Polymer composition and use thereof

Abstract

This invention relates to polymer compositions, and more particularly to biocompatible crosslinkable polymer compositions, methods for their preparation, and their use in the preparation of ophthalmic implants.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Australian Provisional Patent Application No. 2023902079, filed on June 30, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates generally to polymer compositions and their uses. In particular, the present invention relates to biocompatible crosslinkable polymer compositions, methods for their preparation, and their use in the preparation of laminated structures for tissue regeneration (such as ophthalmic implants for treating corneal endothelial dysfunction). Background Technology

[0004] More than eight million people worldwide suffer from corneal diseases, with an increasing trend among the elderly. Corneal endothelial dysfunction is characterized by damage and death of corneal endothelial cells, leading to loss of corneal transparency and ultimately blindness. Currently, the only treatment for corneal endothelial dysfunction is corneal transplantation. However, the technical difficulties in performing this surgery limit its adoption and patient outcomes.

[0005] Surgical options for treating corneal endothelial dysfunction currently include two main techniques: Descemet's membrane automated endothelial keratomileusis (DSAEK) and Descemet's membrane endothelial keratomileusis (DMEK). Both methods involve removing the damaged corneal endothelial cells and the attached Descemet's membrane, replacing them with a donor tissue graft. The graft is typically inserted by the surgeon in a roll-like manner and manually unfolded once positioned. However, unlike the DSAEK graft (which contains stromal cells), the DMEK graft consists only of a thin Descemet's membrane and the attached endothelial layer (15µm), making it difficult to unfold and more prone to tearing during surgery.

[0006] Therefore, although DMEK grafts lead to better patient outcomes, including better visual improvement and lower rejection rates, many surgeons still opt for the simpler DSAEK method, despite associated complications such as poorer optical clarity, aberrations (fluctuations) in the graft due to curvature differences, and hyperopic drift due to curvature differences during cutting (e.g., using a microkeratome).

[0007] Therefore, there is a continued need for improved or alternative methods to treat corneal endothelial dysfunction. Summary of the Invention

[0008] The present invention provides a biocompatible crosslinkable polymer composition comprising: a crosslinkable branched polyether compound having a core portion thereto covalently bonded with at least three polyether arms, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group.

[0009] In another aspect, the present invention provides an aqueous bioadhesive composition comprising the biocompatible crosslinkable polymer composition of the present invention, water, and a biocompatible photoinitiator.

[0010] In another aspect, the present invention provides a kit for adhering a biocompatible hydrogel to a biological substrate, the kit comprising: (a) A first component comprising the biocompatible crosslinkable polymer composition of the present invention; and (b) The second component contains water and a biocompatible photoinitiator.

[0011] In another aspect, the present invention provides a kit for preparing a biological substrate with an adhered cross-linked polymer backbone, the kit comprising: (a) A first component comprising the biocompatible crosslinkable polymer composition of the present invention; and (b) The second component contains water and a biocompatible photoinitiator.

[0012] In another aspect, the present invention provides the use of crosslinkable polymer compositions according to the invention for adhering biocompatible hydrogels to biological substrates.

[0013] In another aspect, the present invention provides a crosslinkable polymer composition according to the invention for preparing a biological substrate with an adhered crosslinked network polymer backbone.

[0014] In another aspect, the present invention provides a process for adhering a biocompatible hydrogel to a biological substrate, the process comprising: (i) Combining the biocompatible crosslinkable polymer composition according to the present invention with water and a biocompatible photoinitiator to form the aqueous bioadhesive composition of the present invention; (ii) Providing an aqueous biogel composition between a biocompatible hydrogel layer and a bio-based substrate layer to form a laminated structure; (iii) Irradiating the stacked structure at a wavelength suitable for activating the biocompatible photoinitiator and initiating crosslinking of the crosslinkable branched polyether compound, the process promoting adhesion between the biocompatible hydrogel and the biological substrate.

[0015] In another aspect, the present invention provides a process for preparing a bio-substrate with an adhered cross-linked polymer network backbone, the process comprising: (i) Combining the biocompatible crosslinkable polymer composition according to the present invention with water and a biocompatible photoinitiator to form the aqueous bioadhesive composition of the present invention; (ii) Providing a layer of the aqueous bioadhesive composition on a biological substrate layer to form a laminated structure; (iii) Irradiating the stacked structure at a wavelength suitable for activating the biocompatible photoinitiator and initiating crosslinking of the crosslinkable branched polyether compound, the process promoting the formation of a crosslinked network polymer backbone that adheres to the biological substrate.

[0016] In another aspect, the present invention provides a layered structure comprising a biocompatible crosslinked network polymer layer between a biocompatible hydrogel layer and a biocompatible substrate layer, wherein the crosslinked network polymer comprises a polymerized crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group.

[0017] In another aspect, the present invention provides a stacked structure comprising a biocompatible crosslinked network polymer layer and a biological substrate layer, wherein the crosslinked network polymer comprises a polymerized crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group.

[0018] In another aspect, the present invention provides an ophthalmic implant having a laminated structure comprising a biocompatible cross-linked polymer network layer between a biocompatible hydrogel layer and a biocompatible substrate layer. The biocompatible crosslinked network polymer layer comprises polymerized crosslinkable branched polyether compound residues, wherein the crosslinkable branched polyether compound comprises a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group. The biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer crosslinked via ester linkages, and The biological basal layer includes a des elastic lamina with a corneal cell layer containing corneal endothelial cells.

[0019] In another aspect, the present invention provides an ocular implant having a laminated structure comprising a biocompatible cross-linked polymer network layer and a biological substrate layer. The biocompatible crosslinked network polymer layer comprises a polymerized, crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group, and The biological basal layer includes a des elastic lamina with a corneal cell layer containing corneal endothelial cells.

[0020] In another aspect, the present invention provides a method for treating corneal endothelial dysfunction in a subject, the method comprising the steps of implanting an ophthalmic implant into the subject's eye, the ophthalmic implant having a laminated structure comprising a biocompatible cross-linked polymer network layer between a biocompatible hydrogel layer and a biocompatible basal layer.

[0021] The biocompatible crosslinked network polymer layer comprises polymerized crosslinkable branched polyether compound residues, wherein the crosslinkable branched polyether compound comprises a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group. The biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer crosslinked via ester linkages, and The biological basal layer is the Descemet's membrane, which is a corneal cell layer containing corneal endothelial cells.

[0022] In another aspect, the present invention provides a method for treating corneal endothelial dysfunction in a subject, the method comprising the steps of implanting an ophthalmic implant into the subject's eye, the ophthalmic implant having a layered structure comprising a biocompatible cross-linked polymer network layer and a biological substrate layer.

[0023] The biocompatible crosslinked network polymer layer comprises a polymerized, crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group, and

[0024] The biological basal layer is the Descemet's membrane, which is a corneal cell layer containing corneal endothelial cells.

[0025] In another aspect, the present invention provides an ophthalmic implant for treating corneal endothelial dysfunction, said ophthalmic implant having a laminated structure comprising a biocompatible cross-linked polymer network layer between a biocompatible hydrogel layer and a biocompatible basal layer. The biocompatible crosslinked network polymer layer comprises polymerized crosslinkable branched polyether compound residues, wherein the crosslinkable branched polyether compound comprises a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group. The biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer crosslinked via ester linkages, and The biological basal layer is the Descemet's membrane, which is a corneal cell layer containing corneal endothelial cells.

[0026] In another aspect, the present invention provides an ophthalmic implant for treating corneal endothelial dysfunction, said ophthalmic implant having a laminated structure comprising a biocompatible cross-linked polymer network layer and a biological substrate layer. The biocompatible crosslinked network polymer layer comprises a polymerized, crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group, and The biological basal layer is the Descemet's membrane, which is a corneal cell layer containing corneal endothelial cells.

[0027] In another aspect, the present invention provides the use of corneal endothelial cells in the manufacture of a medicament for treating corneal endothelial dysfunction, wherein the medicament comprises a layered structure including a biocompatible cross-linked network polymer layer between a biocompatible hydrogel layer and a biological basal layer. The biocompatible crosslinked network polymer layer comprises polymerized crosslinkable branched polyether compound residues, wherein the crosslinkable branched polyether compound comprises a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group. The biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer crosslinked via ester linkages, and The biological basal layer mentioned above is the posterior elastic layer to which corneal endothelial cells are adhered.

[0028] In another aspect, the present invention provides the use of corneal endothelial cells in the manufacture of a medicament for treating corneal endothelial dysfunction, wherein the medicament is an ophthalmic implant having a laminated structure comprising a biocompatible cross-linked polymer network layer and a biological basement layer. The biocompatible crosslinked network polymer layer comprises a polymerized, crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group, and The biological basal layer mentioned above is the posterior elastic layer to which corneal endothelial cells are adhered. Attached Figure Description

[0029] Embodiments of the present invention will now be described with reference to the following accompanying drawings, which are intended to be illustrative only, and wherein: Figure 1 . 1 1H NMR spectrum, showing the chemical characterization of biocollagen component B in CDCl3. Figure 2 Sample preparation of bio-adhesive formulations is shown, including: a) a commercial LED white light panel; b) a sample cured under light; c) a sample prepared in a disposable syringe; and d) an example of the resulting bio-adhesive sample used for testing.

[0030] Figure 3 Dimensions of dog bone-shaped specimens used for tensile mechanical testing, using a cutter C0024 – Type B (ASTM D412-C).

[0031] Figure 4 Overlap shear test of photocrosslinked bioadhesive (glass-bioadhesive-glass).

[0032] Figure 5 a) Schematic diagram of test samples (components A and B, 5:1) photocrosslinked with bio-adhesive for PHF-adhesive-PHF testing.

[0033] Figure 6 Glass slides coated with PHF and amniotic cornea treated with bio-adhesive (50 µL, 30%B).

[0034] Figure 7 Schematic diagrams of the formation of bio-adhesive and PHF. a) GEM synthesis (component B), b) photocrosslinking under light after combination with component A solution, and c) previously reported PHF synthesis (Ozcelik et al., 2014).

[0035] Figure 8 The following ATR-FTIR analyses were performed on: a) glycerol ethoxylate, b) glycerol ethoxytrimethacrylate (GEM, component B), c) aqueous solution of component A, and d) the photocrosslinked bio-adhesive formulation.

[0036] Figure 9Standardized photoinitiator Eosin Y (λ 最大 UV-Vis analysis of absorbance (~520 nm) was used to monitor exposure to white (o) and green (o) light.

[0037] ) and blue ( The degree of photocrosslinking of bioadhesive under LED light. White light (400-700 nm, 1022 mW / cm²) 2 ), green light (λ) 最大 ~520 nm, 2.3 mW / cm 2 ), and blue light (λ) 最大 ~455 nm, 3.8 mW / cm 2 Irradiate the sample at a distance of 2 cm for a given time. The gelation time is indicated by a dashed line, and each light source is indicated by a red label.

[0038] Figure 10 The effect of dilution on gel time, in which the aqueous solution component A of the photoinitiator (containing Eosin Y, triethanolamine, and...) N The ratio of β-vinylcaprolactam to component B (trifunctional glycerol ethoxymethacrylate (GEM)) increases. The inset shows the sample prepared after irradiation with 0.75 mL of white light for 10 minutes.

[0039] Figure 11 . Schematic compressive stress-strain curves up to fracture, for bio-adhesive formulations with different component B concentrations (10-50 v / v% component B (denoted as X%B)).

[0040] Figure 12 Mechanical compression properties of bio-adhesive formulations with varying amounts of component B (10-50 v / v%). Compression tests were performed on three samples (0.75 mL), revealing a) fracture stress (σ); b) fracture strain (ε); and c) Young's modulus at 10% strain. E ); and d) toughness (U T The average value of ).

[0041] Figure 13 The swelling percentage (%) of biogel formulations (entries 1-5, Table 1) containing 10-50 v / v% component B after photocrosslinking and incubation in PBS at 37°C.

[0042] Figure 14 a) Transparency (%), which was prepared by cross-linking bio-glue hydrogels (B-10, 20, 30, 40, and 50%) to a thickness of 0.75 mm. 3ImageJ analysis calculations were performed on photographs of a cylinder (10 mm thick) cast into a 24-hole plate (1.3 mm thick), while b) and c) show photographs of the top of the Koren lens image, respectively.

[0043] Figure 15 UV-Vis analysis of PHF membrane and PHF-biogel samples after exposure to white light (1400 lumens) for t = 0, 5, and 10 minutes.

[0044] Figure 16 Commercial ophthalmic adhesive CoSEAL TM (Based on polyethylene glycol) relative to (i) PHF-bioglue-glass; (ii) PHF-bioglue-PHF; and (iii) PHF-bioglue-tissue overlap shear stress.

[0045] Figure 17 Two batches of bio-adhesive component B in vitro Cytotoxicity analysis: pre-crosslink a) GEM-B1, b) GEM-B2; and c, d) crosslinked bioglue (components A and B).

[0046] Figure 18 . Figure 18 In vitro human HBT studies conducted in Eversight, USA, demonstrated: a) the addition of bioglue to a PHF membrane; b) photoinitiation of the bioglue between the PHF and the human Descemet's membrane under surgical light; c) the HBT graft passing through Endoglide containing Life4C media for the fourth time; and d) intact HBT grafts.

[0047] Figure 19 Ex vivo fabrication of HBT grafts.

[0048] Figure 20 Bio-gel preparations in D2O 1 1H NMR spectra, compared with components A and B individually. Eosin Y is not significant in NMR due to its low concentration.

[0049] Figure 21 White light irradiation (700 lumens, 1022 mW / cm²) 2 Following this, the bio-adhesive formulation (D2O, component A: 80 v / v%, component B: 20 ​​v / v%) 1 H NMR spectrum.

[0050] Figure 22 White light irradiation (1000 lumens, 1460 mW / cm²) 2Following this, the bio-adhesive formulation (D2O, component A: 80 v / v%, component B: 20 ​​v / v%) 1 H NMR spectrum.

[0051] Figure 23 The effect of component B concentration in BSS on the gel time of bio-adhesive formulations.

[0052] Figure 24 The effect of directly casting bio-glue onto an in vitro BT model. A mixed bio-glue (with different ratios of component A and component B) was directly applied to a monolayer of B4G12 human corneal endothelial cells, followed by photocrosslinking for 10 minutes.

[0053] Figure 25 Cell viability of bioglue when applied directly to a monolayer of B4G12 cells. The bioglue showed no toxicity when the percentage of component B was below 12.5% ​​(A:B = 7:1). The green line indicates a cell viability level of 80%, used as a benchmark for endothelial cell viability in effective DMEK grafts from cadaveric donor corneas.

[0054] definition

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0056] Unless otherwise stated, the indefinite articles “a” and “the” used herein include the plural. Thus, for example, “a” refers to a single substance as well as two or more substances; similarly, “composition” or “formulation” refers to a single composition or formulation as well as two or more compositions or formulations; and so on.

[0057] As used herein, the term "alkyl" or "alkyl group" refers to a monovalent ("alkyl") and divalent ("alkylene") straight-chain or branched saturated aliphatic group and is intended to encompass both monovalent and polyvalent alkyl groups. Alkyl groups can have 1 to 12 carbon atoms and are represented as C1. 1-12 Alkyl groups, or those having 1 to 6 carbon atoms, denoted as C2 1-6Alkyl groups, and so on. Examples of suitable alkyl moiety may include, but are not limited to: methyl, ethyl, 1-propyl, isopropyl, 1-butyl, 2-butyl, isobutyl, tert-butyl, pentyl, 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, etc.

[0058] As used herein, the term "heteroalkyl" or "heteroalkyl group" refers to a straight-chain or branched 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 1 to 12 carbon atoms, denoted as C1. 1-12 Heteroalkyl groups, or those having 1 to 6 carbon atoms, denoted as C1 1-6 Heteroalkyl groups, and so on. Examples of suitable heteroalkyl moieties may include, but are not limited to: ethers (e.g., RO-R', where R and R' are independently alkyl groups as defined herein), tertiary amines (e.g., R-NR''-R', where R, R', and R'' are independently alkyl groups as defined herein), secondary amines (e.g., R-NH-R', where R and R' are independently selected from alkyl groups as defined herein), thioethers (e.g., RS-R', where R and R' are independently alkyl groups as defined herein), and so on.

[0059] As used herein, the term "alkenyl" or "alkenyl group" refers to a monovalent ("alkenyl") and divalent ("alkenylyl") straight-chain or branched unsaturated aliphatic hydrocarbon group having at least one double bond anywhere in the chain. Unless otherwise stated, the stereochemistry of each double bond may be independently cis or trans, or E or Z The alkenyl group may have 2 to 12 carbon atoms, denoted as C1. 2-12 Alkenyl groups, or those having 2 to 6 carbon atoms, denoted as C0 2-6Alkenyl, and so on. Examples of suitable alkenyl groups may include, but are not limited to: ethenyl, vinyl, allyl, 1-methylvinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 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, etc.

[0060] As used herein, the term "cycloalkyl" or "cycloalkyl group" refers to a saturated or partially saturated monocyclic, fused, or spirocyclic polycyclic carbon ring, and is intended to encompass both monovalent and polyvalent cycloalkyl groups. The cycloalkyl group may have 3 to 12 carbon atoms per ring, denoted as C1. 3-12 Cycloalkyl. Examples of suitable cycloalkyl groups include, but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, spiro[3.3]heptyl, decahydronaphthalene, and adamantyl.

[0061] As used herein, the term "heterocyclic alkyl" or "heterocyclic alkyl group" refers to a saturated or partially saturated monocyclic, bicyclic, fused, or spirocyclic polycyclic carbon ring, 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 heterocyclic alkyl group may have 2 to 6 carbon atoms per ring, denoted as C1. 2-6 Heterocyclic alkyl groups. Examples of suitable heterocyclic alkyl groups include, but are not limited to: acridine, aziridinyl, pyrrolidinyl, piperidinyl, piperazinyl, quininecycloyl, morpholinyl, diazaspiro[3.3]hexane (e.g., 2,6-diaazaspiro[3.3]hexane), tetrahydrothiophene, tetrahydrofuranyl, and tetrahydropyranyl. The heterocyclic alkyl group may be a terminal group or a bridging group and may be linked by heteroatoms or any carbocyclic atom.

[0062] As used herein, the term "aryl" or "aryl group" refers to a monocyclic or fused-ring polycyclic aromatic carbocyclic ring (i.e., a ring structure in which all ring atoms are carbon atoms), and is intended to encompass both monovalent and polyvalent aryl groups. The aryl group may have 6-12 atoms per ring, denoted as C1. 6-12 Aryl. Examples of suitable aryl groups may include, but are not limited to, phenyl, naphthyl, and phenanthrene. As used herein, the term "aryl" is also intended to cover optionally substituted, partially saturated bicyclic aromatic carbocyclic moieties in which phenyl groups and cycloalkyl or cycloalkenyl groups are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl, or indenyl.

[0063] 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 independently selected from O, N, NH, or S. The heteroaryl group may have 1-12 carbon atoms per ring, denoted as C1. 1-12 Heteroaryl groups. Examples of suitable heteroaryl groups include, but are not limited to: furanyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl (e.g., 1,3-oxazolyl, 1,2-oxazolyl), pyridinyl (e.g., 2-, 3-, 4-pyridinyl), pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrroleyl, tetrazolyl, thiadiazolyl, thiazolyl, thiophene, triazolyl (e.g., 1,2,3-triazolyl, 1,2,4-triazolyl), triazinyl, tetraazinyl, and carbazolyl. Representative examples of bicyclic heteroaryl groups include, but are not limited to: benzimidazolyl, benzofuranyl, benzothiophenyl, benzoxadiazolyl (e.g., 2,1,3-benzoxadiazolyl), zolinyl, dihydroquinolinyl, dihydroisoquinolinyl, furanopyridyl, indazole, indolyl (e.g., 2- or 3-indolyl), isoquinolinyl (e.g., 1-, 3-, 4-, or 5-isoquinolinyl), naphridyl (e.g., 1,5-naphridyl, 1,7-naphridyl, 1,8-naphridyl, etc.), pyrrolopyridyl (e.g., pyrrolo[2,3-b]pyridyl), quinolinyl (e.g., 2-, 3-, 4-, 5-, or 8-quinolinyl), quinoxalinyl, tetrahydroquinolinyl, and thiophenolopyridyl. In one or more embodiments, the heteroaryl group is an N-heteroaryl group having one or more nitrogen heteroatoms, such as one, two, three, or four nitrogen heteroatoms, depending on the specific structure. The N-heteroaryl group may also have heteroatoms other than nitrogen; however, it is characterized by having at least one nitrogen heteroatom. Exemplary N-heteroaryl groups include: imidazolyl, indolyl (e.g., 2- or 3-indolyl), naphridyl, pyrazinyl, pyridyl (e.g., 2-, 3-, or 4-pyridyl), pyrroleyl, pyrimidinyl, quinolinyl (e.g., 2-, 3-, 4-, 5-, or 8-quinolinyl), isoquinolinyl, quinazolinyl, quinoxalinyl, and triazinyl, benzimidazolyl, triazolyl, tetraazinyl, and carbazoleyl. As used herein, the term "heteroaryl" or "heteroaryl group" is also intended to cover optionally substituted, partially saturated bicyclic aromatic heterocyclic moieties, wherein the heterocycle and cycloalkyl or cycloalkenyl groups are fused together to form a ring structure. The heteroaryl group may be a terminal group or a bridging group and may be linked by heteroatoms or any carbocyclic atoms. The invention is also intended to cover salts of N-heteroaryl groups disclosed herein. For example, salts of N-heteroaryl groups may be acid addition salts, such as HCl or HBr addition salts. Non-limiting examples of N-heteroaryl salts include benzimidazonium, imidazonium, triazonium, and pyridinium.

[0064] As used herein, the term "biocompatible" when used in connection with a substance means that the substance is substantially harmless or non-toxic to living tissue. A substance may be biocompatible on its own, or it may be present in amounts that are harmless or non-toxic to living tissue.

[0065] In relation to a condition (e.g., corneal endothelial dysfunction), the term "treat (treating)" refers to the relief or elimination of the cause and / or effect of the condition. As used herein, the term "treat (treatment, and treating)" refers to a reduction or improvement in the progression, severity, and / or duration of a condition, or relief of one or more symptoms of the condition (e.g., one or more identifiable symptoms) resulting from the application of one or more therapies (e.g., ophthalmic implants as described herein) (i.e., "managing" rather than "curing" the condition). In specific embodiments, the term "treat (treatment and treating)" refers to the improvement of at least one measurable bodily parameter of the condition described herein. In other embodiments, the term "treatment" refers to the suppression of the progression of the condition described herein, for example, whether physically by, for example, stabilizing identifiable symptoms, physiologically by, for example, stabilizing physical parameters, or both.

[0066] As used herein, unless otherwise understood in the art, the term “about” generally means ±10% of a nominal value.

[0067] The terms “v / v%” and “volume%” are used interchangeably in this document and have the same meaning.

[0068] Throughout this specification and the claims, unless the context requires otherwise, the word “comprising” or its variations such as “including” and “containing” shall be understood to imply inclusion of the said integer or step or a set of integers or steps, but not to exclude any other integer or step or a set of integers or steps.

[0069] The term "composed of" means "composed of only", that is, including and limited to the integer or step, or the set of integers or steps, and excluding any other integers or steps or the set of integers or steps.

[0070] The term "substantially composed of" means including the said integer or step or a set of integers or steps, but may also include other integers or steps or a set of integers or steps that do not substantially change or contribute to the operation of the invention.

[0071] In this specification, references to any prior art are not, and should not be, considered an admission or an implication in any form that the prior art is part of common general knowledge.

[0072] Other definitions are provided throughout the manual. Detailed Implementation

[0073] This invention provides biocompatible crosslinkable polymer compositions that, once crosslinked, can be used as biocompatible adhesives or "bioglues" to adhere biocompatible hydrogels to biological substrates, or can be used in the absence of hydrogels to provide a suitable framework for biological substrates. The inventors have discovered that the biocompatible crosslinkable polymer compositions disclosed herein can be used to prepare laminated structures suitable for use as ophthalmic implants for treating corneal endothelial dysfunction. Advantageously, use of ophthalmic implants according to the invention can result in higher success rates of corneal endothelial transplantation, reduced procedure time, and / or lower long-term rejection rates.

[0074] The biocompatible crosslinkable polymer composition according to the present invention comprises a crosslinkable branched polyether compound having a core portion covalently bonded to at least three polyether arms, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group. In one or more embodiments, the crosslinkable branched polyether compound is not: or , Where n is an integer ranging independently from about 2 to about 75.

[0075] The biocompatible crosslinkable polymer composition may be provided neatly, or it may be provided as an aqueous solution or suspension. Therefore, the biocompatible crosslinkable polymer composition is soluble in or miscible with an aqueous liquid. In one or more embodiments, the biocompatible crosslinkable polymer is water-soluble. In this case, the biocompatible crosslinkable polymer composition may also be described as a water-soluble biocompatible crosslinkable polymer composition. In other embodiments, the biocompatible crosslinkable polymer is miscible with an aqueous liquid. For example, the biocompatible crosslinkable polymer according to the invention may be suspended in an aqueous solution in the form of a self-assembling polymer, micelles, dispersions, or emulsions.

[0076] The core portion of the crosslinkable branched polyether compounds disclosed herein may be any suitable core portion, including but not limited to: hydrocarbon, carbohydrate, heteroalkyl, heterocycloalkyl, or heteroaryl portions. Those skilled in the art will understand that, in order to covalently bond with at least three polyether arms, the core portion should be trivalent or higher (e.g., trivalent, tetravalent, pentavalent, hexavalent, or higher) core portion.

[0077] In embodiments where the core portion is a hydrocarbon portion, the hydrocarbon portion may be a linear, branched, cyclic, or aryl hydrocarbon portion. It will be understood that the hydrocarbon portion includes any suitable portion consisting of carbon and hydrogen atoms (e.g., alkyl, cycloalkyl, or aryl groups, as defined elsewhere herein). In some embodiments, the hydrocarbon portion contains one carbon atom (i.e., a trimethyl or tetravalent methyl group). In other embodiments, the hydrocarbon portion is a linear, branched, or cyclic hydrocarbon portion containing 2 to 12 carbon atoms (e.g., C12, C23 ... 2-12 Alkyl, C 3-12 cycloalkyl groups, or 3 to 8 carbon atoms (e.g., C14-C24-C ...24). 3-8 Alkyl, C 3-8 cycloalkyl groups, or 3 to 5 carbon atoms (e.g., C14-C5). 3-5 Alkyl, C 3-5 (Cycloalkyl). In other embodiments, the hydrocarbon moiety may comprise, for example, 6 to 12 carbon atoms (C64-C12). 6-12 aryl), or 6 to 10 carbon atoms (C 6-10 The hydrocarbon moiety is an aryl group containing 2 to 12 carbon atoms (e.g., C12). In one or more embodiments, the hydrocarbon moiety is an aryl group containing 2 to 12 carbon atoms (e.g., C12). 2-12 Alkyl groups), or 3 to 8 carbon atoms (e.g., C10). 3-8 Alkyl groups), or 3 to 5 carbon atoms (e.g., C10). 3-5 Linear or branched hydrocarbon moieties of alkyl groups.

[0078] In embodiments where the core portion is a carbohydrate portion, the carbohydrate portion may be a linear, branched, cyclic, or aryl carbohydrate portion. It will be understood that the carbohydrate portion includes any suitable portion 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 include, but are not limited to, glucose, galactose, mannose, fructose, and ribose. In other embodiments, the carbohydrate portion is a polysaccharide (i.e., comprising two or more sugar units), examples of which include, but are not limited to, dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, and hyaluronic acid.

[0079] The core moiety may also be selected from any other suitable core moiety known in the art, including but not limited to heteroalkyl, heterocycloalkyl, or heteroaryl moieties described elsewhere herein. The core moiety (e.g., hydrocarbon, carbohydrate, heteroalkyl, heterocycloalkyl, or heteroaryl moieties disclosed herein) may optionally be substituted with one or more substituents such as hydroxyl (–OH), halogen (–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), amide (–CONR, wherein R is H or alkyl), etc. It will be further understood that any optional substituents will be selected such that the crosslinkable polymer remains biocompatible (i.e., substantially harmless or non-toxic to living tissue). The selection of such optional substituents and the process of incorporating them into the biocompatible crosslinkable polymer compositions disclosed herein will be within the scope of those skilled in the art.

[0080] The polyether walls of the crosslinkable branched polyether compounds disclosed herein comprise polyether segments and polymerizable olefinic unsaturated groups. The at least three polyether arms may be identical or different. Further, each polyether segment may contain the same repeating unit, or it may be a random copolymer or block copolymer containing different repeating units. The polymerizable olefinic unsaturated groups may exist independently suspended on each polyether wall or at the ends of each polyether arm. In one embodiment, the polymerizable olefinic unsaturated groups are present at the ends of each polyether arm.

[0081] In one or more embodiments, the polyether segment is a polyoxyalkylene segment. In the context of polyoxyalkylene, the term "oxyalkylene" as used herein is intended to mean divalent -O(CR) X R Y ) i - Group, where R X and R Y Each is independently selected from hydrogen and optionally substituted alkyl groups, and i is an integer ranging from 1 to 10. Generally, R X and R Y Each is independently selected from hydrogen and optionally substituted C. 1-6 Alkyl group, where i is an integer selected from 2, 3, and 4. When i > 1, each (CR X R Y The units can be the same or different. For example, when the oxoalkylene unit is an oxoethylene unit, R X and R Y Both are hydrogen, and i=2 (i.e., –O(CH2)2-), while when the oxoalkylene unit is an oxopropylene unit, i=2 and the first “i” is R X and RY Both are hydrogen, and the second "i" is R X and R Y The alkylene groups can be hydrogen and methyl (i.e., –OCH2CH(CH3)–). The units within each alkylene group or polyoxyalkylene group can be the same or different. In other words, the polyoxyalkylene group can be a homopolymer or a copolymer (including random or block copolymers). The alkylene unit can be obtained from alkylene oxides, such as ethylene oxide, propylene oxide, or butane oxide. The molecular weight of the polyoxyalkylene segment can range from about 100 to 10,000 Da, about 150 to 5,000 Da, and about 200 to 1,000 Da.

[0082] In one or more embodiments, the polyether segment is selected from poly(alkylene glycols), such as poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(isopropylene glycol), or copolymers or terpolymers 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 poly(propylene glycol) segment. The poly(ethylene glycol) segment or poly(propylene glycol) segment may contain between about 1 and about 100 repeating units, or between 4 and 40 repeating units, or between 6 and 12 repeating units. Other suitable polyether segments will be apparent to those skilled in the art and may include, for example, dioxane-based polyethers, such as poly(1,4-dioxane), poly(4-methyl-1,3-dioxane), etc.

[0083] The polyether arm can be directly bonded to the core portion or covalently linked via a linker portion (e.g., a divalent linker portion). The linker portion can be any suitable linker portion, including but not limited to alkyl groups (e.g., C464). 1-6 Alkyl) or alkoxy (e.g., –O–C) 1-6 Alkyl) linkers. In some embodiments, other linkers such as carbonyl, ester, or amide moieties may also be suitable.

[0084] The polyether arms of the crosslinkable branched polyether compounds disclosed herein all contain polymerizable olefin unsaturated groups. Each polyether arm may contain the same or different polymerizable olefin unsaturated groups. Suitable polymerizable olefin 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-vinylcarbamates, monovalent N-vinylcarbamates, monovalent acrylamides, and monovalent methacrylamides. In one or more embodiments, the polymerizable olefin unsaturated group forms a portion of the following groups: (meth)acryloyl, (meth)acryloyloxy, styrene, vinyl ether, vinyl ester, or (meth)acrylamide group. In one or more embodiments, the polymerizable olefin unsaturated group forms a portion of a (meth)acryloyl group.

[0085] The polymerizable olefinic unsaturated group can be directly bonded or covalently linked to the polyether arm via a linker portion (e.g., a divalent linker portion). The linker portion can be any suitable linker portion, including but not limited to: alkyl groups (e.g., C14, C2 ... 1-6 Alkyl), alkenyl (e.g., C10) 2-6 alkenyl) ethers (e.g., –R–O–R', where each R and R' is independently C). 1-6 Alkyl groups), esters (e.g., –R–C(O)O–R', where each R and R' is independently C). 1-6 Alkyl groups), amides (e.g., –R–C(O)N(R")–R', where each R, R', and R" is independently C 1-6 Alkyl groups), urethane esters (e.g., –R–OC(O)N(R")–R', wherein each R, R', and R" is independently C 1-6 Alkyl), isocyanate, succinimide, or succinimide carbonate linker portion.

[0086] In some embodiments, the crosslinkable branched polyether compound has the following structure: A(BX) n in: A represents the n-valence core portion; BX represents the polyether arm, where B is the polyether segment and X is a polymerizable olefinic unsaturated group; and n is at least 3. In one or more embodiments, A(BX) n no: or , Where n is an independent integer ranging from about 3 to about 75.

[0087] Suitable n-valent core moiety (A), polyether arm (BX), polyether segment (B), and polymerizable olefin unsaturated group (X) are as described above.

[0088] In one or more embodiments, the crosslinkable branched polyether compound is:

[0089] Each n is an independent integer ranging from about 4 to about 40, preferably from about 6 to about 12.

[0090] In one or more embodiments, the crosslinkable branched polyether compound is:

[0091] Each n is an independent integer ranging from about 4 to about 40, or preferably from about 6 to about 12.

[0092] The crosslinkable branched polyether compounds described herein can be prepared by any suitable method known in the art. One such method is illustrated in Scheme 1 below, which involves esterifying a polyether polyol compound (such as a commercially available glycerol ethoxylate) with a suitable electrophile (e.g., a carboxylic acid, acyl chloride, or anhydride) to form an ester bond, wherein the electrophile comprises an olefinically unsaturated compound, such as (meth)acryloyl chloride or (meth)acrylic anhydride. Other suitable methods will be apparent to those skilled in the art and may include, for example, click reactions of thiols with olefins, reactions of isocyanates with amines; click reactions of azo (N3) with olefins, reactions of N-hydroxysuccinimides with alcohols or amines, etc.

[0093] Option 1

[0094] During crosslinking, the biocompatible crosslinkable polymer compositions disclosed herein form a crosslinked network polymer. This crosslinked network polymer is suitable for use as a biocompatible polymeric adhesive (or "bioglue"), for example, for adhering a biocompatible hydrogel to a biological substrate. The crosslinked network polymer is also suitable for use as a framework for a biological substrate in the absence of a hydrogel. Therefore, the present invention also provides crosslinked network polymers comprising residues of the polymerized crosslinkable branched polyether compounds disclosed herein.

[0095] In one or more embodiments, the crosslinked network polymers disclosed herein and the crosslinkable polymer compositions derived therefrom are biodegradable. As used herein, the terms “degradable” and “biodegradable” with respect to a substance mean that the substance is readily degraded, broken down, or fragmented over time under physiological or biological conditions. This degradation, breakage, or fragmentation can occur under selected physiological or biological conditions via the chemical decomposition of suitable unstable portions (e.g., via hydrolysis or reduction). When used with polymeric substances, the terms “degradable” and “biodegradable” indicate that the polymer contains suitably unstable or degradable portions as part of the polymer molecular structure. Breakage or disruption of one or more degradable portions in the polymer results in fragmentation of the polymer, typically into monomers and / or lower molecular weight polymer fragments. Furthermore, if the substance described herein is “biocompatible” and “biodegradable,” it will be understood that the resulting degradation byproducts are also biocompatible, i.e., substantially harmless or non-toxic to living tissue.

[0096] The crosslinking of the crosslinkable branched polyether compounds disclosed herein can be achieved using any suitable method known in the art. In one or more embodiments, the biocompatible crosslinkable polymer composition is achieved by photocrosslinking, for example in the presence of light and optionally one or more photoinitiators. Advantageously, photocrosslinking can be achieved in the presence of biological material (such as cells) without significantly damaging the biological material. In one or more embodiments, crosslinking is achieved in the presence of one or more biocompatible photoinitiators. In some embodiments, the biocompatible photoinitiator is water-soluble. The wavelength of light required to initiate photocrosslinking may depend on the specific photoinitiator and may be visible, infrared, or ultraviolet light. In some embodiments involving crosslinking in the presence of biological material, it may be advantageous for the wavelength of light required to initiate photocrosslinking to be in the visible or infrared spectrum. In some embodiments, the wavelength of light required to initiate photocrosslinking is in the visible spectrum.

[0097] Suitable biocompatible photoinitiators used in this invention may include, but are not limited to: Eosin Y with triethanolamine and vinylcaprolactam; tris(2,2-dipyridyl)ruthenium(II) hexahydrate (Ru(bpy)3) with sodium persulfate, camphorquinone, and N , N -Dimethyl-p-toluidine, 2-ethyl-dimethylbenzoate, or N-Phenylated glycine; 2,2,2,6,6-Tetramethylpiperidine; dl-2,3-dione-1,7,7-trimethylnorbornene (CQ); 1-Phenylated-1,2-propadione (PPD); 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (TPO); bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819); 4,4'-bis(dimethyl... 4,4'-Di(diethylamino)benzophenone; 2-chlorothiazol-9-one; 4-(dimethylamino)benzophenone; phenanthrenequinone; ferrocene; diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide / 2-hydroxy-2-methylphenylacetone (50 / 50 blend); dibenzocycloheptenone; resazurin; halogenated benzoyltrimethylgermanane (Ivocerin®); its derivatives, and combinations thereof.

[0098] In particular, suitable photoinitiators and reaction conditions have been proposed by Teymour et al. (2004). Langmuir, 20 Descriptions in 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 vinylcaprolactam, and / or other additives to promote photocrosslinking. Advantageously, photocrosslinking in the presence of Eosin Y can be carried out under mild conditions using visible light (514 nm), which is compatible with biomaterials. In some embodiments, the photoinitiator is provided as an aqueous solution comprising: Eosin Y at a concentration between about 0.0125 mM and 0.5 mM, or between about 0.025 mM and about 0.15 mM; triethanolamine at a concentration 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 vinylcaprolactam at a concentration 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 one embodiment, the photoinitiator is provided as an aqueous solution comprising: Eosin Y at a concentration of about 0.05 mM; triethanolamine at a concentration of about 0.4% (w / v); and vinylcaprolactam at a concentration of about 0.4% (w / v).

[0099] Forming a crosslinked network polymer according to the invention may comprise combining a biocompatible crosslinkable polymer composition disclosed herein with a photoinitiator in the presence of water. The water may be in any form, with non-limiting examples including distilled water, deionized water (DIW), filtered water, sterile water, balanced salt solution (BSS), and phosphate buffered solution (PBS). In some embodiments where the photoinitiator is Eosin Y with triethanolamine and vinylcaprolactam, the ratio of the photoinitiator to the biocompatible crosslinkable polymer is about 2:1 or less by volume, for example, between about 2:1 and about 10:1 by volume, or between about 2:1 and about 7:1, or between about 2:1 and about 5:1 by volume.

[0100] The biocompatible crosslinkable polymer composition can be combined with a photoinitiator in an aqueous solution and stored (e.g., at low temperatures, such as 4°C, -20°C, or lower) before further use, for example as a bioglue, or it can be used immediately after combination, for example within 24 hours, or within 12 hours, or within 6 hours, or within 2 hours, or within 30 minutes, or within 15 minutes. Therefore, the present invention also provides compositions comprising a biocompatible crosslinkable polymer composition as described herein, a biocompatible photoinitiator, and water. This composition may contain one or more biocompatible additives. For example, the composition may include one or more free radical inhibitors, such as butylated hydroxytoluene (BHT); 4-methoxyphenol, p-methoxyphenol (MEHQ); phenothiazine; 4-tert-butylcatechol (TBC); hydroquinone (HQ); cupferrin (N-nitroso-N-phenylhydroxylamine); 4-tert-butylpyrocatechol; N,N-diethylhydroxylamine; or any combination thereof.

[0101] The biocompatible crosslinkable polymer compositions disclosed herein can be contained in a kit with one or more photoinitiators. The kit may include, for example, the biocompatible crosslinkable polymer composition and the photoinitiator, each individually packaged or formulated, or packaged or formulated in combination. Thus, the kit may comprise a first component and a second component (e.g., a first container and a second container), wherein the biocompatible crosslinkable polymer composition is present in the first component and the photoinitiator is present in the second component, for example, in an aqueous solution. The components (e.g., the containers) may be housed within a package, which may optionally include instructions for combining the biocompatible crosslinkable polymer composition with the photoinitiator, and / or for its subsequent use as a bioadhesive composition for adhering biocompatible hydrogels and bio-substrates, or as a framework for bio-substrates in the absence of a hydrogel. Therefore, in one or more embodiments, the present invention provides a kit comprising: a first component comprising the biocompatible crosslinkable polymer composition disclosed herein; and a second component comprising water and a biocompatible photoinitiator. The kits disclosed herein may optionally contain one or more additives, such as those described above, which may be present in the first component, the second component, or a third or subsequent component of the kit.

[0102] The kit of the present invention can be stored at room temperature or at low temperatures (e.g., 5°C, -20°C). The duration of kit stability depends on the construction of the kit and its components, as well as its storage conditions. In some embodiments, the stability of the kit components can be enhanced by including one or more additives (e.g., free radical inhibitors) in the first component, the second component, or both. In some embodiments, the kit is stable for at least about 2 months, or at least about 6 months, or at least about 12 months.

[0103] The biocompatible crosslinkable polymer composition of the present invention, or a kit containing thereof, can be used to adhere a biocompatible hydrogel to a biological substrate. Specifically, photocrosslinking is initiated by providing a composition comprising the biocompatible crosslinkable polymer composition, a photoinitiator, and water (referred to herein as an "aqueous bioglue composition") between a biocompatible hydrogel layer and a biological substrate layer, and irradiating the resulting laminated structure at a suitable wavelength. The resulting crosslinked network polymer can act as a bioglue, thereby improving the adhesion of the biocompatible hydrogel to the biological substrate. The improved adhesion is characterized by an increased adhesion duration, increased adhesion strength, or both, relative to the adhesion of the hydrogel to a substance (i.e., a crosslinked network polymer) in the absence of a bioglue. In some embodiments, the adhesion duration of the hydrogel to the crosslinked network polymer disclosed herein can be at least about 1 week, 2 weeks, 3 weeks, 1 month, or longer. The adhesion strength of the hydrogel to the substrate can be changed by adjusting the concentration of the bioglue used in this application (e.g., increasing the bioglue concentration can increase the adhesion strength). The adhesion strength can be tested, for example, using an overlap shear test.

[0104] Therefore, the present invention provides a process for adhering a biocompatible hydrogel to a biological substrate. This process may comprise combining a biocompatible crosslinkable polymer composition disclosed herein with water and a biocompatible photoinitiator to form an aqueous biogel composition. The relative amounts of the biocompatible crosslinkable polymer composition, the photoinitiator, and water may depend on the specific polymer composition and / or photoinitiator used. In one or more embodiments, the aqueous biogel composition comprises a crosslinkable polymer composition in an amount of about 10 vol% to about 50 vol%, or about 10 vol% to about 40 vol%, or about 20 vol% to about 50 vol% (based on net composition). As described above, the biocompatible crosslinkable polymer composition may be combined with a photoinitiator in an aqueous solution and stored as a biogel prior to use, or the components may be combined and then used immediately as a biogel (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).

[0105] The aqueous bioadhesive composition thus prepared can be subsequently provided between a biocompatible hydrogel layer and a bio-substrate layer to form a laminated structure. For example, the aqueous bioadhesive composition can be applied to the biocompatible hydrogel layer (e.g., as one or more droplets, or as a homogeneous layer), followed by the application of the bio-substrate layer, or vice versa. In some embodiments, the ratio of the aqueous bioadhesive composition to the biocompatible hydrogel layer is from about 3:1 to about 5:1 by volume (i.e., about 17% to about 25% by volume of the aqueous bioadhesive composition). The resulting laminated structure can then be irradiated at a wavelength suitable for activating the biocompatible photoinitiator and initiating crosslinking of the crosslinkable branched polyether compound (without damaging the bio-substrate), a process that improves the adhesion between the biocompatible hydrogel and the bio-substrate.

[0106] Therefore, the present invention also provides a laminated structure comprising a biocompatible crosslinked network polymer layer between a biocompatible hydrogel layer and a biocompatible substrate layer, wherein the crosslinked network polymer comprises polymerized crosslinkable branched polyether compound residues, the crosslinkable branched polyether compound comprising a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group. In some embodiments, the ratio of the biocompatible crosslinked network polymer layer to the biocompatible hydrogel layer is from about 3:1 to about 5:1 by volume.

[0107] The biocompatible crosslinkable polymer compositions of the present invention, or kits containing them, can also be used as scaffolds for biological substrates. Specifically, by providing a layer of an aqueous bioadhesive composition as described herein on a biological substrate layer, and irradiating the resulting structure at a suitable wavelength to induce photocrosslinking, the resulting crosslinked network polymer can adhere to the biological substrate layer and act as the scaffold of the biological substrate. In some embodiments, the adhesion duration of the crosslinked network polymer to the substrate can be at least about one week, two weeks, three weeks, one month, or longer.

[0108] Therefore, the present invention also provides a process for adhering a bio-substrate with a cross-linked polymer network backbone. The process may include: preparing an aqueous bio-adhesive composition as described above. A layer (e.g., a homogeneous layer) of the thus prepared aqueous bio-adhesive composition may then be applied to a bio-substrate layer to form a laminated structure. The laminated structure may then be irradiated at a wavelength suitable for activating the biocompatible photoinitiator and initiating cross-linking of the cross-linkable branched polyether compound (without damaging the bio-substrate), a process that promotes the formation of a cross-linked polymer network backbone adhering to the bio-substrate.

[0109] Therefore, the present invention also provides a stacked structure comprising a biocompatible crosslinked network polymer layer and a biological substrate layer, wherein the crosslinked network polymer comprises a polymerized crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group.

[0110] The choice of biocompatible hydrogel and / or bio-based substrate in the layered structure of the present invention can be determined depending on its intended application. In this respect, the layered structures disclosed herein can be used particularly as implantable devices for tissue regeneration. For example, the layered structure may include a selected bio-based substrate to replace damaged tissue at the site of infection. Advantageously, the use of biodegradable and / or biocompatible components in the layered structures disclosed herein can provide a scaffold for the bio-based substrate, which degrades over time, producing non-toxic degradation byproducts, thereby preventing the accumulation of foreign material at the site of infection and allowing surrounding tissue to revert to its natural structure. The biodegradation rate can be from several days to several weeks, for example, from 2 weeks to about 20 weeks, or longer.

[0111] In some embodiments of the layered structures disclosed herein, the bio-substrate comprises a Descemet's membrane with an attached corneal cell layer containing corneal endothelial cells. The corneal cell layer may further comprise corneal epithelial cells, corneal stromal cells, or a combination thereof. Typically, when used in endothelial keratoplasty, the Descemet's membrane is derived from a donor and comprises a layer of donor corneal endothelial cells to which it is attached. In some cases, it may be necessary to seed corneal endothelial cells onto the Descemet's membrane (e.g., when using a patient's own Descemet's membrane). It will be understood that the term "Descemet's membrane with an attached corneal cell layer containing corneal endothelial cells" herein is intended to include both cases where corneal cells are naturally present on the Descemet's membrane and cases where corneal cells have been seeded onto the Descemet's membrane. In other embodiments, corneal cells are incorporated into the biocompatible hydrogel layer described herein. For example, corneal cells may be grown on a biocompatible hydrogel. In some embodiments, the corneal cells may be treated with growth factors and / or biological and chemical entities that promote cell growth or proliferation.

[0112] Furthermore, in some embodiments involving biocompatible hydrogels, 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 one specific embodiment, the biodegradable network polymer is a biodegradable and biocompatible polyether network polymer crosslinked via ester linkages. This biodegradable and biocompatible polyether network polymer can be prepared by polymerizing a monomer composition comprising a multifunctional polyether monomer and a multifunctional crosslinking monomer, wherein one of the polyether monomer and the crosslinking monomer contains a hydroxyl functional group, and the other of the polyether monomer and the crosslinking monomer contains a complementary functional group capable of reacting with the hydroxyl functional group to form an ester link, and wherein the polyether monomer is branched.

[0113] According to the laminated structure of the invention, the bio-substrate is a Descemet's membrane with an attached corneal cell layer containing 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, suitable for use as an ophthalmic implant for treating corneal endothelial dysfunction. It will be understood that the bio-adhesive is applied to the Descemet's membrane (which is not attached by corneal cells (i.e., the endothelial side)), and contact between the bio-adhesive and corneal cells should be avoided if possible. Advantageously, the bio-adhesive, optionally together with the hydrogel, provides a framework for the Descemet's membrane (and the attached corneal endothelial cells), which can unfold simultaneously when placed in the eye, thereby reducing the risk of implant damage compared to conventional DMEK grafts. Furthermore, in embodiments involving a hydrogel framework, the improved adhesion of the hydrogel provided by the bio-adhesive to the Descemet's membrane (as opposed to direct application of a biocompatible hydrogel to the Descemet's membrane) reduces the need for repeated implant placement due to delamination of the Descemet's membrane. Furthermore, when applied to the eye, the ophthalmic implants disclosed herein may suitably be transparent.

[0114] Therefore, the present invention also provides an ophthalmic implant having a layered structure comprising a biocompatible crosslinked polymer network layer between a biocompatible hydrogel layer and a biocompatible substrate layer, wherein the biocompatible crosslinked polymer network layer comprises a polymerized crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether polymer network crosslinked via ester linkages, and wherein the biocompatible substrate layer is a descemet's membrane containing corneal endothelial cells. Examples of suitable ophthalmic implants are described in Example 1.

[0115] The present invention also provides an ophthalmic implant having a layered structure comprising a biocompatible crosslinked polymer network layer and a biological substrate layer, wherein the biocompatible crosslinked polymer network layer comprises a polymerized residue of a crosslinkable branched polyether compound, the crosslinkable branched polyether compound comprising a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group, and wherein the biological substrate layer is a descemet's membrane containing corneal endothelial cells. Examples of suitable ophthalmic implants are described in Example 2.

[0116] Ophthalmic implants according to the invention can be prepared and stored before use, or they can be prepared during implantation surgery (e.g., using the patient's own Descemet's membrane). Preferably, the ophthalmic implant is stored at 4°C in the absence of light for up to 14 days. The ophthalmic implant can be stored, for example, in a surgical implantation device such as Busin Glide or Endoglide. TM System. In some embodiments, the ophthalmic implant can be prepared and stored in a unfolded form in a suitable preservation medium. Suitable preservation media are known to those skilled in the art and may include, but are not limited to, PBS, BSS, and Optisol GS. TM (Contains dextran, 2.5% chondroitin sulfate, vitamins, and precursors of adenosine triphosphate (i.e., adenosine, inosine, and adenine)), and Life4C. TM (Glutathione and human insulin, to maintain cell viability at 4°C). In other embodiments, the ophthalmic implant can be prepared and stored in a roll-up form. This roll-up form can be provided with a suitable applicator (such as a Busin glide) for application to the patient's eye.

[0117] It will be understood that the ophthalmic implant according to the invention should be transparent to visible light. For example, the ophthalmic implant should be more than 98% transparent to visible light, or more than 99% transparent, or more than 95.5% transparent. Furthermore, the ophthalmic implant should be of appropriate thickness for easy insertion of the device and for implantation of the device at the desired site in the patient's eye. The thickness of the implant can be adjusted, for example, by adjusting the composition of the biocompatible hydrogel layer and / or the biocompatible cross-linked polymer network (bioglue) layer. This adjustment can be achieved, for example, by adjusting the starting monomer from which the hydrogel and / or biocompatible cross-linked polymer network are obtained; and / or adjusting their amounts. In one embodiment, the thickness of the ophthalmic implant is in the range of: about 100 µm to about 500 µm, or about 100 µm to about 400 µm, or about 150 µm to about 300 µm, or about 150 µm to about 250 µm. The biocompatible cross-linked network polymer (bioglue) layer may comprise approximately 50 µm to approximately 250 µm, approximately 50 µm to approximately 200 µm, or approximately 50 µm to approximately 180 µm of the total thickness of the ophthalmic implant.

[0118] The present invention further provides a method for treating corneal endothelial dysfunction in a subject, the method comprising the steps of implanting an ophthalmic implant as described in the present invention into the subject's eye.

[0119] In some embodiments, the object requiring treatment or prevention of corneal endothelial dysfunction is a mammal. As used herein, the term "mammal" includes: humans, primates, livestock (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.

[0120] The present invention also provides the use of ophthalmic implants as described herein in the treatment of corneal endothelial dysfunction.

[0121] The present invention also provides the use of corneal endothelial cells in the manufacture of a medicament for treating corneal endothelial dysfunction, wherein the medicament comprises a laminated structure comprising a biocompatible crosslinked network polymer layer between a biocompatible hydrogel layer and a biocompatible basal layer, wherein the biocompatible crosslinked network polymer layer comprises a polymerized crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion to which at least three polyether arms are covalently bonded, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer crosslinked via ester linkages, and wherein the biocompatible basal layer is a descemet's membrane to which the corneal endothelial cell layer is attached.

[0122] The present invention also provides the use of corneal endothelial cells in the manufacture of a medicament for treating corneal endothelial dysfunction, wherein the medicament comprises a stacked structure comprising a biocompatible crosslinked network polymer layer and a biological substrate layer, wherein the biocompatible crosslinked network polymer layer comprises a polymerized crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion covalently bonded to at least three polyether arms, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group, and wherein the biological substrate layer is a descemet's membrane to which the corneal endothelial cell layer is attached.

[0123] The drugs described herein may comprise a stacked structure in a rolled-up or unfolded form. The unfolded stacked structure may be suspended in a suitable preservation medium, such as PBS, BSS, or Optisol. TM Or Life4C TM Alternatively, the rolled-up, layered structure can be provided with a suitable applicator (such as Busin Glide) for application to the subject's eye. The drug can be stored (e.g., at a low temperature, such as 4°C) for a period of time (e.g., up to about 14 days) before use.

[0124] Those skilled in the art will recognize that the invention described herein can be subject to variations and modifications other than those specifically described. It should be understood that the invention described herein includes all such variations and modifications. The invention also includes all steps, features, methods, compositions, and compounds individually or collectively mentioned or pointed out in the specification, as well as any two or more, or any and all combinations of said steps or features.

[0125] Some embodiments of the invention will now be described with reference to the following examples, which are for illustrative purposes only and are not intended to limit the scope of the above generalities.

[0126] Example

[0127] Example 1. Preparation of hydrogel-bioglue-tissue (HBT) grafts

[0128] The inventors have proposed a two-component system comprising a photoinitiating solution (component A) [containing Eosin Y, triethanolamine (TEO), and...] N A solution of vinylcaprolactam (VC) in PBS and a PEG-based biocompatible crosslinkable polymer composition (component B) are used to prepare a multifunctional aqueous bioadhesive composition, which is in-situ photocrosslinked to adhere the hydrogel membrane to the post-elastic layer, ready for use in DMEK surgery. The resulting hydrogel-bioadhesive-tissue (HBT) graft allows for simplification of the highly desirable but previously difficult DMEK surgical procedure.

[0129] 1. Materials and Methods

[0130] 1.1 Materials

[0131] Aluminum oxide (Brockmann I, basic, Al2O3), aluminum oxide (Brockmann I, acidic, Al2O3), 4-(dimethylamino)pyridine (DMAP, >99%), Eosin Y (>99%), glycerol ethoxylate (GE <M n ~1000), dried magnesium sulfate (dried MgSO4, >98%), methacrylic anhydride (MAA, >94%), phosphate-buffered saline tablets (PBS, 1 tablet / 200mL), sodium hydroxide (NaOH, >98%), sodium bicarbonate (NaHCO3, >99.5%), triethylamine (TEA, >99%), triethanolamine (TEO, >99%), and N - Vinylcaprolactam (VC, 98%) was purchased from Merck and used without further purification. Acetone (AR grade), acetonitrile (CH3CN, AR grade, Chemsupply), deuterated chloroform (CDCl3, Cambridge Isotope Laboratories), dichloromethane (DCM, Honeywell), balanced salt solution (BSS), aqueous hydrochloric acid solution (aq. HCl, 0.5 M), and human corneal stromal cell regulation mediator (hCSK) were provided by CERA, stored at 4°C, and used within 7 days of delivery.

[0132] 1.2 Equipment

[0133] Unless otherwise stated, NMR spectra were recorded on a Bruker (Ascend 400MHz) spectrometer using deuterated chloroform (CDCl3) as a reference. UV-Vis spectra were performed on a Shimadzu UV-Vis scanning spectrophotometer (UV-2101 PC) using a fast scan rate with 1nm intervals. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra were performed using a Bruker Tensor 27 FTIR and a GladiATR ATR accessory from Pike Technologies. Biogel component B was purified using a Buchi Pure C-850 FlashPrep HPLC system (equipped with a FlashPure™ EcoFlex silica column). Compression tests of the crosslinked biogel samples were performed on an Intron 5848 microforce tester (equipped with a 50N force sensor). Tensile tests of the polyethylene glycol-based hydrogen film (PHF) were performed in PBS using an Instron 5949 microforce tester (equipped with a 50N force sensor).

[0134] 1.3 Preparation of photoinitiator solution - Component A

[0135] When forming a solid biogel, a solution containing Eosin Y and a photoinitiator that initiates visible light is used to crosslink the trifunctional prepolymer (component B). The photoinitiator solution (component A) is prepared by mixing Eosin Y (1.72 mg, 0.265 mM), triethanolamine (82.3 µL, 62.3 µM), and... N -Vinyl-caprolactam (62.6 mg, 45.0 µm) was dissolved in phosphate-buffered saline (PBS) (10 mL). The stock solution was divided into 0.5 mL aliquots and stored in 1.5 mL black microcentrifuge tubes at 4°C, protected from light, until needed.

[0136] 1.4 Synthesis and purification of biocompatible crosslinkable polymers, bioglue – Component B

[0137] Add glycerol ethoxylate (GE) (16.0 g, MW 1 kDa, 16.0 mmol) to an oven-dried and cooled round-bottom flask (RBF) (500 mL), weighed on a 3-position balance. Weigh 4-dimethylaminopyridine (DMAP) (0.64 g, 5.24 mmol) on weighing paper using a 4-position balance and transfer it to the flask. Then add triethylamine (10.4 mL, 0.075 mmol). The RBF is equipped with a magnetic stir bar and a diaphragm (Suba-Seal) and placed on a magnetic stirring plate (100 rpm). Transfer anhydrous DCM (100 mL) to the flask via an oven-dried needle and a disposable syringe (100 mL). Purge the flask with argon gas via the needle for 30 minutes. Treat the reaction mixture 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) was added via a 20 mL plastic Luer-lock syringe and needle. The entire flask was covered with household aluminum foil to block light from reaching the reaction. The mixture was stirred at room temperature under UHP argon atmosphere for 72 hours.

[0138] Transfer the contents of the reaction flask to a 2L separatory funnel housed in a ring holder attached to an iron stand. Dilute the mixture with 200 mL of DCM (AR grade). Add 100 mL of saturated aqueous NaHCO3 solution, mechanically shake, depressurize, and return the flask to the stand. Collect the bottom organic layer into a conical flask (1000 mL). Return the organic phase to the separatory funnel and repeat the washing step twice with additional NaHCO3 (aq) (2 x 100 mL). Return the DCM extract to the separatory funnel and wash with subsequent amounts of aqueous HCl solution (0.5 M, 3 x 100 mL), and finally with saturated aqueous NaCl solution (1 x 100 mL). Dry the collected DCM extract with approximately 5 g of anhydrous MgSO4, or until the solution becomes clear. The solution was filtered through a Buchner funnel equipped with Whatman 542 filter paper and a side-arm conical flask under house vacuum. The solution was transferred to a clean conical flask and treated with basic alumina (Al₂O₃, 5 g), stirred for 5 minutes, and then filtered as described above. The solution was then treated with acidic alumina (5 g), stirred, and filtered as described above. The DCM phase was transferred to 500 mL RBF and concentrated to dryness in a rotary evaporator at 20°C under house vacuum (~20 mmHg). The clear, pale yellow oil (15.0 g, 78% yield) was collected and stored at -20°C until further purification.

[0139] Use with FlashPure TM Crude oil was purified using a Buchi Pure C-850 FlashPrep HPLC system with an EcoFlex silica column. Crude oil (10 g / pass) in 10 mL of DCM was loaded onto a 120 g silica 50 mm column connected to the LC system. The system used a gradient mixture of solvent A: DCM (100%) and solvent B: DCM (90%) / methanol (10%) at a flow rate of 60 mL / min. The initial fraction was discarded (~7 min), and the major fraction was collected at approximately 10 min when the A:B solvent ratio was 1:1. These fractions were combined and washed with water (3 x 100 mL) to remove methanol. The organic phase was dried over MgSO4 (~5 g), filtered (using #542 filter paper), and concentrated to dryness on a rotary evaporator at 6 mbar for 2 h. 1 H NMR analysis ( Figure 1 The presence of <1% DCM was indicated. The purified, biocompatible, crosslinkable polymer oil (component B) was stored at -20°C in aluminum foil to prevent light exposure until needed.1 H NMR (400MHz, CDCl3, δ): 6.13 (s, 3H; CH), 5.57 (s, 1H; -CH), 5.30 (s, 2H, CH2Cl2), 4.30 (t, 6H, J = 6.0 Hz, -CH2O-CO), 3.75 (t, 6H, J = 6.0 Hz, -CH2-CH2O-), 3.7-3.6 (m, 90H), 1.95 (s, 3H, CH3). ATR-FTIR: 3582, 2865 (CH), 1717 (C=O), 1633 (C=C), 1452 (CH), 1294, 1098.

[0140] 1.5 Preparation of bio-adhesive formulations and cross-linked samples

[0141] Biogel samples for UV-Vis analysis were prepared by combining crosslinkable macromonomers and photoinitiator solutions in different proportions (i.e., component A to component B by volume was 1, 2, 3, 4, 5, 7.5, and 10:1). A standard curve of Eosin Y in PBS was prepared (taken from λ). 最大 = Absorbance at 520 nm) and used to determine the concentration of the bio-adhesive crosslinked sample. A sample (100 µL) of the bio-adhesive formulation was placed in a quartz cell with a channel length of 1 mm, irradiated with light (white, green, and blue) for a given time, and its UV-Vis spectrum was recorded. 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 that did not flow within the cell.

[0142] Biogel samples for mechanical testing, biodegradation, and swelling tests were prepared by dissolving different concentrations of component B (10, 20, 30, 40, and 50 v / v%) in solution A (Table 1). In short, the mixed homogeneous biogel formulation solution was injected into the end of a 3 mL disposable syringe (with the end cut off). Figure 2 c). Subsequently, the sample array was illuminated with white light from a 1400-lumen (20W) LED flat panel (105 mm x 80 mm), with a distance of 1 cm between the light and the sample. Figure 2 ab). Once cured, the sample was removed and stored at 4°C prior to biodegradation and compression testing. Figure 2 d).

[0143] 1.6 Mechanical Testing

[0144] Compression tests of the crosslinked bio-adhesive composition were performed using an Instron 5848 microforce tester (Instron, USA) (equipped with a 50N force sensor). Prior to testing, cylindrical photocrosslinked bio-adhesive samples were placed between two compression plates with the flat surface parallel to the plates. Compression tests were conducted according to ASTM standard F2255-05 (2015) at a strain rate of 5 mm / min. The initial height and diameter of the samples were measured and recorded using calipers prior to compression. The final stress (σ) and strain (σ) were recorded at the failure point. ε The slope of the stress-strain curve at 10% strain is obtained and reported as Young's modulus. E The area under the stress-strain curve before fracture is calculated and reported as toughness (U). T ).

[0145] Tensile tests of PHF were performed using an Instron 5944 microforce tester (Instron, USA) equipped with a 50N force sensor. Dog-bone shaped samples of the PHF were prepared using a Type B C0024 punching machine (RDM Test Equip. UK., 6 mm diameter). Figure 3 Prior to testing, the samples were pre-swollen in PBS and subjected to hydration and swelling tests in a PBS-containing water bath. Tensile tests were performed at a rate of 5 mm / s. The initial displacement and the dimensions of the test area were measured prior to testing.

[0146] 1.7 In vitro swelling test

[0147] Crosslinked biogel samples were prepared in the same manner as the mechanical tests according to Table 1. The weight of each sample was measured after crosslinking and after incubation in PBS at 37°C for 24 hours. Three samples were prepared for each formulation, and their swelling ratios were calculated according to Equation 1, where the value measured directly after preparation is (S0) and the value measured after swelling in PBS for t = 1, 2, 5, and 24 h is (S...). t ).

[0148] Swelling rate % (S) t ) = (S t – S0) / S t x 100% (1)

[0149] 1.8 Transparency Test

[0150] Prepare 0.75 cm according to Table 1. 3The transparency of photocrosslinked bio-adhesive formulation samples was analyzed using a photographic method (Gonzalez-Andrades et al., 2015), as previously described. Samples were placed on a Koren 2003 lens test chart, and photographs were taken of each sample. Subsequently, the photographs were analyzed using ImageJ software, with each image defined as a 5 mm circular region of interest (ROI), according to Equation 2, by the maximum (I... 最大 ) and minimum (I 最小 Pixel intensity determines transparency.

[0151] Transparency (T%) = (2)

[0152] 1.9 In vitro degradation test of photocrosslinked bio-adhesive

[0153] Biogel samples were prepared according to Table 1 (items 1-5) and as described in Section 3.4. After photocrosslinking, the samples (n = 6 for each formulation) were weighed (D0), then incubated in PBS or hCSK conditioning medium at 37°C. Samples were periodically removed, dried, and weighed (D0). t The solution was refreshed with fresh medium every seven days. Degradation was monitored until the bioglue was no longer visible in the solution. Time t The remaining weight after degradation is defined as D. t And calculate according to Equation 3.

[0154] Remaining weight (D) t ) = D t / D0x 100% (3)

[0155] 1.10 Preparation of PHF membrane

[0156] PHF was prepared using a modified procedure based on that of Gonzalez-Andrades et al., 2015, in the standard process. 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 (pre-punctured with a small needle). Once the PCL was completely dissolved, sebacate chloride (SbCl, 0.137 mL, 0.641 mmol) was gently added via a 100 mL pipette until the solution became homogeneous. The solution was poured into pre-dried glass petri dishes (90 mm in diameter) and placed in a preheated oven (60 °C) under low vacuum for 30 minutes. A full vacuum was then applied to the oven by fully opening the vacuum line and closing the drain valve, and maintained for another 60 minutes. The vacuum was then released, and the culture was immediately placed in deionized water for 30 minutes. The entire culture was then transferred to 10%, 25%, and 50% acetone aqueous solutions, each for 30 minutes. The gel floated completely off the culture dish and was transferred to a beaker of deionized water to remove unreacted monomers. The water was replaced three times over three days to remove any unadulterated material. The gel was then cut into discs using a hollow circular 12mm shear punch. The PHF discs (8 discs / bottle) were placed in a container containing 10 mL of BSS, sealed with Parafilm®, labeled, and sent for gamma irradiation. Upon receiving the irradiated PHF sample, it was stored at 4°C before use.

[0157] 1.11 UV-Vis Evaluation of PHF-Biogel

[0158] The transmittance of the photocrosslinked bio-adhesive was evaluated using aliquots of bio-adhesive added to an 8 mm PHF disk. Hydrated PHF was placed in BSS solution for 1 h, followed by UV-Vis evaluation. The PHF was then dried and treated with 20 μL of bio-adhesive component A:B (5:1), sufficient to cover the PHF surface. The samples were then photocrosslinked using 1400 lumens of light at a distance of 2 cm from the surface. The transmittance of the PHF-bio-adhesive samples (n=3) was determined as follows: after irradiation at 25°C for t=0, 5, and 10 minutes, the UV and visible spectra (300–700 nm) were determined.

[0159] 1.12 Evaluate the adhesion of bioadhesive to HBH and HBT grafts.

[0160] All bioadhesive adhesion tests were performed using an Instron 5944 microforce meter (Instron, USA) equipped with a 50N force sensor. Stress-strain curves for each sample were recorded at a rate of 5 mm / min until fracture. The stress (N) / area (m²) ratio was used. 3 Determine the stress (kPa), where 1 N / m 3 = 1 kPa.

[0161] 1.13 Study on the Adhesion of Hydrogel-Biogel-Hydrogel (HBH)

[0162] Use bio-adhesive to treat glass coverslips (24 x 75 mm) to cover... l 1 cm x d : 2.5 cm area. Distribute the formulation (50 µL, component B, 30 v / v%, component A 70 v / v%) onto the coverslip, and place the second slide on top. Figure 4 The sample was irradiated with white light (1400 lumens) for 5 minutes to achieve complete curing. Tensile tests were performed at a rate of 5 mm / s.

[0163] The PHF solvent sample, cast onto a glass coverslip (24 x 75 mm), was treated with bio-glue and covered. l 1cm x d : 2.5 cm area. Distribute equal portions (50 µL, 30% B%) of the bio-adhesive onto a PHF-coated coverslip, and place a second PHF-coated coverslip on top. Figure 5 The sample was irradiated with white light (1400 lumens) for 5 minutes to achieve complete curing. Tensile tests were performed at a rate of 5 mm / s.

[0164] 1.14 Study on the adhesion of bioadhesive-tissue (HBT)

[0165] To determine the adhesion strength of the bio-adhesive formulation to the amniotic cornea tissue (sclera), a series of overlapping bio-adhesive-tissue samples were prepared. Glass slides coated with PHF and amniotic cornea (using a commercially available cyanoacrylate-based super-adhesive) were treated with bio-adhesive (50 µL, 30%B), as shown below. Figure 6 As shown in the image. Light was shone through a transparent glass slide (1400 lumens, 5 min, 2 cm). l 1cm x d The test area is 2.5 cm.

[0166] 1.15 Assembling the HBT graft (sheep model)

[0167] HBT grafts are prepared by combining components A and B and then applying them to the surface of a PHF graft, followed by the addition of a post-elastic layer (T). The preparation process is described in more detail below.

[0168] Component A consists of the following: photoinitiator Eosin Y (1.72 mg, 0.265 mM), and reagents triethanolamine (82.3 µL, 62.3 µM) in BSS (10 mL). N -Vinyl-caprolactam (62.6 mg, 45.0 µm). The stock solution was aliquoted for combination with component B prior to the preparation of HBT grafts.

[0169] Preparation of thinning media

[0170] On the HBT assembly day, glucose is added to the thinning media matrix, glucose dissolution is accelerated by shaking in a 37 °C water bath, and the thinning media is sterilely filtered using a 2 µm filter before use.

[0171] Corneal preparation

[0172] The cornea was dissected and transferred to a dilution medium (Gibco Essential Flex 1:1 DMEM:F12 (Thermo Fisher Scientific, USA), insulin-transferrin-selenium (5 μg / ml-5 μg / ml-5 ng / ml, Thermo Fisher Scientific, USA), 2% fetal bovine serum, antibiotics / antifungals (penicillin 100 U / ml, streptomycin 100 μg / ml, amphotericin 250 ng / ml (Thermo Fisher Scientific, USA)), and 5% dextran 500 (MW 500000, Pharmacosmos, Denmark) for 16 h, after which the corneal endothelium was dissected. Eyeballs from fresh Merino sheep carcasses were obtained from local slaughterhouses (Westside Meats, Australia) or experimental cadavers and placed at 4°C in antibiotics / antifungals (penicillin 100 U / ml, streptomycin 100 μg / ml, amphotericin 250 ng / ml (Thermo Fisher Scientific, USA)). μg / ml amphotericin B 250 ng / ml (Thermo Fisher Scientific, USA) phosphate-buffered saline (PBS). Before corneal detachment, the eyeball was 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 then rinsed with antibiotic / antifungal agent in PBS.

[0173] Preparation of post-elastic layer

[0174] Inside the laminar flow hood, the culture dish is placed upside down on the dissecting microscope stage. Inside the hood, 2.5 mL of sterile air is drawn into a syringe. The cornea is placed with the endothelium facing up, and a drop of dilution medium is added. With the cornea held at the edge of the culture dish with the aid of forceps, a needle is inserted 180 degrees parallel to the culture dish, directly below the endothelium and at the limbus. The needle is inserted until the insertion length is twice the size of the pore. The full volume (2.5 mL) of air is steadily injected into the cornea via the syringe, and then the needle is removed once the Descemet's membrane has separated from the stroma. Complete separation should minimize the amount of lamellar fibers on the endothelium. The swollen cornea is transferred to a new culture dish, endothelium facing up, and a drop of dilution medium is added. Using a 15-degree needle, two 1 cm incisions are made, one at the top of the swollen area next to the limbus and one at the bottom, forming a double-opening 'envelope'.

[0175] Preparation of bio-glue

[0176] The bioglue was prepared on the same day as the HBT assembly. In a laminar flow hood, an equal part of bioglue component B (5 µL) was added to bioglue component A (15 µL) in a black, light-proof Eppendorf tube. The solution was well mixed by repeatedly pumping the mixture into and out of the tube. Once mixed, the tube was closed and stored on ice or in a refrigerator at 4°C prior to assembly.

[0177] Prepare PHF for HBT assembly

[0178] Add 20 µL of sterile trypan blue staining solution to the BSS (5 mL) in the petri dish. Transfer the γ-irradiated PHF dish (12 mm dish, in BSS) to the solution and incubate at ambient temperature for 10 min. Once the PHF dish is fully stained blue, it is ready for use.

[0179] Assemble HBT grafts

[0180] Using an eye spear, absorb all the diluent from the two openings adjacent to the 'capsule'. Lift one 'capsule opening' with forceps and further dry the inside of the corneal 'capsule' using the eye spear. Remove the PHF disc from the 'blue stain' solution and briefly dry it using the eye spear. Again using forceps, lift the 'capsule opening', insert the PHF into the 'capsule', and smooth the endothelium from the edges to reduce air bubbles. Using a pipette, apply 10 µL of mixed bioglue between the Descemet's membrane and the PHF. Move the entire cornea onto the fixator of the 8.5 mm trephine and penetrate the cornea by pressing firmly. Add 20 µL of diluent to the endothelium in the center of the punch and keep the trephine in place. Activate the bioglue by placing a light source on top of the trephine and crosslink at room temperature for 10 min. Remove the trephinated portion of the cornea using a guarded punch, and carefully detach the stroma from the PHF side of the HBT graft under a dissecting microscope. Transfer the assembled HBT graft to fresh, diluted media and store at room temperature prior to transplantation.

[0181] 2 Results and discussion

[0182] 2.1 Synthesis and physical characterization of bio-glue

[0183] Component B is prepared by chemically modifying a PEG-based trifunctional glycerol ethoxylate to form a photocrosslinkable glycerol ethoxytrimethacrylate (GEM). In dichloromethane (DCM), in the presence of dimethylaminopyridine (DMAP) and triethylamine (Et3N), an excess of methacrylic anhydride (MAA) is used to functionalize the glycerol ethoxylate (1 kDa). Figure 7 The crude material was purified by preparative liquid chromatography, then separated and evaporated to dryness to obtain a clear, colorless oil. 1 ¹H NMR analysis showed that the methacrylate esterification functional group was successfully introduced, located at δ 6.13, 5.57 and 1.95 ppm ( Figure 1 All batches of GEM (referred to herein as component B) should be stored at -20°C in the dark until required.

[0184] In common adhesive applications, components A and B are premixed in various volume ratios to obtain a low-viscosity bio-adhesive, which is then applied immediately. Figure 7 b) The bio-adhesive is applied to a polymeric hydrogel membrane (PHF) having another PHF membrane added for mechanical testing; or, in in vitro or in vivo studies, having a post-elastic layer. In this case, white light is irradiated onto the sample to achieve photocrosslinking and gelation of the bio-adhesive, thereby forming a bio-adhesive layer between the two materials.

[0185] The photocrosslinking of bio-adhesive formulations (component A:B ratio of 5:1) is achieved through... 1 H NMR and UV-Vis evaluations were performed to determine the optimal time required for gelation in the bulk upon exposure to various light sources. Biogel formulations under white light... 1 1H NMR analysis directly indicates that prior to gelation, as crosslinking proceeds, the polymer undergoes methacrylate esterification conversion, with a significant decrease in signal at δ 6.13 and 5.57 ppm. Figure 8 However, at higher conversion rates, the signal caused by methylene groups even at δ 3.7 ppm disappears due to sample solidification.

[0186] Perform ATR-FTIR analysis and summarize the results. Figure 8 middle, Figure 8 The following spectra are shown: a) glycerol ethoxylate, b) glycerol ethoxytrimethacrylate (GEM, component B), c) solution of component A, and d) photocrosslinked bio-adhesive formulation after crosslinking (component B, 50 v / v%). The functionalization of glycerol ethoxylate (GE) to form a GEM shows a 3463 cm⁻¹. -1 The OH group at the location showed reduced stretching, and at 1717 cm⁻¹-1 A strong ester signal appears at the (C=O stretching) point. Figure 8 a, b). At 1633-1642cm -1 The signal within the specified range is due to both aliphatic (CH) and conjugated (C=C) stretching of each sample. When component A ( Figure 8 c) and component B are combined and photoinitiated using light, resulting in the final FTIR (Flattened Fibre Induction Reflectance) of the biocollagen. Figure 8 d) shows 1717 cm -1 The relatively reduced C=O signal at the location indicates that cross-linking of methacrylates has occurred, although the cross-linking is incomplete.

[0187] Indirect UV-Vis analysis was used to monitor exposure to visible light (white light, 1022 mW / cm²). 2 (400-700 nm), blue light (3.8 mW / cm²) 2 ~455 nm), and green light (2.3 mW / cm²). 2 The gelation time at ~520 nm and the photoinitiator EosinY (λ) 最大 Absorbance decreases at ~520 nm. UV-Vis analysis ( Figure 9 The results show the relationship between light source intensity and wavelength and gelation time. Relatively high-intensity white light showed a rapid decrease in Eosin Y absorbance, with a gelation time of 3 min. Green LED light, with significantly lower intensity but a narrower wavelength matching the Eosin Y wavelength, showed a slower decrease in Eosin Y absorbance, with a gelation time of 7 min. Blue LED light of similar intensity showed that even after 20 min of irradiation, Eosin Y absorbance decreased moderately, indicating limited gelation.

[0188] Laboratory tests and in vitro In the study, broad-spectrum white light generated by commercial LEDs, bicycle lights, newer iPhone models, and operating table lights was sufficient to achieve biogel gelation.

[0189] In practice, the application of bioglue is expected to be carried out in various aqueous media, such as phosphate-buffered saline (PBS), balanced salt solution (BSS), or commercial ophthalmic solutions, such as Optisol GS. TM Or Life4C TM Staining solutions such as trypan blue are commonly used to visualize ophthalmic implants during DMEK / DSAEK surgery. For this reason, the dilution of the bio-glue and its impact on its mechanical strength are important considerations. Figure 10 As shown, a series of bio-adhesive formulations containing premixed components A and B were prepared by using components A and B in different volume ratios.

[0190] Prepare a sample (0.75 mL) and test it under white light (1022 mW / cm²). 2 Irradiate at a distance of 2 cm. Figure 10 The effect of dilution of the biogel formulation relative to its gelation time is shown, where a ratio of 1:1 gels within 1 min, while a ratio of 10:1 gels at approximately 21 min, where the sample appears soft and fragile (inset). Figure 10 ).

[0191] To test the mechanical properties of the in-bulk biogel, samples (0.75 mL samples according to Table 1) were prepared in a disposable syringe and irradiated with white light until complete gelation occurred.

[0192] Table 1. Summary of formulations, compression test results, and swelling properties of cross-linked bio-adhesive samples, where component B is 10-50 v / v%. Swelling rate was determined in BSS.

[0193]

[0194] The volume fraction of the crosslinking component (component B) was increased from 10 v / v% to 50 v / v%. Compression measurements were determined using an Instron 5848 instrument, and mechanical properties are shown as an average of three samples, with only one sample from entry 1 being successful. Representative pre-fracture stress-strain curves for samples containing between 10 and 50 v / v% of component B are shown in the supporting information ( Figure 11 The results of mechanical tests on component B at different concentrations (10-50 v / v%) are summarized in Table 1 and shown below. Figure 12 middle.

[0195] The samples have relatively similar elasticity, and the fracture strain is ( ε Between 0.22 and 0.32 mm / mm Figure 12 a). Increasing the concentration of component B from 10 v / v% to 50 v / v% resulted in an increase in overall strength, with fracture stresses ranging from 0.04 to 2.47 MPa, reaching a maximum of 3.31 MPa at 40%. Similarly, increasing the crosslinking component from 10 v / v% to 50 v / v% resulted in an increase in Young's modulus ( E The toughness of the sample increased from 0.2 MPa to 8.1 MPa. U T The strength increased from 0.01 MPa to 0.43 MPa. The strength of the bioglue indicates its ability to achieve cross-linking and shows the effect of dilution during application on the mechanical strength of the bioglue. However, in practice, the adhesion properties with both the hydrogel PHF substrate and the tissue sample are more important.

[0196] The swelling ratio of each biogel formulation was determined after incubation in PBS at 37°C for 1, 2, 5, and 24 hours. Figure 13 The results showed that all samples continued to swell during the initial 5 hours, stabilizing after being kept overnight (24 hours). Furthermore, increasing the crosslinking component from 10 vol% to 50 vol% resulted in a decrease in the swelling percentage of the resulting bio-glue samples from 19.9 ± 2.8% to 12.3 ± 1.7%, respectively. Increasing the concentration of component B showed an increase in the crosslinking density and strength of the subsequent bio-glue, with a decrease in the swelling of the resulting samples.

[0197] The transparency of samples prepared for mechanical testing was also analyzed. In applications where the bioglue is used to adhere the Descemet membrane to PHF, the expected total thickness will vary between 100–250 µm. Although this constitutes a minor component of the HBT graft, the transparency of the bioglue remains important. It has been reported that combinations of hydrophobic gelatin with hydrophilic polymers can lead to loss of transparency during heating (Sani et al., 2019; Jumelle et al., 2021), although the authors caution that patient vision may have been impaired during healing. In this case, using hydrophilic PEG-based PHF and the bioglue, both components maintained high clarity. Samples prepared as thin cross-linked membranes (0.2 mL and 1.3 mm thick) in standard 24-well plates showed transparency between 98–100%. Figure 14 a). Even thicker 10 mm biogel samples showed a transparency ratio of approximately 70-85%, and were still clearer than previously reported gelatin-based hydrogels (Sani et al., 2019; Jumelle et al., 2021). Figure 14 b and c show the cylindrical biogel sample before mechanical testing and the thinner sample prepared in a 24-well plate, respectively. Note also that the thicker test sample appears yellow; however, this is not observed in the thinner 1.3 mm sample, and it is expected to be colorless when applied as a 50 µL sample to HBT grafts.

[0198] The transmittance of the cross-linked bioglue was also evaluated using UV-Vis spectroscopy. In the preparation of HBT grafts for surgery, PHF was treated with 10–30 µL of bioglue solution, with the post-elastic layer placed on top of both, and irradiated with light to achieve cross-linking of the bioglue. To determine the effect of the bioglue on the clarity of the HBT graft, PHF samples were treated with aliquots of the bioglue formulation (components A:B, 5:1, 20 µL). UV-Vis spectra of PHF; PHF and unreacted bioglue; and subsequent hydrogel-bioglue (HB) structures were performed. Figure 15The orange HB before photocrosslinking showed strong absorption at ~522 nm, consistent with the photoinitiator Eosin Y. After irradiating the sample with light for 5 and 10 minutes (1400 lumens, distance = 2 cm), the signal decreased significantly, resulting in a clear and colorless HB. Transmittance was shown to be between 90% and 100% in the visible range of 380–700 nm.

[0199] The adhesion of the bio-adhesive formulation to PHF and tissue (amniotic sclera) samples was determined using an overlap shear test. The hydrogel PHF samples were cast onto microscope slides. Tissue samples were adhered to the slides using a commercially available super-strong adhesive (cyanoacrylate). Overlap shear tests were performed on (i) PHF-bio-adhesive-slide; (ii) PHF-bio-adhesive-PHF; and (iii) PHF-bio-adhesive-tissue samples, with an overlap area of ​​approximately 2.5 x 1.5 cm. 50 μL of the bio-adhesive formulation (30%B) was dropped onto the PHF surface, and a second slide was inverted and placed on top of the sample. The two slides were inverted, and white light (1400 lumens) was irradiated through the glass and PHF for 10 minutes to achieve cross-linking of the bio-adhesive. Tensile tests were performed on each series using an Instron 5949 microforce meter equipped with a 50 N force sensor, in triplicate. Figure 16 A summary of publicly available commercially available ophthalmic adhesives (Dermabond) TM Evicel TM GelCORE TM and CoSEAL TM The results for three series were based on the overlap shear stress. These adhesives are designed to close 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 the PHF-bioglue-glass sample showed failure at 45 ± 23 kPa, indicating stronger adhesion with the gel than with the glass. The lowest adhesion strength was observed in amniotic corneal sclera adhered to PHF using bioglue, at 35 ± 15 kPa. Note that in some samples, tissue delamination from the glass occurred prior to testing. Nevertheless, overlap shear adhesion of PHF to corneal tissue via bioglue was observed.

[0200] 2.2 In vitro studies of bio-adhesive toxicity

[0201] The cytotoxicity of two separate batches of bioglue component GEM (component B) – denoted as GEM-B1 and GEM-B2 – was investigated in B4G12 cells on DMEK grafts. After 72 hours of incubation, concentrations were observed at most 1 / 1500, comparable to the control. Figure 17 a,b).

[0202] The cytotoxicity of the cross-linked formulation (5:1 component A: B) in B4G12 cells was also investigated. After 72 hours of incubation with a maximum sample size of 45 µL, successful but gradually decreasing cell counts were observed. Figure 17 (c, d). Data indicate that the cross-linked bio-adhesive has no in vitro cytotoxicity to DMEK grafts.

[0203] Similar results were obtained for cross-linked bioadhesive corneal stroma (Primary hCSK, human corneal stroma cells) and renal epithelium (HEK, human endothelial corneal cells), with both proliferation and metabolism confirmed as viable.

[0204] 2.3 PHF Preparation

[0205] According to the procedure described by Gonzalez-Andrades et al., 2015, PHF samples were prepared ( Figure 7 c). The challenges of reproducibility and scalability were addressed by reducing the amount of sebacyl chloride in the original formulation. PHF containing 0, 5, and 10% mol excess SbCl and 5 wt% PCL was prepared, resulting in stable and reproducible gel samples prepared in 15 mm diameter petri dishes, enabling quantitative preparation of PHF samples. PHF was prepared by reacting glycerol ethoxylate and polycaprolactone with sebacyl chloride in dichloromethane at 60°C. After cooling, the hydrogels were floated using increasingly concentrated aqueous solutions of acetone (0, 25, and 50 wt%), followed by washing in deionized water for several days. A 10 mm diameter disc was then punched out using a metal trephine saw and stored in a BSS before γ-irradiation (25 Gy) (Steritech Aus.).

[0206] 2.4 In vitro human tissue HBT test

[0207] The experiment was conducted at Eversight USA, using donor human tissue, PHF, and a bio-glue formulation. A PHF sample stored in PBS was placed on a trephine table, and ~100 mL of premixed bio-glue (components A:B, 5:1) was added via syringe. Figure 18 a). Subsequently, the donor's posterior elastic layer was added, with the cells facing down. The intact HBT graft was then irradiated with white light (surgical scope light) for 4 minutes. Figure 18 b). Yellow HBT grafts were cut into 8mm pieces and transferred to BusinGlide using forceps. Figure 18 The HBT graft in cell medium was pulled through a glide stage four times, and the HBT graft did not detach and showed good preservation. Figure 18 e). Similar results were achieved using a simple iPhone light, demonstrating the versatility of photocrosslinkable bioglue.

[0208] In the second experiment, an intact human cornea with the stromal layer was flipped and partially cut with a trephine saw, allowing the Descemet's membrane to peel off to one side while still remaining attached to the cornea through a small contact area. A photosensitive epidermal graft (PHF) was placed on top of the stromal layer and dried. Then, an equal amount (~10 µL) of bio-adhesive was added to the surface of the PHF via a micropipette, and the Descemet's membrane was peeled back onto the bio-adhesive and PHF. Subsequently, the cornea was irradiated with white light from a surgical lamp for 4 minutes to facilitate adhesion between the PHF and the Descemet's membrane. Once cross-linked, the high-density stromal graft (HBT) was punched using a trephine saw blade to obtain a circular HBT graft, which was then separated from the remaining unattached stromal layer.

[0209] Here, it has been shown that HBT grafts were successfully formed in vitro from human posterior elastic layer and were successfully and repeatedly passed through the Endoglide device, demonstrating the use of photocrosslinked polyethylene-based bioglue to achieve rigidity of the bond between PHF and posterior elastic layer.

[0210] Optical coherence tomography (OCT) scans were performed on the HBT grafts. The typical thickness of the human posterior elastic lamina is between 10 µm and 20 µm, depending on the patient. The estimated thickness of the PHF membrane is approximately 121 µm, while the thickness of the cross-linked bio-glue varies, ranging from 50 to 180 µm. The large variation in the thickness of the cross-linked bio-glue layer depends on the bio-glue concentration and the method of application.

[0211] 2.5 Ex vivo evaluation of HBT grafts

[0212] In vitro assembly of sheep descemet's membrane (containing residual stroma, cross-linked with bioglue to facilitate PHF adhesion) was performed, and the effects of dilution, cross-linking time, and evidence of delamination were recorded. PHF, bioglue, and descemet's membrane tissue grafts were termed HBTs. Note the evidence of adhesion; the constructed HBT grafts remained intact after several months of storage in PBS at 4°C. Cell viability (N=4) of HBT grafts to individual DMEK grafts (N=4) was determined using the MTT assay. Figure 19 ).

[0213] 2.6 In vivo assessment

[0214] The following methods were used to test DMEK in sheep: a gamma-irradiated PHF membrane and a fresh sheep des Ossae layer prepared by photoinitiated crosslinking using a bioadhesive formulation (components A:B, volume ratio 3:1 or 5:1). Both the PHF and des Ossae layer were dried according to practice before adding the bioadhesive to the PHF surface. Bioadhesive dilution should be minimized when handling the two materials before bonding or during implantation, as dilution indicates that it reduces the bioadhesive's ability to crosslink and thus produce adhesion. This initial method of peeling off the des Ossae layer led to difficulties in handling and subsequent cell viability. An improved method was developed involving the preparation of a capsule in the sheep cornea, resulting in improved surgical handling of HBT grafts.

[0215] 3 Supporting Information

[0216] 3.1 Pass 1 H NMR analysis of bio-collagen component B

[0217] 1 ¹H NMR was performed in deuterated CDCl₃ and the signals were located at δ 6.13, 5.57, and 1.95 ppm, consistent with the methacrylate methine and methyl groups a, b, and c in the trimethacrylated PEG of component B, respectively. Figure 1 Trace amounts of dichloromethane were observed at δ 5.30 ppm and quantified by comparison with the signal at δ 6.30 ppm. Functionalization was determined using the integral of signal a with respect to the polyethylene repeating unit (3.8–3.6 ppm).

[0218] 3.2 Photocrosslinking of biocollagen from component A and component B

[0219] The bioadhesive formulation for adhering PHF to corneal tissue (human or animal) consists of a photoinitiator solution component A, combined with a crosslinkable polymer component B. Photoinitiator solution component A consists of the photocatalyst Eosin Y, vinylcaprolactam (VC) in an aqueous solution, and triethanolamine (TEO). The concentration of component A and the component A:B ratio are important for the successful adhesion of HBT grafts. In a typical procedure, 20 wt% (w / w) of component B is combined with 80 wt% of component A solution. An equal part (30 μL) of the homogeneous mixture is added to the PHF, and the post-elastic lamina tissue is placed on both surfaces. Light is irradiated at a distance of 2 cm for 10 minutes to achieve photocrosslinking of the bioadhesive and adhesion between the PHF and the tissue layer.

[0220] The effects of light intensity and light source were studied in order to determine the curing time of the bio-adhesive.

[0221] Using D2O 1¹H NMR analysis was used to determine the conversion of unreacted acrylate groups in the biocollagen under white light exposure. UV-Vis was used to determine the decrease in the photoinitiator Eosin Y signal over time at ~520 nm.

[0222] Figure 20 The image shows proton NMR analysis of components A and B in D2O solvent prior to photoinitiation. NMR failed to detect a signal for Eosin Y (0.23 mM) in component A due to its low concentration in the biogel formulation. Signals associated with the catalysts VC and TEA were observed at δ 7.2 and 2.4 ppm, respectively. Separate samples of component A (0.80 mL, in D2O) and component B (0.20 mL, aliquot oil) were prepared in NMR tubes and analyzed by light (700 lumens, 1022 mW / cm²). 2 Irradiate for a specific time. Before analysis, note the gelation of each sample.

[0223] Figure 21 The image shows samples irradiated for different durations. 1 1H NMR spectroscopy. Before gelation, the sample showed a gradual decrease in acrylate signals a and b at δ 6.1 and 5.7 ppm, respectively. After 10 minutes of irradiation, the sample gelled, and due to the lack of polymer mobility, no signals were observed that were caused by the acrylate moieties a and b, or the PEG backbone signal eg (3.7–3.5 ppm).

[0224] When using higher intensity light, the effect of light intensity on crosslinking time was observed to occur within 3 minutes of light irradiation. This was observed when using 1000 lumens, 1460 mW / cm². 2 The light source replaces 700 lumens (1022 mW / cm²) 2 When the gel time was reduced from 10 min to 3 min, the gel time was reduced from 10 min to 3 min. 1 1H NMR analysis showed the effect of gelation due to the inability of polymer chains to move on the NMR signal; however, signals of TEA and solvent D2O at δ 2.7 and 4.7 ppm, respectively, were still observed. Figure 22 ).

[0225] 3.3 Crosslinking analysis by UV-Vis spectroscopy

[0226] UV-Vis spectroscopy was used to monitor the concentration of the photoinitiator Eosin Y after light irradiation. 最大 The correlation between the concentration of Eosin Y peak at 520 nm and the sample gelation time, and the relationship between the concentration of Eosin Y peak at 520 nm and the gelation time of the sample. 1 The samples prepared by ¹H NMR analysis were correlated. Eosin Y at certain concentration ranges with λ 最大= 520 nm to generate a standard curve. The standard curve is used to monitor the progress of photocrosslinking of the bio-adhesive formulation and to determine the normalized concentration of Eosin Y before gelation. Bio-adhesive samples (component A: 80 wt% in BSS; component B: 20 ​​wt%) were prepared and added to quartz vials with a 1 mm channel length for analysis by UV-Vis. The samples were irradiated with light at different time intervals (a. white light (400-700 nm, 1022 mW / cm²)). 2 b. Green LED light, λ 最大 ~520nm, 2.3mW / cm 2 Or blue LED light: λ 最大 ~455 nm, 3.8 mW / cm 2 ), and record its UV-Vis spectrum. Figure 22 The relative absorbance of samples irradiated with white, green, and blue light is shown, demonstrating the effect of wavelength and intensity on biocollagen crosslinking. Using white light with higher intensity but a wider wavelength range resulted in a decrease in Eosin Y concentration and gelation of the sample within 3 minutes. Directional green light with a wavelength matching Eosin Y but lower intensity still successfully crosslinked the biocollagen sample, but only after 7 minutes of irradiation. For samples using blue light with an intensity higher than green light but a wavelength unlikely to activate Eosin Y, gelation still occurred, but only after 20 minutes of irradiation. It is also shown that natural light leads to spontaneous gelation of the sample, and this is the reason why the biocollagen can be delivered as two separate formulations in the absence of light.

[0227] During HBT graft fabrication, surgeons typically use a diluent to aid in handling soft, fragile tissue. This dilution has the potential to influence cross-linking (or gelation) time, the strength of the bioadhesive formulation, and adhesion. A series of experiments were designed to investigate the effect of bioadhesive dilution on these properties, thereby determining the parameters in which the HBT fabrication will successfully or likely to delaminate.

[0228] Samples containing different concentrations of bio-gel components were prepared and tested under white light (700 lumens, 1022 mW / cm²). 2The crosslinking time was determined by irradiation at a distance of 2 cm. Standard solutions of component A (80 wt% in BSS, [Eosin Y] = 0.26 mM) and component B (20 wt%, [Component B] = 17 mM) were prepared. Equal portions of this solution were diluted with BSS to reduce the overall concentration of photoinitiator, catalyst, and crosslinking polymer according to Table 2. The gel time was recorded as the point where no mobile phase was observed when the sample was inverted. UV-Vis analysis was used to determine the relative concentration (mM) of Eosin Y after irradiation with white light (700 lumens) and before gelation of the sample. When the sample was diluted ~10 times (Table 1, Sample 4), no gelation occurred even after 30 minutes of irradiation.

[0229] Table 2. Biogel formulations of component A (containing Eosin Y, mM) and component B (mM) in BSS This indicates that the observed sample was completely gelled.† Sample 4 showed no gelation signal even after 30 minutes.

[0230] Figure 23 The effect of component B concentration on the gel time of the bio-adhesive formulation is shown. For practical application of the bio-adhesive during HBT graft formation, dilution by surgical media should be kept at a minimum of a 3:1 dilution factor (i.e., Table 2, Sample 3), and light irradiation should be prolonged to ensure successful cross-linking of the bio-adhesive. Alternatively, a more concentrated sample containing a higher proportion of component B (relative to component A) can be prepared prior to HBT formation.

[0231] To evaluate the strength of the bioadhesive formulation at different concentrations, compression tests were performed on irradiated cylindrical samples (1 cm diameter x 1 cm height). The tests were repeated three times, and the standard deviations for each sample were recorded (Table 1).

[0232] Example 2. Preparation of bioglue-tissue (BT) grafts

[0233] The inventors have further discovered that the bioglue of the present invention allows for the preparation of bioglue-tissue (BT) grafts suitable for DMEK surgery in the absence of hydrogels. The BT graft is prepared by applying a mixture of components A and B according to Example 1 to human DMEK tissue using a non-adhesive silicone patch as an assembly platform. The resulting bioglue-tissue (BT) graft also allows for the simplification of the highly desirable but previously difficult DMEK surgical procedure.

[0234] 1. Method

[0235] The cadaveric donor cornea was transferred to thinning media overnight and maintained at 37 °C, 5% CO2 until the BT assembly day. On the assembly day, DMEK tissue was prepared by manually dissecting the Descemet's membrane from the corneal stroma (removing 85% of the entire graft) using a 10 mm (diameter) circular trephine saw (#17212D1000, Moria, France) for circumferential scoring, followed by dissection using fine-tipped surgical tools, including DMEK stripping-peeling forceps (J2892E, Fogla, Italy) and cleavage hooks (J2891E, Janach, Italy).

[0236] After the DMEK tissue was peeled off, a sterile 11mm x 11mm medical-grade non-adhesive silicone patch (MED82-5010-10 0.010” (0.25mm), Polymer Systems Technology Limited, UK) was placed on the stroma at the center of the cornea. Using a micropipette, 10μL of a mixture of bioadhesive components A and B (mixing ratio A:B = 7:1) was added to the center of the patch (adapted to room temperature by refrigeration, component A at 4°C and component B at 20°C). Subsequently, using fine-tipped forceps, the peeled DMEK tissue was carefully placed on top of the bioadhesive, starting from the periphery of the endothelium. The complex was then removed from the rest of the cornea and placed in a sterile culture dish. A droplet (10μL) of OCM (organic culture medium) was added to the endothelium to provide the necessary moisture for cell viability.

[0237] A sterile ring-shaped platform, measuring 13 mm in diameter and 2 cm in height, was placed on the assembly complex. A light source (1000 lumens LED bicycle light) was then placed on top of the ring and turned on for 10 minutes to allow for photocrosslinking of the bioglue (OCM droplets (10 μL) were added to the endothelium every 2 minutes to maintain cell viability). The assembled BT graft was carefully removed from the silicone patch, starting from the periphery. The BT graft was maintained in OCM at 37 °C (5% CO2 incubator) for 2 hours. Subsequently, the assembled BT graft was stained with trypan blue (0.4%, sterile) for easier visualization and surgical implantation. Alternatively, the bioglue can be added directly to the endothelial side of the DMEK tissue, followed by assembly and photocrosslinking as described above.

[0238] 2. Assessing BT cell viability in vitro using CAM-PI

[0239] To ensure that the chemical reactions resulting from the crosslinking of bioglue components A and B do not affect the viability of endothelial cells in DMEK tissue, the mixed bioglue was applied directly to a monolayer of B4G12 (immortalized human endothelial) cells, which served as an in vitro model. Cell viability was then assessed using a CAM-PI (calcein AM-propidium iodide)-based staining and imaging protocol.

[0240] B4G12 cells were used in a 12-well tissue culture plate on a plastic tray (13 mm in diameter) at a density of 2000 cells / mm. 2 A confluent monolayer (inoculated) was used as the test subject (used as DMEK tissue). After washing the cell monolayer with sterile BSS to remove cell debris, 13 μL equal parts of a biogel mixture with different volume ratios of component A and component B were directly applied to the cells.

[0241] To test the effect of crosslinking of bioglue mixtures on B4G12 cell viability, three test groups were set up. These included no treatment, bioglue added but not photocrosslinked, and bioglue photocrosslinked.

[0242] For the test group of bio-linked gel, after applying the mixed bio-gel, the transparent cap of the 12-well plate was placed on top of the plate, and a 1000-lumen LED white light was placed on top and turned on for 10 minutes. After the photocrosslinking process, the cell monolayer was washed in sterile BSS, and 500 μL of warm OCM was added to the wells, allowing the cells to recover at 37°C and 5% CO2 for 3 hours.

[0243] For the non-photocrosslinked test group, a mixed biogel (with different volume ratios of components A and B, as described above) was applied to the cells for the same duration of 10 minutes, followed by aspiration of the biogel and washing with BSS. Cells were then allowed to recover at 37°C and 5% CO2 for 3 hours, as described above. After the 3-hour recovery period, OCM was removed from the wells, and the cells were washed with BSS.

[0244] After aspirating the BSS, add 500 μL of CAM solution (2.5 μM, #C3100MP, Invitrogen, USA) to each well and incubate in the dark at room temperature for 30 minutes. Aspirate the chlorophyll solution and wash the wells with BSS. Add 500 μL of PI solution (2.5 μM, #P4170-10MG, Merck, USA) to each well and incubate in the dark at room temperature for 5 minutes. Subsequently, wash the wells with BSS and remove the tissue culture plastic (TCP) trays from the wells.

[0245] Mount the TCP disk on a glass slide, cell side down, for visualization using a fluorescence microscope at wavelengths of 488 nm (for CAM) and 560 nm (for PI). Green dots indicating CAM uptake indicate live cells, and red dots indicating PI uptake indicate dead cells. Cell viability is calculated as the number of green dots relative to the total number of green and red dots.

[0246] 3 Results

[0247] A series of bio-glue mixing ratios (with decreasing percentage of component B) were tested: A:B = 1:1 (50% B), A:B = 1.5:1 (40% B), up to A:B = 8:1 (11.1% B). As the proportion of bio-glue component B in the mixture decreased, an increase in the number of viable cells [indicated by positive CAM staining (green fluorescence)] in the B4G12 cell monolayer was observed. Simultaneously, a decrease in the number of dead cells [indicated by positive PI staining (red fluorescence)] was observed. Figure 24 Bright-field images showed that B4G12 cells maintained a polygonal morphology when components A and B of the bioglue were mixed at an A:B ratio of 7:1, compared to the untreated and untreated controls. Negligible percentage of dead cells (indicated by red positive PI staining) were observed in untreated cells and controls treated with uncrosslinked bioglue (each photocrosslinking protocol lasting the same 10-minute duration). Increased cell viability was observed in B4G12 cells as the mixing ratio of component B decreased, indicated by higher CAM uptake and lower PI uptake. For example, only 20% of cells remained viable after direct casting of the bioglue and crosslinking with 50% of component B (A:B = 1:1); while more than 80% of viable cells were observed after direct casting of the bioglue and crosslinking with 12.5% ​​(A:B = 7:1) and lower components B. Figure 25 The 80% cell viability is noteworthy because traditional DMEK grafts should have at least 80% surviving corneal endothelial cells to be considered effective for corneal transplantation.

[0248] 4. Assessing BT cell viability in vitro using MTT assay

[0249] To ensure corneal endothelial cell viability after BT assembly, a cell viability assay based on MTT (3-(4,5-dimethyltriazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed on assembled BT grafts and untreated DMEK grafts as controls. Live cells were treated with MTT and cultured to generate formazan. This amount of insoluble formazan was then dissolved in acidified isopropanol, and cell viability was quantified using a plate reader at 570 μm.

[0250] After dissecting the DMEK or BE graft assembly, the graft was carefully transferred to each well of a 24-well TC plate. The graft was carefully washed once with 500 μL of BSS at RT, followed by aspiration of the BSS. 500 μL of MTT (0.5 mg / mL, #M2128-250MG, Merck, USA) was added to each well containing the graft. The plate was incubated at 37°C and 5% CO2 for 3 hours.

[0251] Subsequently, MTT was aspirated, and 500 μL of acidified isopropanol (100% isopropanol with 0.25 M acetic acid, Merck, USA) was added to each well. Using a micropipette, the purple formazan crystals produced by the living cells were gently dissolved by pipetting the solution around the graft for 3 minutes. For each graft, three 100 μL aliquots of the mixture were transferred to each well in the 96-well plate. The plate was read at an absorption wavelength of 570 nm.

[0252] References

[0253] Gonzalez-Andrades, J.; Cardona, de la., C.; Ionescu, AM; Mosse, CA; Brown, RA, Photographic-Based Optical Evaluation of Tissues andBiomaterials Used for Corneal Surface Repair: A New Easy-Applied Method. PLoS ONE October 2015, e0142099

[0254] Ozcelik, B.; Brown, KD; Blencowe, A.; Ladewig, K.; Stevens, GW;Scheerlinck, JPY; Abberton, K.; Daniell, M.; Qiao, GG, Biodegradable and Biocompatible Poly(Ethylene Glycol)-based Hydrogel Films for theRegeneration of Corneal Endothelium. Adv Healthc Mater 2014, 3, (9), 1496-1507

[0255] Sani, ES; Kheirhah , A. ; Rana, D.; Sun, Z.; Foulsham , W. ; Sheikhi,A.; Khademhosseini, A.; Dana, R.; Prophet, N., Sutureless repair of cornealinjuries using naturally derived bioadhesive hydrogels. Sci. Adv. 2019, 5,eaav1

[0256] Jumelle, C.; Sani, ES; Taketani, Y.; Yung, A.; Gantin , F. ;Chauhan , SK ; Prophet, N.; Dana, R. Growth factor-eluting hydrogels formation of corneal defects. Materials Science and Engineering: C 2021,120, 111790

Claims

1. A biocompatible crosslinkable polymer composition comprising: a crosslinkable branched polyether compound having a core moiety and at least three polyether arms covalently bonded to the core moiety, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group.

2. The biocompatible crosslinkable polymer composition according to claim 1, wherein the crosslinkable branched polyether compound has the following structure: A(BX) n in: A represents the n-valence core portion; BX represents the polyether arm, where B is the polyether segment and X is a polymerizable olefinic unsaturated group; and n is at least 3.

3. The biocompatible crosslinkable polymer composition according to claim 1 or claim 2, wherein the core portion is a hydrocarbon, carbohydrate, heteroalkyl, heterocycloalkyl, or heteroaryl portion.

4. The biocompatible crosslinkable polymer composition according to claim 3, wherein the core portion comprises 1 to 12 carbon atoms, or 3 to 8 carbon atoms, or 3 to 5 carbon atoms.

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

6. The biocompatible crosslinkable polymer composition according to any one of claims 1 to 5, wherein the polymerizable olefinic unsaturated group forms part of a (meth)acryloyl group, (meth)acryloyloxy group, styrene group, vinyl ether group, vinyl ester group, or (meth)acrylamide group.

7. The biocompatible crosslinkable polymer composition according to any one of claims 1 to 6, wherein the crosslinkable branched polyether compound is: Each n is an independent integer ranging from approximately 4 to approximately 40.

8. The biocompatible crosslinkable polymer composition according to any one of claims 1 to 7, wherein it is biodegradable.

9. An aqueous bioadhesive composition comprising a biocompatible crosslinkable polymer composition according to any one of claims 1 to 8, water, and a biocompatible photoinitiator.

10. The aqueous bio-adhesive composition according to claim 9, wherein the biocompatible photoinitiator is selected from: Eosin Y with triethanolamine and vinylcaprolactam; tris(2,2-dipyridyl)ruthenium(II) hexahydrate (Ru(bpy)3) with sodium persulfate, camphorquinone, and N , N -Dimethyl-p-toluidine, 2-ethyl-dimethylbenzoate, or N -Phenylan; 2,2,2,6,6-Tetramethylpiperidine; dl-2,3-dione-1,7,7-trimethylnorbornene (CQ); 1-Phenylan-1,2-propanedione (PPD); 2,4,6-Trimethylbenzoyl-diphenylphosphine oxide (TPO); bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819); 4,4'-bis(dimethylamino)diphenyl Methyl ketone; 4,4'-bis(diethylamino)benzophenone; 2-chlorothioxanth-9-one; 4-(dimethylamino)benzophenone; phenanthrenequinone; ferrocene; diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide / 2-hydroxy-2-methylphenylacetone (50 / 50 blend); dibenzocycloheptenone; resazurin; halogenated benzoyltrimethylgermanane (Ivocerin®); its derivatives, and combinations thereof.

11. The aqueous bioadhesive composition according to claim 9 or claim 10, comprising about 10% to about 50% by volume of a biocompatible crosslinkable polymer composition.

12. A kit for adhering a biocompatible hydrogel to a biological substrate, the kit comprising: (a) A first component comprising the biocompatible crosslinkable polymer composition as described in any one of claims 1 to 8; and (b) The second component contains water and a biocompatible photoinitiator.

13. A kit for preparing a bio-substrate with an adhered cross-linked polymer backbone, the kit comprising: (a) A first component comprising the biocompatible crosslinkable polymer composition as described in any one of claims 1 to 8; and (b) The second component contains water and a biocompatible photoinitiator.

14. Use of the crosslinkable polymer composition according to any one of claims 1 to 8, the aqueous biogel 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 crosslinkable polymer composition according to any one of claims 1 to 8, the aqueous bioadhesive composition according to any one of claims 9 to 11, or the kit according to claim 13 for preparing a biosubstrate with an adhered crosslinked polymer backbone.

16. A process for adhering a biocompatible hydrogel to a biological substrate, the process comprising: (i) Combining the biocompatible crosslinkable polymer composition according to any one of claims 1 to 8 with water and a biocompatible photoinitiator to form an aqueous bioadhesive composition according to any one of claims 9 to 11; (ii) Providing an aqueous biogel composition between a biocompatible hydrogel layer and a bio-based substrate layer to form a laminated structure; (iii) Irradiating the stacked structure at a wavelength suitable for activating the biocompatible photoinitiator and initiating crosslinking of the crosslinkable branched polyether compound, the process of which promotes adhesion between the biocompatible hydrogel and the bio-substrate.

17. The process of claim 16, wherein the biocompatible hydrogel comprises a biodegradable and biocompatible polyether network polymer crosslinked via ester linkage.

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

19. A process for preparing a bio-substrate with an adhered cross-linked polymer network backbone, the process comprising: (i) Combining the biocompatible crosslinkable polymer composition according to any one of claims 1 to 8 with water and a biocompatible photoinitiator to form an aqueous bioadhesive composition according to any one of claims 9 to 11; (ii) Providing a layer of the aqueous bioadhesive composition on a biological substrate layer to form a laminated structure; (iii) Irradiating the stacked structure at a wavelength suitable for activating the biocompatible photoinitiator and initiating crosslinking of the crosslinkable branched polyether compound, the process promoting the formation of a crosslinked network polymer backbone that adheres to the biological substrate.

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

21. The process according to any one of claims 16 to 20, wherein the biological substrate is a posterior elastic lamina with a corneal cell layer containing membrane endothelial cells adhered to it.

22. A layered structure comprising a biocompatible crosslinked network polymer layer between a biocompatible hydrogel layer and a biocompatible substrate layer, wherein the crosslinked network polymer layer comprises a polymerized residue of a crosslinkable branched polyether compound, the crosslinkable branched polyether compound comprising a core portion and at least three polyether arms covalently bonded to the core portion, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group.

23. The laminated structure of claim 22, wherein the biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer crosslinked via ester linkage.

24. A layered structure comprising a biocompatible crosslinked network polymer layer and a bio-based substrate layer, wherein the crosslinked network polymer comprises a polymerized crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion and at least three polyether arms covalently bonded to the core portion, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group.

25. The laminated structure according to any one of claims 22 to 24, wherein the biological base layer is a des elastic layer with a corneal cell layer containing corneal endothelial cells adhered to it.

26. An ophthalmic implant having a layered structure, said layered structure comprising a biocompatible cross-linked polymer network layer between a biocompatible hydrogel layer and a biocompatible substrate layer. The biocompatible crosslinked network polymer layer comprises a polymerized, crosslinkable branched polyether compound residue, wherein the crosslinkable branched polyether compound comprises a core portion and at least three polyether arms covalently bonded to the core portion, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group. The biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer crosslinked via ester linkages, and The biological basal layer is the Descemet's membrane, which is a corneal cell layer containing corneal endothelial cells.

27. An ophthalmic implant having a layered structure comprising a biocompatible cross-linked polymer network layer and a biological substrate layer. The biocompatible crosslinked network polymer layer comprises a polymerized, crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion, at least three polyether arms covalently bonded to the core portion, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group, and The biological basal layer includes a des elastic lamina with a corneal cell layer containing corneal endothelial cells.

28. A method for treating corneal endothelial dysfunction in a subject, the method comprising the steps of: implanting an ophthalmic implant having a layered structure into the eye of the subject, the layered structure comprising a biocompatible cross-linked polymer network layer between a biocompatible hydrogel layer and a biocompatible basal layer. The biocompatible crosslinked network polymer layer comprises a polymerized, crosslinkable branched polyether compound residue, wherein the crosslinkable branched polyether compound comprises a core portion and at least three polyether arms covalently bonded to the core portion, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group. The biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer crosslinked via ester linkages, and The biological basal layer is the Descemet's membrane, which is a corneal cell layer containing corneal endothelial cells.

29. A method for treating corneal endothelial dysfunction in a subject, the method comprising the steps of: implanting an ophthalmic implant into the eye of the subject, the ophthalmic implant having a layered structure comprising a biocompatible cross-linked polymer network layer and a biological substrate layer, The biocompatible crosslinked network polymer layer comprises a polymerized, crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion, at least three polyether arms covalently bonded to the core portion, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group, and The biological basal layer is the Descemet's membrane, which is a corneal cell layer containing 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 layered structure comprising a biocompatible cross-linked polymer network layer between a biocompatible hydrogel layer and a biocompatible basal layer. The biocompatible crosslinked network polymer layer comprises a polymerized, crosslinkable branched polyether compound residue, wherein the crosslinkable branched polyether compound comprises a core portion and at least three polyether arms covalently bonded to the core portion, wherein each of the at least three polyether arms comprises a polymerizable olefinically unsaturated group. The biocompatible hydrogel layer comprises a biodegradable and biocompatible polyether network polymer crosslinked via ester linkages, and The biological basal layer mentioned above is the posterior elastic layer to which corneal endothelial cells are adhered.

31. Use of corneal endothelial cells in the manufacture of a medicament for treating corneal endothelial dysfunction, wherein the medicament is an ophthalmic implant having a layered structure comprising a biocompatible cross-linked polymer network layer and a biological basement layer. The biocompatible crosslinked network polymer layer comprises a polymerized, crosslinkable branched polyether compound residue, the crosslinkable branched polyether compound comprising a core portion, at least three polyether arms covalently bonded to the core portion, wherein each of the at least three polyether arms comprises a polymerizable olefinic unsaturated group, and The biological basal layer mentioned above is the posterior elastic layer to which corneal endothelial cells are adhered.

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