound as disclosed herein.

[0089] In one or more embodiments, the cross-linked network polymers disclosed herein, and the cross-linkable polymer compositions from which they are derived, are biodegradable. As used herein, the terms “degradable” and “biodegradable” in relation to a substance means that the substance is susceptible to degradation, cleavage or fragmentation over time under physiological conditions or in a biological environment. Such degradation, cleavage or fragmentation may occur via chemical decomposition (e.g., via hydrolysis or reduction) of suitably labile moieties under selected physiological or biological conditions. When used in relation to a polymer substance, the terms “degradable" and “biodegradable” indicate that the polymer comprises suitably labile or degradable moieties as part of the molecular structure of the polymer. The cleavage or breakdown of one or more degradable moieties in the polymer leads to fragmentation of the polymer, generally into monomers and / or lower molecular weight polymer fragments. Further, where a substance described herein is both “biocompatible” and “biodegradable”, it is to be understood that the degradation by-products produced should also be biocompatible, i.e., not substantially harmful or toxic to living tissue.

[0090] Cross-linking of the cross-linkable branched polyether compounds disclosed herein may be achieved using any suitable method known in the art. In one or more embodiments, the biocompatible cross-linkable polymer composition is formed by photo-cross-linking, e.g., in the presence of light and, optionally, one or more photoinitiators. Advantageously, photo- cross-linking may be achieved in the presence of biological materials, such as cells, without substantially damaging the biological material. In one or more embodiments, cross-linking is achieved in the presence of one or more biocompatible photoinitiators. In some embodiments, the biocompatible photoinitiators are aqueous soluble. The wavelength of the light required to initiate photo-cross-linking may depend on the specific photoinitiator and may be visible,infrared light or ultraviolet light. In certain embodiments involving cross-linking in the presence of biological materials, it may be advantageous for the wavelength of the light required to initiate photo-cross-linking to be in the visible or infrared spectrum. In some embodiments, the wavelength of the light required to initiate photo-cross-linking is in the visual spectrum.

[0091] Suitable biocompatible photoinitiators for use in the present invention may include, but are not limited to, Eosin Y with triethanolamine and vinyl caprolactam; tris(2,2- bipyridyl)dichlororuthenium(II) hexahydrate (Ru(bpy)s) with sodium persulfate; camphorquinone and / V, / V-dimethyl-p-toluidine, 2-ethyl-dimethylbenzoate, or N- phenylglycine; 2,2,2,6,6-tetramethylpiperidine; dl-2,3-diketo-l,7,7-trimethylnorcamphane (CQ); l-phenyl-l,2-propadione (PPD); 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO); bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819); 4,4’- bis(dimethylamino)benzophenone; 4,4’-bis(diethylamino)benzophenone; 2- chlorothioxanthen-9-one; 4-(dimethylamino)benzophenone; phenanthrenequinone; ferrocene; diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide / 2-hydroxy-2- methylpropiophenone (50 / 50 blend); dibenzosuberenone; resazurin; resorufin; benzoyltrimethylgermane (Ivocerin®); derivatives thereof, and combinations thereof.

[0092] In particular, a suitable photoinitiator and reaction conditions have been described by Teymour et al. (2004), Langmuir, 20, 8652-8658, the entire contents of which are incorporated herein by cross-reference. Thus, in one or more embodiments, the photoinitiator comprises Eosin Y, and triethanolamine. The photoinitiator may further comprise vinyl caprolactam and / or other additives to facilitate photo-cross-linking. Advantageously, photo-cross-linking in the presence of Eosin Y may be carried out under mild conditions using visual light (514 nm), which are compatible with biological materials. In some embodiments, the photoinitiator is provided in the form of an aqueous solution comprising: Eosin Y at a concentration of between about 0.0125 mM and about 0.5 mM, or between about 0.025 mM and about 0.15 mM; triethanolamine at a concentration of between about 0.05% (w / v) and about 1.5% (w / v), or between about 0.2 % (w / v) and about 1.5% (w / v); and vinyl caprolactam at a concentration of between about 0.05% (w / v) and about 1.5% (w / v), or between about 0.09% (w / v) and about 0.8% (w / v). In an embodiment, the photoinitiator is provided in theform of an aqueous solution comprising: Eosin Y at a concentration of about 0.05 mM, triethanolamine at a concentration of about 0.4 % (w / v), vinyl caprolactam at a concentration of about 0.4 % (w / v).

[0093] Formation of a cross-linked network polymer according to the invention may comprise combining the biocompatible cross-linkable polymer compositions disclosed herein with a photoinitiator in the presence of water. The water may be any suitable form of water, nonlimiting examples of which include distilled water, deionised water (DIW), filtered water, sterilised water, balanced salt solution (BSS) and phosphate buffer solution (PBS). In certain embodiments in which the photoinitiator is Eosin Y with triethanolamine and vinyl caprolactam, the ratio of photoinitiator to biocompatible cross-linkable polymer is about 2:1 or lower by volume, e.g., between about 2:1 and about 10:1, or between about 2:1 and about 7: 1 , or between about 2 : 1 and about 5 : 1 by volume.

[0094] The biocompatible cross-linkable polymer composition may be combined in aqueous solution with the photoinitiator and stored (for example, at low temperature, e.g., 4 °C, -20 °C, or less) before further use, e.g., as a bioglue, or they may be combined immediately prior use, e.g., within 24 hours, or within 12 hours, or within 6 hours, or within 2 hours, or within 30 minutes, or within 15 minutes. Thus, the present invention also provides a composition comprising a biocompatible cross-linkable polymer composition as described herein, a biocompatible photoinitiator and water. Such compositions may comprise one or more biocompatible additives. For example, one or more radical inhibitors may be included in the composition, such as butylated hydroxy toluene (BHT); 4-methoxylphenol, mequinol (MEHQ); phenothiazine; 4-tert-butylcatechol (TBC); hydroquinone (HQ); cupferrin (N- nitroso-N-phenylhydroxylamine); 4-t-nutylpyrocatechol; N,N-diethylhydroxylamine; or any combination thereof.

[0095] The biocompatible cross-linkable polymer composition disclosed herein may be contained in a kit with one or more photoinitiators. The kit may include, for example, the biocompatible cross-linkable polymer composition and the photoinitiator(s), each packaged or formulated individually, or packaged or formulated in combination. Thus, the kit may comprise a first and second part (e.g., a first and second container), wherein the biocompatiblecross-linkable polymer composition is present in the first part and the photoinitiator is present in the second part, e.g., in the form of an aqueous solution. The parts (e.g., containers) may be placed within a package, and the package can optionally include instructions for combining the biocompatible cross-linkable polymer composition and the photoinitiator and / or their subsequent use as a bioglue composition for adhering a biocompatible hydrogel to a biological substrate or as a scaffold for a biological substrate in the absence of a hydrogel. Thus, in one or more embodiments, the present invention provides a kit comprising: a first part comprising the biocompatible cross-linkable polymer composition as disclosed herein; and a second part comprising water and a biocompatible photoinitiator. The kits disclosed herein may optionally comprise one or more additives, such as those described above, which may be present in the first part, the second part, or a third or subsequent part of the kit.

[0096] The kits of the present invention may be stored at ambient temperature or at low temperature (e.g., 5 °C, -20 °C). The duration of stability of the kits may depend on the makeup of the kits and their individual parts and the conditions under which they are stored. In some embodiments, the stability of the individual parts of the kit may be enhanced by including one or more additives, for example, a radical inhibitor, in the first part, the second part, or both. In some embodiments, the kits are stable for at least about 2 months, or at least about 6 months, or at least about 12 months.

[0097] The biocompatible cross-linkable polymer compositions of the present invention, or kits comprising the same, may be used for adhering a biocompatible hydrogel to a biological substrate. In particular, by providing a composition comprising the biocompatible crosslinkable polymer composition, a photoinitiator and water (referred to herein as a an “aqueous bioglue composition”) between a biocompatible hydrogel layer and a biological substrate layer and irradiating the resulting laminate structure at a suitable wavelength to initiate photocross-linking, the resulting cross-linked network polymer may act as a bioglue to improve adherence of the biocompatible hydrogel to the biological substrate. An improvement in adherence may be characterised by an increase in the duration of adherence, an increase in the strength of adherence, or both, of the biocompatible hydrogel to the biological substrate relative to the adherence of the hydrogel to the substance in the absence of the bioglue (i.e., the cross-linked network polymer). In certain embodiments, the duration of adherence of ahydrogel to the substrate using the cross-linked network polymers disclosed herein may be at least about 1 week, 2 weeks, 3 weeks, 1 month, or more. The strength of adherence of a hydrogel to the substrate may be modified by adjusting the concentration of bioglue used in the application (e.g., increasing the concentration of bioglue may increase the adherence strength). The adherence strength may be tested, for example, using a lap shear test.

[0098] Thus, the present invention provides a process for adhering a biocompatible hydrogel to a biological substrate. The process may comprise combining a biocompatible cross-linkable polymer composition as disclosed herein with water and a biocompatible photoinitiator to form an aqueous bioglue composition. The relative amounts of biocompatible cross-linkable polymer composition, photoinitiator and water may depend on the specific polymer composition and / or photoinitiator used. In one or more embodiments, the aqueous bioglue composition comprises the cross-linkable polymer composition in an amount of from about 10 vol% to about 50 vol%, or from about 10 vol% to about 40 vol%, or from about 20 vol% to about 50 vol% (based on the neat composition). As described above, the biocompatible cross-linkable polymer composition may be combined in aqueous solution with the photoinitiator and stored prior to use as a bioglue, or the components may be combined immediately prior to use as a bioglue (e.g., within 24 hours, or within 12 hours, or within 6 hours, or within 2 hours, or within 30 minutes, or within 15 minutes).

[0099] The aqueous bioglue composition so prepared may then be provided between a biocompatible hydrogel layer and a biological substrate layer to form a laminate structure. For example, the aqueous bioglue composition may be applied (e.g., in one or more droplets or in a homogenous layer) to the biocompatible hydrogel layer, and the biological substrate layer subsequently applied to the aqueous bioglue composition, or vice versa. In some embodiments, the ratio of the aqueous bioglue composition to the biocompatible hydrogel layer is from about 3:1 to about 5:1 by volume (i.e., from about 17 vol% to about 25 vol% of the aqueous bioglue composition). The resulting laminate structure may then be irradiated at a wavelength suitable to activate the biocompatible photoinitiator and initiate cross-linking of the cross-linkable branched polyether compound (and that does not damage the biological substrate), the process of which promotes adhesion between the biocompatible hydrogel and the biological substrate.

[0100] Thus, the present invention also provides a laminate structure comprising a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the cross-linked network polymer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three poly ether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group. In some embodiments, the ratio of the biocompatible cross-linked network polymer layer to the biocompatible hydrogel layer is from about 3 : 1 to about 5 : 1 by volume.

[0101] The biocompatible cross-linkable polymer compositions of the present invention, or kits comprising the same, may also be used as a scaffold for a biological substrate. In particular, by providing a layer of an aqueous bioglue composition as described herein on a biological substrate layer and irradiating the resulting structure at a suitable wavelength to initiate photo-cross-linking, the resulting cross-linked network polymer may adhere to the biological substrate layer and act as a scaffold for the biological substrate. In certain embodiments, the duration of adherence of a cross-linked network polymer to the substrate may be at least about 1 week, 2 weeks, 3 weeks, 1 month, or more.

[0102] Thus, the present invention also provides a process for preparing a biological substrate having adhered thereto a cross-linked network polymer scaffold. The process may comprise preparing an aqueous bioglue composition as described above, A layer (e.g., a homogenous layer) of the aqueous bioglue composition so prepared may then be applied to a biological substrate layer to form a laminate structure. The resulting laminate structure may then be irradiated at a wavelength suitable to activate the biocompatible photoinitiator and initiate cross-linking of the cross-linkable branched polyether compound (and that does not damage the biological substrate), the process of which promotes formation of a cross-linked network polymer scaffold adhered to the biological substrate.

[0103] Thus, the present invention also provides a laminate structure comprising a biocompatible cross-linked network polymer layer and a biological substrate layer, wherein the cross-linked network polymer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at leastthree polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group.

[0104] The choice of biocompatible hydrogel and / or biological substrate in the laminate structures of the invention may depend on its intended application. In that respect, the laminate structures disclosed herein may be particularly useful as implantable devices for use in tissue regeneration. For example, the laminate structure may comprise a biological substrate selected so as to replace damaged tissue at a site within a subject. Advantageously, the use of biodegradable and / or biocompatible components in the laminate structures as disclosed herein may provide a scaffold for a biological substrate that degrades over time to produce non-toxic degradation by-products, thereby preventing foreign material from accumulating at the implantation site and allowing the surrounding tissue to revert to its native structure. The rate of biodegradation may vary from days to weeks, e.g., about 2 weeks to about 20 weeks, or more.

[0105] In certain embodiments of the laminate structures disclosed herein, the biological substrate comprises Descemet's membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells. The layer of corneal cells may further comprise comeal epithelial cells, comeal stromal cells, or a combination thereof. Typically, when used in endothelial keratoplasty, Descemet's membrane is obtained from a donor and comprises a layer of the donor's corneal endothelial cells already attached thereto. In some cases, it may be necessary to seed corneal endothelial cells onto Descemet’s membrane (e.g., where the patient’s own membrane is used). It is to be understood that reference herein to Descemet’s membrane having attached thereto layer of corneal cells comprising comeal endothelial cells is intended to include circumstances in which the comeal cells are naturally occurring on the membrane and in which the corneal cells have been seeded onto the membrane. In other embodiments, corneal cells are incorporated into the biocompatible hydrogel layer as described herein. For example, comeal cells may be grown on the biocompatible hydrogel. In some embodiments, the corneal cells may be treated with growth factors and / or other biological and chemical entities that facilitate the growth or proliferation the cells.

[0106] Furthermore, in certain embodiments involving a biocompatible hydrogel, the biocompatible hydrogel comprises a biodegradable network polymer as described in WO2014 / 165917, the entire contents of which are incorporated herein by cross-reference. In a particular embodiment, the biodegradable network polymer is a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages. Such biodegradable and biocompatible polyether network polymer may be prepared by polymerising a monomer composition comprising a multifunctional polyether monomer and a multifunctional crosslinking monomer, wherein one of the polyether monomer and the cross-linking monomer comprises a hydroxy functional group and the other of the polyether monomer and the crosslinking monomer comprises a complementary functional group capable of reacting with the hydroxy functional group to form an ester linkage, and wherein the polyether monomer is branched.

[0107] A laminate structure in accordance with the present invention, in which the biological substrate is Descemet's membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells, the cross-linked network polymer is as described herein and the optional biocompatible hydrogel comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages, may be suitable for use as an ocular implant for treating corneal endothelial dysfunction in a subject. It is to be understood that the bioglue is applied to the Descemet’s membrane that does not have corneal cells attached thereto (i.e., the endothelial side) and that contact of the bioglue with the corneal cells should be avoided where possible. Advantageously, the bioglue, optionally together with the hydrogel, provide a scaffold for Descemet's membrane (and the attached corneal endothelial cells) that may spontaneously unscroll when placed in the eye, thereby reducing the risk of damage to the implant compared to traditional DMEK grafts. Further, in embodiments involving a hydrogel scaffold, the improved adherence of the hydrogel to the membrane provided by the bioglue (relative to when the biocompatible hydrogel is applied directly to Descemet's membrane) may reduce the need for repeated positioning of the implant due to delamination of the membrane. Furthermore, the ocular implants disclosed herein may be suitably transparent when applied to the eye.

[0108] Thus, the present invention also provides an ocular implant having a laminate structure comprising a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages, and wherein the biological substrate layer is Descemet's membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells. An example of a suitable ocular implant is described in Example 1.

[0109] The present invention also provides an ocular implant having a laminate structure comprising a biocompatible cross-linked network polymer layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, and wherein the biological substrate layer is Descemet's membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells. An example of a suitable ocular implant is described in Example 2.

[0110] An ocular implant according to the present invention may be prepared and stored prior to use, or it may be prepared during implant surgery (e.g., using the patient’s own Descemet's membrane). Preferably, the ocular implant is stored at 4 °C in the absence of light for up to 14 days. The ocular implant may be stored, for example, in a surgical implantation device, such as a Busin Glide or Endoglide™ system. In some embodiments, the ocular implant may be prepared and stored in unscrolled form in a suitable preservation medium. Suitable preservation media will be known to those skilled in the art and may include, but are not limited to, PBS, BSS, Optisol GS™ (comprising dextran, 2.5% chondroitin sulfate, vitamins, and precursors of adenosine triphosphate (i.e., adenosine, inosine and adenine)) and Life4C™ (glutathione and human insulin to maintain cell viability at 4 °C). In otherembodiments, the ocular implant may be prepared and stored in scrolled form. The scrolled form may be provided in a suitable applicator for administration to the eye of a patient, such as a Busin glide.

[0111] It is to be understood that an ocular implant according to the present invention should be transparent to visual light. For example, the ocular implant should be greater than 98%, or greater than 99%, or greater than 95.5% transparent to visible light. Furthermore, the ocular implant should be of a suitable thickness that facilitates manipulation of the device and implantation of the device in a desired site in an eye of a subject. The thickness of the implant may be adjusted, for example, by altering the composition of the biocompatible hydrogel layer and / or the biocompatible cross-linked network polymer (bioglue) layer. Such adjustments may be achieved, for example, by altering the starting monomers from which the hydrogel and / or biocompatible cross-linked network polymer are made and / or the quantity thereof. In an embodiment, the ocular implant has a thickness in the range of from about 100 pm to about 500 pm, or about 100 pm to about 400 pm, or about 150 pm to about 300 pm, or about 150 pm to about 250 pm. The biocompatible cross-linked network polymer (bioglue) layer may comprise from about 50 pm to about 250 pm, or about 50 pm to about 200 pm, or about 50 pm to about 180 pm of the total thickness of the ocular implant.

[0112] The present invention further provides a method for treating comeal endothelial dysfunction in a subject, the method comprising a step of implanting an ocular implant as described herein in an eye of the subject.

[0113] In some embodiments, the subject in need of treatment or prevention of comeal endothelial dysfunction is a mammal. The term “mammal” as used herein includes humans, primates, livestock animals (e.g., horses, cattle, sheep, pigs, donkeys), laboratory test animals (e.g., mice, rats, guinea pigs), companion animals (e.g., dogs, cats) and captive wild animals (e.g., kangaroos, deer, foxes). In one or more embodiments, the mammal is a human.

[0114] The present invention also provides an ocular implant as described herein for use in treating corneal endothelial dysfunction.

[0115] The present invention also provides use of corneal endothelial cells in the manufacture of a medicament for treating corneal endothelial dysfunction, wherein the medicament comprises a laminate structure comprising a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three poly ether arms, wherein the at least three poly ether arms each comprise a polymerisable ethylenically unsaturated group, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages, and wherein the biological substrate layer is Descemet's membrane having attached thereto a layer of the corneal endothelial cells.

[0116] The present invention also provides use of corneal endothelial cells in the manufacture of a medicament for treating corneal endothelial dysfunction, wherein the medicament comprises a laminate structure comprising a biocompatible cross-linked network polymer layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, and wherein the biological substrate layer is Descemet's membrane having attached thereto a layer of the corneal endothelial cells.

[0117] The medicaments described herein may comprise the laminate structure in scrolled or unscrolled form. The unscrolled laminate structure may be suspended in a suitable preservation medium, such as PBS, BSS, Optisol™ or Life4C™. Alternatively the scrolled laminate structure may be provided in a suitable applicator for administration to the eye of a subject, such as a Busin glide. The medicament may be stored (for example, at low temperature, e.g., 4 °C) for a period of time (e.g., up to about 14 days) prior to use.

[0118] Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The inventionalso includes all such steps, features, methods, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0119] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.EXAMPLESExample 1. Preparation of hydrogel-bioglue-tissue (HBT) graft

[0120] The present inventors proposed a two component system consisting of a photoinitiating solution (Part A) containing Eosin Y, triethanolamine (TEO) and N- vinylcaprolactam (VC) in PBS, and a PEG-based biocompatible cross-linkable polymer composition (Part B) to prepare a multifunctional aqueous bioglue composition that is photocross-linked in situ to adhere a hydrogel film to Descemet’s membrane in preparation for DMEK surgery. The resulting hydrogel-bioglue-tissue (HBT) graft may allow for the simplification of the highly desirable yet previously difficult DMEK surgical procedure.1 Materials and methods1.1 Materials|00121 | Aluminium oxide (Brockmann 1, basic, AI2O3), aluminium oxide (Brockmann 1, acidic, AI2O3), 4-(dimethylamino)pyridine (DMAP, >99%), Eosin Y (>99%), glycerol ethoxylate (GE< Mn~ 1000), dried magnesium sulfate (dried MgSO4, >98%), methacrylic anhydride (MAA, >94%), phosphate buffered saline-tablet (PBS, 1 per 200 mL), sodium hydroxide (NaOH, >98%), sodium bicarbonate (NaHCOs, >99.5%), triethylamine (TEA, >99%), triethanolamine (TEO, >99%), and V-viny Icaprolaclam (VC, 98%) were all purchased from Merck and used without further purification. Acetone (AR grade), Acetonitrile (CH3CN, AR Grade, Chemsupply), deuterated chloroform (CDCI3, Cambridge Isotope Laboratories), dichloromethane (DCM, Honeywell), Balanced Salt Solution (BSS), aqueous hydrochloric acid (aq. HC1, 0.5 M), human cornea stromal keratocytes conditioned media (hCSK) was supplied by CERA, stored at 4 °C and used within 7 days of delivery.1.2 Instrumentation

[0122] Nuclear magnetic resonance spectra were recorded on a Broker (Ascend 400MHz) spectrometer using deuterated chloroform (CDCh) as reference unless otherwise stated. UV- Vis Spectroscopy was performed on a Shimadzu UV-Vis Scanning Spectrophotometer (UV- 2101 PC) with a fast scanning rate of 1 nm intervals. Attenuated Total Reflection-Fouriertransform infrared (ATR-FTIR) spectroscopy was carried out using a Bruker Tensor 27 FTIR, with a GladiATR ATR attachment obtained from Pike Technologies. Bioglue Part B was purified using a Buchi Liquid Chromatography Pure C-850 FlashPrep system fitted with a FlashPure™ EcoFlex Silica cartridge. Compression testing of cross-linked bioglue samples were performed on an Intron 5848 mirotester fitted with a 50N load cell. Tensile testing of polyethylene glycol based hydrogen films (PHFs) were performed in PBS using an Instron 5949 microtester fitted with a 50N load cell.1.3 Formulation of photoinitiator solution - Part A

[0123] A solution containing Eosin Y, a visible light- initiated photoinitiator was used to cross-link the trifunctional pre-polymer (Part B) in the formation of solid bioglue. The photoinitiator solution (Part A) was prepared by dissolving Eosin Y (1.72 mg, 0.265 mM), triethanolamine (82.3 pL, 62.3 pM) and A-vinyl-caprolactam (62.6 mg, 45.0 pM) in phosphate buffer saline (PBS) (10 mL). The stock solution was divided into 0.5 mL aliquots and stored at 4 °C in 1.5 mL Black microcentrifuge tubes and protected from light until required.1.4 Synthesis and purification of biocompatible cross-linkable polymer, bioglue - Part B

[0124] To an oven dried and cooled round bottom flask (RBF) (500 mL) was added glycerol ethoxylate (GE) (16.0 g, MW 1 kDa, 16.0 mmol) weighed on a 3-figure weighing balance. 4- Dimethylamino pyridine (DMAP) (0.64 g, 5.24 mmol) was weighed on weighing paper using a 4-figure balance and transferred to the flask. Triethylamine (10.4 mL, 0.075 mmol) was then added. The RBF was fitted with a magnetic stirrer bar and suba-seal, placed above a magnetic stirrer plate (100 rpm). Anhydrous DCM (100 mL) was transferred to the flask via an oven dried needle and disposable syringe (100 mL). The flask was purged via a needle with argon for 30 min. The reaction mixture was treated with Methacrylic anhydride (MA) (9.60 mL,64.4 mmol) via a plastic disposable syringe and needle. Anhydrous triethylamine (TEA) (10.4 mL, 74.6 mmol) added via a 20 mL plastic luer-lock syringe and needle. The whole flask was covered with a sheet of domestic aluminium foil to block light from the reaction. The mixture was stirred for 72 hrs at ambient temperature under UHP argon.

[0125] The contents of the reaction flask were transferred to a 2 L separating funnel held within a ring holder mounted to a retort stand. The mixture was diluted with 200 mL of DCM. (AR Grade). Aqueous saturated NaHCOs solution (100 mL) was added, and mechanically shaken, releasing the pressure and return the flask to the stand. The bottom organic layer was collected into a conical flask (1000 mL). The organic phase was returned to the separating funnel and the washing step was repeated twice with more NaHCCL (aq) (2 x 100 mL). The DCM extracts were returned to the separating funnel and washed with subsequent amounts of aqueous HC1 (0.5 M, 3 x 100 mL), and finally saturated aqueous NaCl (1 x 100 mL). The collected DCM extracts were dried with approximately 5 g of anhydrous MgSCh or until the solution became clear. The solution was filtered through a Buchner funnel fitted with a Whatman 542 filter paper and a side arm Erlenmeyer flask under house vacuum. The solution was transferred to a clean conical flask and treated with basic alumina (AI2O3, 5 g) stirred for 5 min followed by filtration as above. The solution was then treated with acidic alumina (5 g), stirred and filter as above. The DCM phase was transferred to a 500 mL RBF and reduced to dryness on a rotary evaporator at 20 °C, under house vacuum (~20 mmHg). The clear paleyellow oil (15.0 g, 78 % yield) was collected and stored at -20 °C until further purification.

[0126] The crude oil was purified using a Buchi Liquid Chromatography Pure C-850 FlashPrep system fitted with a FlashPure™ EcoFlex Silica cartridge. Crude oil (10 g per pass) in 10 mL DCM, was loaded onto an 120 g silica 50 mm column attached to the LC system. The system uses a gradient mixture of Solvent A: DCM (100%) to solvent B: DCM (90%) / methanol (10%) with a flow rate of 60 mL / min. The initial fraction (~7 min) was discarded with the collection of the major fraction at around 10 min when the A:B solvent ratio is 1 :1. These fractions were combined and washed with water (3 x 100 mL) to remove the methanol. The organic phase was dried over MgSCL (~5 g), filtered (#542 filter paper) and reduced to dryness on a rotary evaporator for 2 h at 6 mbar.]H NMR analysis (Figure 1) indicated <1% DCM was present. The purified biocompatible cross-linkable polymer oil (Part B) was stored at -20 °C wrapped in Aluminium foil to exclude light until required. H NMR (400 MHz, CDCh, 8): 6.13 (s, 3H; CH), 5.57 (s, 1H; -CH), 5.30 (s, 2H, CH2CI2), 4.30 (t, 6H, J =6.0 Hz, -CH2O-CO), 3.75 (t, 6H, 7 =6.0 Hz, -CH2-CH2O-), 3.7-3.6 (m, 90H), 1.95 (s, 3H, CH3). ATR-FTIR: 3582, 2865 (C-H), 1717 (C=O), 1633 (C=C), 1452 (C-H), 1294, 1098.1.5 Bioglue formulation and cross-linked sample preparation

[0127] Bioglue samples for UV-Vis analysis were prepared by combining varying ratios of cross-linkable macromonomer and photoinitiator solution (i.e., 1, 2, 3, 4, 5, 7.5 and 10: 1 by volume of Part A relative to Part B). A standard curve was prepared for Eosin Y (taken from the absorbance of kmax = 520 nm) in PBS and used to determine the concentration of bioglue cross-linked samples. Samples (100 pL) of the bioglue formulation were placed in 1 mm path length quartz cells, shone with light (white, green, blue) for a given time and their UV-Vis spectra recorded. The intensity of the absorbance at 520 nm was used to determine the change in Eosin Y concentration. Gelation was recorded as the time taken to produce a visually solid gel which did not flow in the cell.

[0128] Bioglue samples for mechanical testing, biodegradation and swelling experiments were prepared by dissolving various concentrations of Part B (10, 20, 30, 40 and 50 v / v%) in Part A solution (Table 1). Briefly, solutions of mixed homogeneous bioglue formulations injected into the end of 3 mL disposable syringes with the ends cut off (Figure 2c). The array of samples were then shone with white light from a 1400 Lumen (20W) LED flat plate (dimensions: 105 mm x 80 mm) at a distance of 1 cm between the light and the samples (Figure 2a-b). Once cured, the samples were removed and stored at 4 °C prior to biodegradation and compression testing (Figure 2d).1.6 Mechanical tests

[0129] Compression testing of cross-linked bioglue compositions were determined using an Instron 5848 microtester (Instron, USA) fitted with a 50N load cell. Prior to the test the cylindrical photo-cross-linked bioglue samples were placed between the two compression plates with the flat surfaces horizontal to the plate. The compression test was performed at a strain rate of 5 mm / min in accordance with ASTM standard F2255-05 (2015). The initial height and diameter of the samples were measured and recorded using callipers prior to compression. The ultimate stress (o) and strain (e) were recorded at the point of failure. The slope of the stress strain curves at 10% strain was obtained and reported as the Young’s modulus (E). The area under the stress-strain curve prior to break was calculated and reported as the toughness (UT).

[0130] Tensile testing of PHFs were performed using an Instron 5944 microtester (Instron, USA) fitted with a 50N load cell. Dogbone samples of PHF samples were prepared using a Type B C0024 dye cutter (RDM Test Equip. UK., 6 mm diam.) (Figure 3). Samples were pre-swollen in PBS prior to testing and tested hydrated and swollen in a water bath containing PBS. The tensile test was performed at a rate of 5 mm / s. Initial displacement and dimensions of the test area were measured prior to testing.1.7 In vitro swellability tests

[0131] Cross-linked bioglue samples were prepared the same as for mechanical testing according to Table 1. The weight of each sample was measured following cross-linking and after incubation in PBS at 37 °C up to 24 hr. Three samples were prepared for each formulation and their swelling ratio calculated according to Equation 1, where the samples weighed directly after preparation (So) and after swelling in PBS (St) after time t = 1, 2, 5 and 24 h.Swelling Ratio % (St) = (St - So) / St x 100% (1)1.8 Transparency tests

[0132] The transparency of 0.75 cm3photo-cross-linked samples of bioglue formulations were prepared according to Table 1. These were analysed using a photographic method as previously described (Gonzalez- Andrades et al., 2015). Samples were placed on a Koren 2003 lens test chart and photographs of each sample taken. Images were then analysed using ImageJ software from a circular 5 mm region of interest (ROI) and the transparency determined from the maximum (Imax) and minimum (Imin) pixel intensities according to Equation 2. mTransparency1.9 In vitro degradation tests of photo-cross-linked bioglue

[0133] Bioglue samples were prepared according to Table 1 (Entry 1-5) and as described in Section 3.4. After photo-cross-linking sample (n = 6 per formulation) were weighed (Do) then incubated in either PBS or hCSK conditioned media at 37 °C. The samples were periodically removed, dabbed dry and weighed (Dt). The solutions were refreshed every 7 days with freshSUBSTITUTE SHEET (RULE 26)media. The degradation monitoring progressed until the bioglue was not visible in the solution. The weight remaining under degradation after time t, was defined as Dtand calculated according to Equation 3.Weight Remaining (Dt) = Dt / Do x 100% (3)1.10 Preparation of PHF membranes

[0134] In a typical procedure PHFs are prepared based on an adaptation of a procedure by Gonzalez-Andrades et al., 2015. Polycaprolactone diol (PCL, 0.03 g, 0.015 mmol) and glycerol ethoxylate (GE, 0.417 g, 0.417 mmol) were weighed into a glass vial. Anhydrous DCM (10 mL) was added via syringe. The vial was immediately sealed with a cap (prepunctured with a small needle). Once the PCL had fully dissolved, sebacoyl chloride (SbCl, 0.137 mL, 0.641 mmol) was added via a 100 mL pipette and gently until the solution became homogeneous. The solution was poured into pre-dried glass petri-dish (diam. 90 mm) and placed in a pre-heated oven (60 °C) under slight vacuum for 30 min. Full vacuum was applied to the oven by opening the vacuum line fully and closing the bleed valve and left for a further 60 min. The vacuum was then released, and the dish placed immediately into deionised water for 30 min. The whole dish was then transferred into solutions of a. 10%, 25%, and 50% acetone in water for 30 min each. The gels floated cleanly off the petri-dish and was transferred to a beaker of deionised water to removed unreacted monomers. The water was replaced three times over three days to remove any unincorporated material. The gel was then cut into circular discs using a hollow circular 12 mm cutting punch. The PHF discs (8 per vial) were placed in containers containing BSS (10 mL), sealed with Parafilm®, labelled and sent for gamma irradiation. Upon receipt of the irradiated PHF samples they were stored at 4 °C prior to use.1.11 UV-Vis evaluation of PHF-bioglue

[0135] Light transmittance evaluation of photo-cross-linked bioglue were performed on an aliquot of bioglue added to an 8 mm PHF disc. Hydrated PHF was placed in BSS solution for 1 h and then subjected to UV-Vis evaluation. The PHF was dabbed dry and treated with 20 LIL of bioglue Part A:B (5:1), sufficient for coverage of the PHF surface. Sample were then photo-cross-linked with 1400 Lumen light at a distance of 2 cm from the surface. Light transmittance of the PHF-bioglue samples (n=3) were determined after t = 0-, 5- and 10-min irradiation at 25 °C, over the UV and visible spectrum (300-700 nm).1.12 Evaluation of bioglue Adhesion on HBH and HBT grafts

[0136] All bioglue adherence tests were carried out using an Instron 5944 microtester (Instron, USA) fitted with a 50N load cell. The stress-strain curves of each sample was recorded until break at a rate of 5 mm / min. Stress (kPa) was determined from Force (N) / Area (m3), where 1 N / m3= 1 kPa.1.13 Hydrogel-bioglue-hydrogel (HBH) Adhesion studies

[0137] Glass cover slips (24 x 75 mm) were treated with bioglue covering an area of / : 1 cm x d: 2.5 cm. Formulations (50 pL, Part B, 30 v / v%, Part A 70 v / v%) were dispensed onto a cover slip and a second slip placed on top (Figure 4) White light (1400 Lumen) was shone on the sample for 5 min to effect full curing. The tensile test was performed at a rate of 5mm / s.

[0138] Samples of PHF solvent cast onto glass cover slips (24 x 75 mm) were treated with bioglue covering an area of Z:lcm x d: 2.5 cm. Aliquots of bioglue (50 pL, 30%B%) were dispensed onto one PHF coated cover slip and a second PHF coated cover slip placed on top (Figure 5). White light (1400 Lumen) was shone on the sample for 5 min to effect full curing. The tensile test was performed at a rate of 5mm / s.1.14 Bioglue-tissue (HBT) adhesion studies

[0139] In order to determine the adhesion strength of the bioglue formulations for adhering hydrogel to sheep corneal tissue (schlera) a series of overlapping bioglue-tissue samples were prepared. Glass slides coated with PHF and sheep cornea (adhered using commercial cyanoacrylate -based superglue) were treated with bioglue (50 pL, 30%B) as shown in Figure 6. Test areas of / :1cm x d: 2.5 cm, were shone with light through the transparent glass slide (1400 Lumen, 5 min, 2 cm).1.15 Assembly of HBT graft (sheep model)

[0140] The HBT graft is prepared by combining Part A and B formulations which is then applied to the surface of a PHF, followed by addition of Descemet’s membrane (T). The procedure for preparation in more detail is given below.

[0141] Part A consists of photoinitiator Eosin Y (1.72 mg, 0.265 mM), and reagents triethanolamine (82.3 pL, 62.3 pM) and A-vinyl-caprolactam (62.6 mg, 45.0 pM) in BSS (10 mL). The stock solution is divided into aliquots for combining with part B prior to preparation of the HBT graft.Preparation of thinning media

[0142] Add dextran to thinning media-base on the day of HBT assembly, accelerate dissolution of dextran by shaking in a 37 °C water bath and sterile filter the thinning media using a 2 pM filter prior to use.Corneal preparation

[0143] Corneas were dissected and transferred to thinning medium (Gibco Essential Flex 1:1 DMEM:F12 (Thermo Fisher Scientific, USA), insulin-transferrin-selenium (5 pg / ml- 5 pg / ml-5 ng / ml, Thermo Fisher Scientific, USA), 2% fetal calf serum, antibiotic / antimycotic (penicillin 100 U / ml, streptomycin 100 pg / ml, amphotericin 250 ng / ml (Thermo Fisher Scientific, USA)), and 5% dextran 500 (MW 500000, Pharmacosmos, Denmark) for 16 h before proceeding with dissection of the corneal endothelium. Ovine globes from fresh cadavers of merino sheep were obtained from a local abattoir (Westside Meats, Australia) or scavenged from experimental cadavers and placed in antibiotic / antimycotic (penicillin 100 U / ml, streptomycin 100 pg / ml, amphotericin 250 ng / ml (Thermo Fisher Scientific, USA)) phosphate buffered saline (PBS) at 4 °C. Before dissection of the cornea, globes were washed with povidone 1:50, for 8 min, methanol 20% v / v for 60 s, Peracetic acid 0.1% v / v pH 7.4 (Sigma-Aldrich, USA) for 5 min, and rinsed in PBS with antibiotic / antimycotic.Preparation of'Descemet’s membrane

[0144] Within a laminar flow hood, set up a petri dish upside down on the stage of a dissection microscope. Draw 2.5 mL of sterile air into the syringe within the laminar flow hood. Position the cornea with endothelium side up and add a drop of thinning media. With the help of forceps pinning the cornea to the edge of the petri dish, insert the needle just below the endothelium at the limbus at 180 degrees parallel to the petri dish. Insert the needle until the length of the inserted section is twice the size of the hole. Steadily push an entire volume (2.5 mL) of air into the cornea via the syringe and then remove the needle once the Descemet membrane is separated from stroma. A complete separation ought to have minimal lamella fibre on the endothelium. Transfer the inflated cornea to a new petri dish with endothelium facing up and add a drop of thinning media. Using the 15-degree stab, make two 1 cm incisions, one on the top side of the inflation next to the limbus and one on the bottom - forming a double opening ‘envelope’.Preparation of bioglue

[0145] The bioglue is prepared on the same day as the HBT assembly. Within a laminar flow cupboard, an aliquot of bioglue Part B (5 pL) is added to bioglue Part A (15 pL) in a black light-proof Eppendorf tube. The solution is mixed well by withdrawing and expelling the mixture repeatedly into the tube. Once mixed the tube is closed and stored either over ice or at 4 °C in a fridge prior to assembly.Preparation ofPHF for HBT assembly

[0146] Sterile trypan blue staining solution (20 pL) is added to a BSS (5 mL) in a petri dish. Gamma irradiated PHF discs (12 mm discs in BSS) were transferred to this solution and left to incubate at ambient temperature for 10 min. The PHF discs were ready to use once they had been sufficiently stained blue.Assembly of HBT graft

[0147] Using an eye spear, absorb all thinning media adjacent to the two openings of the ‘envelope’. Using forceps lift one ‘envelope opening’, further dry the interior of the corneal'envelop' with an eye spear. Remove a PHF disc from the ‘bluing’ solution and dry it briefly using an eye spear. Once again using forceps, lift an ‘envelope opening’, insert the PHF inside the ‘envelope’ and flatten the endothelium from the edges to reduce air bubbles. Using a pipette, apply 10 pL of mixed bioglue between the Descemet membrane and the PHF. Transfer the entire cornea to the holder of an 8.5 mm trephine and punch the cornea thorough through with firm pressure. Add 20 u L of thinning media to the endothelium in middle of the punch and leave the trephine in place. Activate the bioglue by positioning the light source on top of the trephine and allow 10 mins of cross-linking at room temperature. Remove the trephined corneal section from the guarded punch and carefully peel the stroma away from the PHF side of HBT graft under a dissection microscope. Transfer the assembled HBT graft to fresh thinning media and store at room temperature prior to transplantation.2 Results and discussion2.1 Synthesis and physical characterisation of bioglue

[0148] Part B was prepared by chemical modification of PEG-based trifunctional glycerol ethoxylate to form a photo-cross-linkable glycerol ethoxyl trimethacrylate (GEM). Glycerol ethoxylate (1 kDa) was functionalized with excess methacrylic anhydride (MAA) in the presence of dimethylaminopyridine (DMAP) and triethylamine (Et?N) in dichloromethane (DCM) (Figure 7). Purification of the crude material was achieved by preparative liquid chromatography, followed by isolation and evaporation to dryness to produce a clear colorless oil.]H NMR analysis indicated successful incorporation of methacrylated functional groups at 5 6.13, 5.57 and 1.95 ppm (Figure 1). Batches of GEM (referred to herein as Part B), were stored in the absence of light and at -20 °C until required.

[0149] In a typical gluing application, various ratios by volume of Part A and B were premixed to produce low-viscosity bioglue, immediately prior to application (Figure 7b). The bioglue was applied to polymeric hydrogel films (PHFs), with either: another PHF membrane added for mechanical testing; or in the case of in vitro or in vivo studies a Descemet’ s membrane. In each case, white light was shone on the samples to effect photo-cross-linking and gelation of the bioglue to form a bioglue layer between both materials.

[0150] The photo-cross-linking of bioglue formulations (Part A:B ratio of 5: 1), were assessed by]H NMR and UV-Vis to determine the optimal time required for gelation in bulk, when exposed to various light sources, ' f l NMR analysis of the bioglue formulation under white light, directly showed the conversion of methacrylated polymer as the cross-linking progressed, prior to gelation, with an obvious reduction in the signals at 5 6.13, 5.57 ppm (Figure 8). However, at higher conversions, due to the sample solidifying, even the signals due to methylene signals at 6 3.7 ppm was absent.

[0151] ATR-FTIR analysis was performed and summarised in Figure 8 showing spectra of a) glycerol ethoxylate, b) glycerol ethoxy trimethacrylate (GEM, Part B), c) Part A sol. and d) photo-cross-linked bioglue formulation (Part B, 50 v / v%) after cross-linking. Functionalisation of glycerol ethoxylate (GE) to form GEM showed a reduction in the O-H stretch at 3463 cm’1and the appearance of a strong ester signal at 1717 cm’1(C=O stretch) (Figure 8a, b). The signals in the range between 1633-1642 cm’1are due to both aliphatic (C- H) and conjugated (C=C) stretching from each sample. After combining Part A (Figure 8c) and Part B, and photoinitiating with light, the FTIR (Figure 8d) of the final bioglue showed a relative reduction in the C=O signal at 1717 cm’1indicating methacrylate cross-linking had occurred, albeit incomplete.

[0152] Indirect UV-Vis analysis was used to monitor the reduction in absorbance of the photoinitiator Eosin Y (kmax ~ 520 nm) and the gelation time when subjected to visible light (white, 1022 mW / cm2, 400-700 nm), blue (3.8 mW / cm2, ~ 455 nm) and green (2.3 mW / cm2, ~ 520 nm) light. UV-Vis analysis (Figure 9) showed a correlation between the intensity and wavelength of the light source with the gelation times. White light of relatively high intensity showed a rapid decrease in Eosin Y absorbance with a gelation time of 3 min. Green LED light with a much lower intensity but with a narrow wavelength matching that of Eosin Y showed a slower reduction in Eosin Y absorbance with a gelation time of 7 min. Blue LED light with a similar intensity showed moderate reduction in Eosin Y absorbance and limited gelation, even after 20 min irradiation.

[0153] In laboratory trials and in vitro studies the broad spectrum white light produced by commercial LEDs, bicycle torches and newer model iPhones as well as a surgeons stage light were sufficient to effect gelation of the bioglue.

[0154] In practice, the bioglue application is expected to proceed in various aqueous media such as phosphate buffer solution (PBS), balanced salt solution (BSS) or commercial ocular solutions such as Optisol GS™ or Life4C™. Staining solutions such as Trypan Blue are used routinely to visualise ocular implants in DMEK / DSAEK surgery. For this reason, dilution of the bioglue and the effect on its mechanical strength is an important consideration. As shown in Figure 10 a series of bioglue formulations containing premixed Part A and B, were prepared with varying ratios by volume of Part A to B.

[0155] Samples (0.75 mL) were prepared and irradiated with white light (1022 mW / cm2) at a distance of 2 cm. Figure 10 shows the effect of dilution of bioglue formulations relative to their gelation time with a ratio of 1: 1 gelating within 1 min, while at a ratio of 10: 1 gelation occurs around 21 min, with the sample appearing soft and weak (insets Figure 10).

[0156] To test the mechanical properties of the bioglue in bulk, samples were prepared in disposable syringes, as small 0.75 mL samples according to Table 1 and irradiated with white light until complete gelation occurred.Table 1. Summary of the formulations, compressive testing results and swelling properties of cross-linked bioglue samples with Part B, 10-50 v / v%. Swelling ratios were determined in BSS.

[0157] Here the volume amount of cross-linking component (Part B) was increased from 10 v / v% to 50 v / v%. The compressive measurements were determined using an Instron 5848 instrument and the mechanical properties shown as an average of three samples, with only one sample from Entry 1 successful. Representative stress-strain curves prior to break, for samples comprising of between 10 and 50 vol% Part B are shown in the supporting information (Figure 11). The results of the mechanical testing are summarised in Table 1 and shown in Figure 12 for varying concentrations of Part B (10-50 v / v%).

[0158] The elasticity of the samples was relatively similar, with a strain at break (e) of between 0.22 and 0.32 mm / mm (Figure 12a). An increase in the concentration of Part B from 10 to 50 v / v%, led to an increase in the overall strength, with a stress at break of 0.04 to 2.47 MPa respectively with a maximum at 40% of 3.31 MPa. Similarly, an increase in the cross-linking component from 10 to 50 v / v%, led to increases in the Young’s Modulus (E) from 0.2 to 8.1 MPa and the toughness of the samples UT) from 0.01 to 0.43 MPa respectively. The strength of the bioglue is an indication of the ability to effect cross-linking of the bioglue and gives a clear indication as to the effect of dilution during application in the mechanical strength of the bioglue. However, in practice the adhesion properties to both hydrogel PHF substrate and tissue samples are more important.

[0159] The swelling ratio of each bioglue formulation was determined after incubation in PBS at 37 °C for 1, 2, 5, and 24 hr (Figure 13). Results showed that all the samples continued to swell for the first 5 hr and were stabilised once left overnight (24 hr). In addition, an increase in the cross-linking component from 10 to 50 vol% led to a decrease in the swelling ratio of the subsequent bioglue samples from 19.9 ± 2.8% to 12.3 + 1.7% respectively. Increasing the concentration of Part B is shown to increase the cross-linking density and strength of the subsequent bioglue with a reduction in the swellability of the resulting sample.

[0160] Samples prepared for mechanical testing were also analysed for transparency. In the application as a bioglue for the adhesion of Descemet’ s membrane to PHF, the expected total thickness will vary from between 100-250 pm. Despite this contributing to a minor component of the HBT graft, the transparency of the bioglue is nonetheless important. It has been reported that the combination of hydrophobic gelatin with hydrophilic polymer can lead to a loss intransparency during the healing process (Sani et al., 2019; Jumelle et al., 2021), although the authors noted the patients vision is already compromised during healing. In the present case using a hydrophilic PEG-based PHF and bioglue, the clarity of both components remained high. Samples were prepared as thin cross-linked films (0.2 mL and 1.3 mm thick) in standard 24-well plates were shown to be between 98-100% transparent (Figure 14a). Even thicker 10 mm samples of bioglue showed transparency ratios in the order of 70-85 % and were still much clearer than gelatin based hydrogels previously reported (Sani et al., 2019; Jumelle et al., 2021). Figures 14b and c show photographs of the cylindrical bioglue samples prior to mechanical testing and thinner samples prepared in 24-well plates respectively. It is noted that thicker test samples appeared yellow, however this was absent from the thinner 1 .3 mm samples and would be expected to be colorless when applied as a 50 L sample in the HBT graft.

[0161] The light transmittance of the cross-linked bioglue was also assessed using UV-Vis spectrometry. When preparing the HBT graft for surgery a PHF is treated with 10-30 L of bioglue solution, a Descemet’s membrane placed on top of both and irradiated with light to effect cross-linking of the bioglue. In order to determine the effect of the bioglue on the clarity of the HBT graft, a sample of PHF was treated with an aliquot of bioglue formulation (Part A:B, 5:1, 20 pL). UV-Vis spectroscopy of the PHF; PHF and unreacted bioglue; and the subsequent hydrogel-bioglue (HB) construct were performed (Figure 15). The orange HB prior to photo-cross-linking showed a strong absorbance at -522 nm consistent with the photoinitiator Eosin Y. After shining the samples with light for 5 and 10 min (1400 Lumen, dist. = 2 cm), this signal was greatly reduced resulting in a reduction in this signal and resulting in a clear and colourless HB. Light transmittance over the visible range from 380-700 nm showed between 90 and 100 %.

[0162] Adherence of bioglue formulations to PHF and tissue (sheep schlera) samples were determined using lap shear testing. Hydrogel PHF samples were cast onto glass microscope slides. Tissue samples were adhered to glass slides using commercial superglue (cyanoacrylates). Lap shear tests were performed with an overlap area of approx. 2.5 x 1.5 cm for (i) PHF-bioglue- glass slide; (ii) PHF-bioglue-PHF; and (iii) PHF-bioglue-tissue samples. A 50pL aliquot of bioglue formulation (30%B) was dropped onto the PHF surface and thesecond slide placed inverted on top of the sample. The two slides were inverted, and white light (1400 Lumen) shone through the glass and PHF for 10 min to effect cross-linking of the bioglue. Tensile testing was performed for each series in triplicate using a Instron 5949 microtester fitted with a 50N load cell. Figure 16 summarises the results from the three series benchmarked against the published lap shear stress of commercial ocular adhesives (Dermabond™, Evicel™, GelCORE™ and CoSEAL™. These adhesives are designed for the closure of open ocular wounds or surgical incisions. HBT samples prepared between two PHF samples showed moderate adhesion with a shear stress strength of 85 ± 59 kPa, while adhesion of the PHF-bioglue-glass sample showed failure at 45 ± 23 kPa demonstrating a greater adhesion to gel over glass. Sheep corneal schlera adhered to PHF with bioglue resulted in the lowest adhesion strength of 35 ± 15 kPa noting delamination of tissue from the glass prior to testing occurred in some samples. Nonetheless, the lap shear adhesion of PHF to cornea tissue via bioglue was observed.2.2 In vitro studies of bioglue toxicity

[0163] Cytotoxicity studies of two separate batches of bioglue component GEM (Part B) - termed GEM-B1 and GEM-B2 - were performed in B4G12 cells on DMEK graft. 72 hr incubation showed concentration up to 1 in 1500 comparable to control (Figure 17a, b).

[0164] Cytotoxicity studies of cross-linked formulations (5:1 Part A: B) in B4G12 cells were also studied. After 72 h incubation and up to 45 L of sample, successful but gradual reduction in cell count was observed (Figure 17c, d). Data demonstrated no cytotoxicity of the crosslinked bioglue on the DMEK graft in vitro.

[0165] Similar results were obtained for cross-linked bioglue corneal stroma (Primary hCSK, human corneal stroma keratocytes) and kidney epithelium (HEK, human endothelium keratocytes) with proliferation and metabolism all determined to be viable.2.3 PHF preparation

[0166] Samples of PHFs were prepared in accordance with the procedure of Gonzalez- Andrades et al., 2015 (Figure 7c). Difficulties with reproducibility and efficiency of scale were addressed by reducing the amount of sebacoyl chloride in the original formulations.Preparation of PHFs containing 0, 5, and 10% mol excess of SbCl and 5 wt% of PCL resulted in robust and reproducible gel samples prepared within 15mm diam. Petri dishes, resulting in quantitative preparation of PHF samples. PHFs were prepared by the reaction of glycerol ethoxylate and polycaprolactone with sebacoyl chloride in dichloromethane at 60 °C. After cooling the hydrogels were floated using increasing concentrations of aq. acetone (0, 25, and 50 wt%) prior to washing in deionised water over several days. Circular discs (diam. 10 mm) were then punched out using a metal trephine and the discs stored in BSS prior to gamma irradiation (25 Gy) (Steritech Aus.).2.4 In-vitro human tissue HBT trials

[0167] Trials were conducted at Eversight USA with donor human tissue, PHF and bioglue formulations. PHF samples stored in PBS were placed on a trephine stage and -100 mL of premixed bioglue (Part A: B, 5: 1) was added via syringe (Figure 18a). Human donor Descemet's membrane was then added with cells facing down and the whole HBT graft was shone with white light (surgical scope light) for 4 min (Figure 18b). The yellow HBT graft was cut to 8 mm and transferred via forceps to a Busin glide (Figure 18c-d). The HBT graft in cell media was pulled through the glide 4 times without delamination of the HBT graft and shown to remain intact (Figure 18e). Similar results were achieved using a simple iPhone light demonstrating the versatility of the photo-cross-linkable bioglue.

[0168] In a second trial a human cornea complete with stromal layer was inverted and partially cut with a trephine, to allow for Descemet's membrane to peel to one side, yet still held to the cornea by a small attachment. A PHF was placed on top of the stromal layer and dabbed dry. An aliquot of bioglue (-10 pL) was then added via micropipette to the surface of the PHF and the Descemet's membrane peeled back over the bioglue and PHF. The cornea was then shone with white light from a surgical scope light for 4 min to effect adherence of the PHF with the Descemet's membrane. Once cross-linked the HBT was punched using a trephine blade to produce a circular HBT graft which was separated from the remaining unattached stromal layer.

[0169] Here the successful in vitro formation of a HBT graft using human Descemet’s membrane has been shown, which is capable of successful repeatable passage through anEndoglide apparatus demonstrating the rigidity of the binding between the PHF and Descemet's membrane using the photo-cross-linked polyethylene based bioglue.

[0170] An Optical Coherence Tomography (OCT) scan was performed on the HBT graft. Typical thicknesses of human Descemet’s membrane are between 10 and 20 pm depending on the patient. The thickness of the PHF membrane was estimated to be approximately 121 pm, with a varying thickness of cross-linked bioglue ranging from 50 - 180 pm in thickness. The large variation in the cross-linked bioglue layer thickness will be dependent on the concentration of the bioglue and the means of application.2.5 Ex vivo evaluation of HBT graft

[0171] Ex vivo assembly of sheep Descemet's membrane containing remnant stroma, crosslinked using bioglue to attach to PHF, was developed and the effect of dilution, cross-linking times and evidence of delamination were recorded. The PHF, bioglue and Descemet’s membrane tissue graft is referred to as HBT. Evidence of adhesion was noted and the constructed HBT graft remained intact for several months when stored in PBS at 4 °C. The cell viability of HBT grafts (n = 4) against DMEK grafts alone (n = 4) were assessed using MTT colorimetric assay (Figure 19).2.6 In vivo evaluation

[0172] The in vivo DMEK on sheep was trialled using gamma irradiated PHF membranes, and fresh sheep Descemet's membrane prepared by photoinitiated cross-linking using the bioglue formulation (Part A: B at either 3: 1 or 5:1 ratios by volume). PHF and Descemet's membrane were dried as was practical prior to addition of the bioglue to the PHF surface. Dilution of the bioglue either in manipulation of the two materials prior to gluing or during insertion was kept to a minimum as this was shown to reduce the ability of the bioglue to cross-link causing adhesion. The initial method of peeling the Descemet's membrane led to difficulties in handling and subsequent cell viability. An improved method was developed that involved an envelope in the sheep cornea being prepared and resulted in greater surgical manipulation of the HBT graft.3 Supporting information3.1 Analysis of bioglue Part B by1H NMR

[0173] 'H NMR were performed in deuterated CDCh and indicated signals at 5 6.13, 5.57 and 1.95 ppm consistent with methacrylate methine and methyl groups a, b and c respectively of the trimethacrylated PEG of Part B (Figure 1). Trace amounts of dichloromethane at 8 5.30 ppm were observed and quantified by comparison with the signal at 6 6.30 ppm. Integration of signal a relative to the polyethylene repeat units (3.8-3.6 ppm) was used to determine the degree of functionalization.3.2 Photo-cross-linking of bioglue from Part A and Part B

[0174] The bioglue formulation used to adhere PHF to corneal tissue (human or animal) consists of a photoinitiator solution Part A, combined with the cross-linkable polymer Part B. The photoinitiator solution Part A consists of photocatalyst Eosin Y, vinylcaprolactam (VC) and triethanolamine (TEO) in an aqueous solution. The concentration of Part A components and the proportion of Part A: B are crucial to the successful adhesion of the HBT graft successfully adhering to one-another. In a typical procedure 20 wt% of Part B with combined with 80 wt% Part A solution. An aliquot (30 pL) of the homogenous mixture is added to the PHF and the Descemet’s membrane tissue is placed over the surface of both. Light is shone for 10 min at a distance of 2 cm, to effect photo-cross-linking of the bioglue and adhesion of both PHF and Tissue layers.

[0175] The effect of light intensity and the source of light were investigated to determine the curing time of the bioglue.

[0176] *H NMR analysis in D2O was used to determine conversion of unreacted acrylate groups of the bioglue under exposure to white light. UV-Vis was used to determine the reduction in the photoinitiator Eosin Y signal at -520 nm over time.

[0177] Proton NMR analysis of Part A and B in D2O solvent are shown in Figure 20 prior to photoinitiation. NMR was unable to detect the signals of Eosin Y in Part A (0.23 mM) due to its low concentration in the bioglue formulation. Signals associated with catalysts VC andTEA were observed at 5 7.2 and 2.4 ppm respectively. Individual samples of Part A (0.80 mL in D2O) and Part B (0.20 mL, neat oil) were prepared in NMR tubes and irradiated with light (700 Lumen, 1022 mW / cm2) for specified times. Gelation was noted for each sample prior to analysis.

[0178] The1H NMR spectra of samples irradiation for various lengths of time are shown in Figure 21. Prior to gelation the samples showed a gradual reduction in the acrylate signals a and b at 8 6.1 and 5.7 ppm respectively. After 10 min irradiation the sample gelled and due to the lack of polymer mobility neither the signals due to the acrylate moieties a and b or the PEG backbone signals e-g (3.7-3.5 ppm) were observed.

[0179] The effect of light intensity on the cross-linking time was shown to occur within 3 min of light irradiation when a higher intensity light was used. When a light source of 1000 Lumen, 1460 mW / cm2was used instead of 700 Lumen (1022 mW / cm2) the gelation time was reduced from 10 min to 3 min.1H NMR analysis showed the effect of gelation on the NMR signals due to immobility of the polymer chains, however the signals of TEA and the solvent D2O at 8 2.7 and 4.7 ppm respectively are still observed (Figure 22).3.3 Cross-linking analysis by UV-Vis spectroscopy

[0180] UV-Vis spectrometry was used to monitor to concentration of photoinitiator Eosin Y when irradiated with light. A correlation between the concentration of Eosin Y peak at kmax = 520 nm after irradiation of light and the gelation time of the samples, correlated with the samples prepared for 'H NMR analysis. A standard curve was generated from the Xmax = 520 nm of Eosin Y at a range of concentrations. This standard curve was used to monitor the progression of photo-cross-linking of bioglue formulations and determine the standardised concentrations of Eosin Y prior to gelation. Samples of bioglue were prepared (Part A: 80 wt% in BSS; Part B: 20 wt%) and added to a 1 mm path length quartz vial for analysis by UV-Vis. Samples were irradiated with light (a. white light (400-700 nm, 1022 mW / cm2; b. green LED light, X max 520 nm, 2.3 mW / cm2or blue LED light: Xmax ~ 455 nm, 3.8 mW / cm2) at various time intervals and their UV-Vis spectra recorded. Figure 22 shows the relative absorbance of samples irradiated with white, green and blue light and demonstrated the effect of wavelength and intensity on the bioglue cross-linking. Using the higher intensity white light yet a broadwavelength range led to a reduction in Eosin Y concentration and gelation of the sample within 3 min. Targeted green light with a wavelength matching that of Eosin Y yet with a lower intensity still successfully cross-linked the bioglue sample yet after 7 min irradiation. For the sample using blue light of a higher intensity than green, yet at a wavelength unlikely to activate Eosin Y, gelation still occurred yet after 20 min irradiation. Natural light was also shown to cause natural gelation of the samples and is the reason the bioglue will be delivered in the absence of light and as two separate formulations.

[0181] During preparation of the HBT graft surgeons typically use diluents to help with the manipulation of the soft fragile tissue. This dilution has the potential to affect the cross-linking (or gelation) time, strength and adherence of the bioglue formulation. A series of experiments were designed to study the effect of bioglue dilution on these properties to determine the parameters in which the HBT fabrication will be successful or potentially delaminate.

[0182] Samples containing various concentrations of bioglue components were prepared and irradiated with white light (700 Lumen, 1022 mW / cm2at 2 cm) to determine their crosslinking times. A standard solution of Part A (80 wt% in BSS, [Eosin Y] = 0.26 mM) and Part B (20 wt%, [Part B] = 17 mM) was prepared. Aliquots of this solution were diluted with BSS to reduce the overall concentration of photoinitiator, catalysts and cross-linking polymer according to Table 2. Gelation times were recorded as the point where no mobile phase was observed upon inversion. UV-Vis analysis was used to determine the relative concentration of Eosin Y (mM) after irradiation with white light (700 Lumen) prior to gelation of the samples. When the sample was diluted by a factor of ~10 (Table 1, sample 4), gelation did not occur even after 30 min irradiation.Table 2. Bioglue formulations of Part A (containing Eosin Y, mM) and Part B (mM) in BSS.♦Represents samples that were observed to be fully gelated, f sample 4 showed no sign of gelation even after 30 min.

[0183] Figure 23 shows the effect of Part B concentration on gelation times of the bioglue formulations. For the practical application of bioglue in the formation of the HBT graft, dilution with surgical media should be kept to a minimum of a 3:1 dilution factor (i.e., Table 2, sample 3) with an extended irradiation with light to ensure successful cross-linking of the bioglue. Alternatively, more concentrated samples containing a greater proportion of Part B relative to Part A can be prepared prior to HBT formation.

[0184] To evaluate the strength of the bioglue formulations at varying concentrations, compression testing of irradiated cylindrical samples (1 cm diam x 1 cm height) were prepared. Samples were tested in triplicate and the standard deviation for each sample recorded (Table 1).Example 2. Preparation of bioglue-tissue (BT) graft

[0185] The present inventors have further found that the bioglue of the present invention may allow for the preparation of a bioglue-tissue (BT) graft in the absence of a hydrogel, which BT graft is suitable for use in DMEK surgery. Such BT graft is prepared by applying a mixture of Part A and B formulations in accordance with Example 1 to the human DMEK tissue using a non-adherent silicone patch as the assembly platform. The resulting bioglue- tissue (BT) graft may also allow for the simplification of the highly desirable yet previously difficult DMEK surgical procedure.1 Methods

[0186] Cadaveric donor cornea was transferred to thinning media overnight and kept at 37 °C 5% CO2 incubator prior to the day of BT assembly. On the day of assembly, the DMEK tissue was prepared by manually dissecting the Descemet’s membrane from the comeal stroma (85% peel of the whole graft) using a 10 mm (in diameter) circular trephine (#17212D1000, Moria, France) for peripheral scoring, followed by separation using fine tipped surgical instruments including DMEK stripping-peeling forceps (J2892E, Fogla, Italy) and cleavage hook (J2891E, Janach, Itay).

[0187] Following the peeling of the DMEK tissue, a sterile 11 mm x 11 mm medical grade non-adherent silicone patch (MED82-5010-10 0.010” (0.25 mm), Polymer systems technology limited, UK) was positioned atop the stroma in the centre of the cornea. A 10 pL mixture of bioglue Part A and Part B (at mixing ratio of A:B = 7:1) (acclimatized to room temperature from cold storage, Part A at 4 °C and Part B at 20 °C) was added to the centre of the patch using a micropipette. The peeled DMEK tissue was then gently positioned atop the bioglue from the peripheral of the endothelium using fine tipped forceps. This complex was then removed from the rest of the cornea and positioned in a sterile petri dish. A drop (10 pL) of OCM (organ culture media) was added atop the endothelium to maintain moisture needed for cell viability.

[0188] A sterile ring stand measuring 13 mm in diameter and 2 cm in height was positioned atop the assembly complex and a light source (1000 lumen LED bike light) was then positioned atop the ring and switched on for 10 min to photo-crosslink the bioglue (a drop (10 pL) of OCM is added the endothelium every two minutes to maintain cell viability). The assembled BT graft was gently removed from the silicone patch from the peripheral. The BT graft was kept for 2 hr in OCM at 37 °C (5% CO2 incubator). The assembled BT graft can then be subjected to Trypan blue staining (0.4%, sterile) for easier visualization and surgical implantation. Alternatively, bioglue can be directly added to the endothelial side of the DMEK tissue, followed by assembly and photo-crosslinking as mentioned above.2 BT cell viability assessment by CAM-PI in vitro

[0189] To ensure the chemical reaction from bioglue Part A and Part B crosslinking does not affect the viability of the endothelial cells on the DMEK tissue, the mixed bioglue was directly applied on a monolayer of B4G12 (immortalized human endothelial) cells as an in vitro model, followed by assessment of cell viability using CAM-PI (Calcein AM-Propidium iodide) based staining and imaging protocol.

[0190] A confluent monolayer of B4G12 cells (seeded at 2000 cells / mm2) on tissue culture plastic discs (13 mm in diameter) in a 12-well tissue culture plate was used as the test subject (as the DMEK tissue). Following a wash of the cell monolayer with sterile BSS to remove cell debris, a 13 pL aliquot of bioglue mixture with various ratios by volume of Part A to Part B was applied directly to the cells.

[0191] To test the effect of the bioglue mixture crosslinking on B4G12 cell viability, three test groups were set up. This included non-treatment, bioglue added but not photo-crosslinked and bioglue photo-crosslinked.

[0192] For the bioglue photo-crosslinked test group, following application of the mixed bioglue, the transparent lid of the 12-well plate was replaced atop the plate and a 1000 lumen LED white light was positioned atop and switched on for 10 min. Following the photocrosslinking process, the monolayer of cells were washed in sterile BSS and 500 pL of warm OCM was added to the wells to allow cell recovery at 37 °C 5%COz for 3 hr.

[0193] For the non-photo-crosslinked test group, mixed bioglue (with Part A and B at different ratios by volume as specified above) was applied atop the cells for the same duration of 10 min, following by aspiration of the bioglue and washing with BSS. As mentioned above, cells were allowed to recover at 37 °C 5%COz for 3 hr. Following the 3 hr recovery period, OCM was removed from the wells and cells were washed with BSS.

[0194] Following aspiration of BSS, 500 pL of CAM solution per well (2.5 pM, #C3100MP, Invitrogen, USA) was added to the wells and incubated away from light at room temperature for 30 mins. Calcein solution is aspirated, and cells washed with BSS. 500 pL of PI (2.5 pM, #P4170-10MG, Merck, USA) solution per well was added to the wells and incubated awayfrom light at room temperature for 5 mins. Cells were then washed using BSS and the tissue culture plastic (TCP) discs removed from the wells.

[0195] TCP discs were mounted with the cell side down on a glass slide for visualization using a fluorescence microscope at 488 nm (for CAM) and 560 nm (for PI) wavelength. Green dots indicating CAM uptake indicated viable cells and red dots indicating PI uptake indicated dead cells. Cell viability was calculated as the number of green dots over the total number of green and red dots.3 Results

[0196] A series of (decreasing Part B %) bioglue mixing ratio from A:B = 1 : 1 (50% B), A: B = 1.5 :1 (40%B), to A: B = 8: 1 (11.1%B) were tested. As the ratio of bioglue Part B is reduced in the mixing formulation, an increase in the number of viable cells indicated by positive CAM staining (green fluorescence) is observed in the monolayer of B4G12 cells. Meanwhile, a decrease in the number of dead cells indicated by positive PI staining (red fluorescence) is observed (Figure 24). Brightfield images demonstrated the polygonal morphology of B4G12 cells is maintained when Part A and Part B of bioglue was mixed at A:B=7:1 as compared to untreated control and non-crosslinked control. Control groups of untreated cells and cells treated with non-crosslinked bioglue (for the same duration of 10 minutes per photo-crosslinking protocol) showed negligible percentage of dead cells (indicated by positive PI staining in red). As the mixing ratio of Part B decreased, an increase in cell viability demonstrated by more CAM uptake and less PI uptake was observed in B4G12 cells. For example, only 20% of cells remained viable following bioglue direct casting and crosslinking using Part B at 50% (A: B = 1: 1); while more than 80% of viable cells were observed following bioglue direct casting and crosslinking using Part B at 12.5% (A:B = 7:1) and lower (Figure 25). This 80% cell viability was significant to note as a traditional DMEK graft ought to have at least 80% viable corneal endothelial cells to be considered an effective graft for corneal transplantation purpose.4 BT cell viability assessment by MTT ex vivo

[0197] To ensure the corneal endothelial cells remain viable following the BT assembly process, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) based cell viability assay was carried out on the assembled BT grafts and untreated DMEK grafts as control. Viable cells produce insoluble formazan upon MTT treatment and incubation. The amount of insoluble formazan was then dissolved in acidified isopropanol and quantified as the level of cell viability using a plate reader at 570 pm wavelength.

[0198] Following the dissection of a DMEK graft or the assembly of a BT graft, the grafts were gently transferred to individual wells in a 24 well TC plate. The grafts were gently washed once with BSS (500 pL) at RT, with the BSS aspirated after. 500 pL of MTT (0.5 mg / mL, #M2128-250MG, Merck, USA) was added to each well containing a graft. The plate was incubated at 37 °C 5%CO2 for 3 hr.

[0199] Following this, MTT was aspirated and 500 pL of acidified isopropanol (0.25 M acetic acid in 100% isopropanol, Merck, USA) was added to each well. Using a micropipette, the purple formazan crystal produced by viable cells was gently dissolved by pipetting the solution around the grafts for 3 minutes. Three 100 pL aliquots of the mixture per graft was transferred to each well of a 96 well plate. The plate was read at an absorbance wavelength of 570 nm.ReferencesGonzalez-Andrades, J.; Cardona, de la., C.; lonescu, A. M.; Mosse, C. A.; Brown, R. A., Photographic-Based Optical Evaluation of Tissues and Biomaterials Used for Comeal Surface Repair: A New Easy-Applied Method. PLoS ONE 2015, 10, e0142099Ozcelik, B.; Brown, K. D.; Blencowe, A.; Ladewig, K.; Stevens, G. W.; Scheerlinck, J. P. Y.; Abberton, K.; Daniell, M.; Qiao, G. G., Biodegradable and Biocompatible Poly(Ethylene Glycol)-based Hydrogel Films for the Regeneration of Corneal Endothelium. Adv Healthc Mater 2014, 3, (9), 1496-1507Sani, E. S.; Kheirhah, A.; Rana, D.; Sun, Z.; Foulsham, W.; Sheikhi, A.; Khademhosseini, A.; Dana, R.; Annabi, N., Sutureless repair of corneal injuries using naturally derived bioadhesive hydrogels. Sci. Adv. 2019, 5,eaavl281Jumelle, C.; Sani, E. S.; Taketani, Y.; Yung, A.; Gantin, F.; Chauhan, S. K.; Annabi, N.; Dana, R. Growth factor-eluting hydrogels for management of comeal defects. Materials Science and Engineering: C 2021, 120, 111790

Claims

CLAIMS1. A biocompatible cross-linkable polymer composition comprising: a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded to at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group.

2. The biocompatible cross-linkable polymer composition according to claim 1, wherein the cross-linkable branched polyether compound has a structureA(BX)nwherein:A is an n-valent core moiety;BX is a polyether arm, wherein B is a polyether segment and X is a polymerisable ethylenically unsaturated group; and n is at least 3.

3. The biocompatible cross-linkable polymer composition according to claim 1 or claim 2, wherein the core moiety is a hydrocarbon, carbohydrate, heteroalkyl, heterocycloalkyl or heteroaryl moiety.

4. The biocompatible cross-linkable polymer composition according to claim 3, wherein the core moiety comprises from 1 to 12 carbon atoms, or from 3 to 8 carbon atoms, or from 3 to 5 carbon atoms.

5. The biocompatible cross-linkable polymer composition according to any one of claims 1 to 4, wherein the at least three polyether arms each comprise a poly(ethylene glycol) segment or a poly(propylene glycol) segment.

6. The biocompatible cross-linkable polymer composition according to any one of claims 1 to 5, wherein the polymerisable ethylenically unsaturated group forms part of a (meth)acryloyl, (meth)acryloyloxy, styrenyl, vinyl ether, vinyl ester or (meth)acrylamide group.

7. The biocompatible cross-linkable polymer composition according to any one of claims 1 to 6, wherein the cross-linkable branched polyether compound is:where each n is an integer independently ranging from about 4 to about 40.

8. The biocompatible cross-linkable polymer composition according to any one of claims 1 to 7, which is biodegradable.

9. An aqueous bioglue composition comprising the biocompatible cross-linkable polymer composition according to any one of claims 1 to 8, water and a biocompatible photoinitiator.

10. The aqueous bioglue composition according to claim 9, wherein the biocompatible photoinitiator is selected from: Eosin Y with triethanolamine and vinyl caprolactam; tris(2,2-bipyridyl)dichlororuthenium(II) hexahydrate (Rufbpyfa) with sodium persulfate, camphorquinone and A,A-dimethyl-p-toluidine, 2-ethyl- dimethylbenzoate, or A-phenylglycine; 2,2,2,6,6-tetramethylpiperidine; dl-2,3- diketo-l,7,7-trimethylnorcamphane (CQ); l-phenyl-l,2-propadione (PPD); 2,4,6- trimethylbenzoyl-diphenylphosphine oxide (TPO); bis(2,6-dichlorobenzoyl)-(4- propylphenyl)phosphine oxide (Ir819); 4,4'-bis(dimethylamino)benzophenone; 4,4'- bis(diethylamino)benzophenone; 2-chlorothioxanthen-9-one; 4-(dimethylamino)benzophenone; phenanthrenequinone, ferrocene; diphenyl(2,4,6- trimethylbenzoyl)phosphine oxide I 2-hydroxy-2- methylpropiophenone (50 / 50 blend); dibenzosuberenone; resazurin; resorufin; benzoyltrimethylgermane (Ivocerin®); derivatives thereof, and combinations thereof.

11. The aqueous bioglue composition according to claim 9 or claim 10 comprising the biocompatible cross-linkable polymer composition in an amount of from about 10 vol% to about 50 vol%.

12. A kit when used for adhering a biocompatible hydrogel to a biological substrate, the kit comprising:(a) a first part comprising the biocompatible cross-linkable polymer composition of any one of claims 1 to 8; and(b) a second part comprising water and a biocompatible photoinitiator.

13. A kit when used for preparing a biological substrate having adhered thereto a crosslinked network polymer scaffold, the kit comprising:(a) a first part comprising the biocompatible cross-linkable polymer composition of any one of claims 1 to 8; and(b) a second part comprising water and a biocompatible photoinitiator.

14. Use of the cross-linkable polymer composition according to any one of claims 1 to 8, the aqueous bioglue composition according to any one of claims 9 to 11, or the kit according to claim 12, for adhering a biocompatible hydrogel to a biological substrate.

15. Use of the cross-linkable polymer composition according to any one of claims 1 to 8, the aqueous bioglue composition according to any one of claims 9 to 11, or the kit according to claim 13, for preparing a biological substrate having adhered thereto a cross-linked network polymer scaffold.

16. A process for adhering a biocompatible hydrogel to a biological substrate comprising:(i) combining the biocompatible cross-linkable polymer composition according to any one of claims 1 to 8 with water and a biocompatible photoinitiator to form an aqueous bioglue composition according to any one of claims 9 to 11;(ii) providing the aqueous bioglue composition between a biocompatible hydrogel layer and a biological substrate layer to form a laminate structure;(iii) irradiating the laminate structure at a wavelength suitable to activate the biocompatible photoinitiator and initiate cross-linking of the cross-linkable branched polyether compound, the process of which promotes adhesion between the biocompatible hydrogel and the biological substrate.

17. The process according to claim 16, wherein the biocompatible hydrogel comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages.

18. The process according to claim 16 or claim 17, wherein the ratio of the aqueous bioglue composition to the biocompatible hydrogel layer is from about 3:1 to about 5:1 by volume.

19. A process for preparing a biological substrate having adhered thereto a cross-linked network polymer scaffold comprising:(i) combining the biocompatible cross-linkable polymer composition according to any one of claims 1 to 8 with water and a biocompatible photoinitiator to form an aqueous bioglue composition according to any one of claims 9 to 11 ;(ii) providing a layer of the aqueous bioglue composition on a biological substrate layer to form a laminate structure;(iii) irradiating the laminate structure at a wavelength suitable to activate the biocompatible photoinitiator and initiate cross-linking of the cross-linkable branched polyether compound, the process of which promotes formation of a cross-linked network polymer scaffold adhered to the biological substrate.

20. The process according to any one of claims 16 to 19, wherein step (i) is performed immediately prior to step (ii).

21. The process according to any one of claims 16 to 20, wherein the biological substrate is Descemet's membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells.

22. A laminate structure comprising a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three poly ether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group.

23. The laminate structure according to claim 22, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages.

24. A laminate structure comprising a biocompatible cross-linked network polymer layer and a biological substrate layer, wherein the cross-linked network polymer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group.

25. The laminate structure according to any one of claims 22 to 24, wherein the biological substrate layer is Descemet's membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells.

26. An ocular implant having a laminate structure comprising a biocompatible crosslinked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages, and wherein the biological substrate layer is Descemet's membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells.

27. An ocular implant having a laminate structure comprising a biocompatible crosslinked network polymer layer and a biological substrate layer,wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, and wherein the biological substrate layer comprises Descemet's membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells.

28. A method for treating corneal endothelial dysfunction in a subject, the method comprising a step of implanting an ocular implant into an eye of the subject, the ocular implant having a laminate structure comprising a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages, and wherein the biological substrate layer is Descemet's membrane having attached thereto a layer of corneal cells comprising comeal endothelial cells.

29. A method for treating corneal endothelial dysfunction in a subject, the method comprising a step of implanting an ocular implant into an eye of the subject, the ocular implant having a laminate structure comprising a biocompatible cross-linked network polymer layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three poly ether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, andwherein the biological substrate layer is Descemet's membrane having attached thereto a layer of corneal cells comprising corneal endothelial cells.

30. Use of corneal endothelial cells in the manufacture of a medicament for treating corneal endothelial dysfunction, wherein the medicament comprises a laminate structure comprising a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer cross-linked via ester linkages, and wherein the biological substrate layer is Descemet's membrane having attached thereto a layer of the corneal endothelial cells31. Use of corneal endothelial cells in the manufacture of a medicament for treating corneal endothelial dysfunction, wherein the medicament is an ocular implant having a laminate structure comprising a biocompatible cross-linked network polymer layer and a biological substrate layer, wherein the biocompatible cross-linked network polymer layer comprises polymerised residues of a cross-linkable branched polyether compound comprising a core moiety to which is covalently bonded at least three polyether arms, wherein the at least three polyether arms each comprise a polymerisable ethylenically unsaturated group, and wherein the biological substrate layer is Descemet's membrane having attached thereto a layer of the corneal endothelial cells.

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