Ha-dendrimer cornea formulation for regeneration of corneal tissue

Abstract

A dendrimer-hyaluronic acid hydrogel including a methacrylate-functionalized hyaluronic acid ("HA") and a methacrylate-functionalized hydroxyl dendrimer [poly(amidoamine) can be easily applied over or into a corneal wound or a partially removed cornea to form new corneal tissue. The HA crosslinked hydrogel slowly degrades and facilitates regeneration of the cornea over a 6–10-month period, resulting in a tissue structure with structural and functional elements of the original cornea. The HA promotes corneal tissue regeneration, while the dendrimer component allows the ideal modulus within the hydrogel network, preserving corneal surface integrity.

Description

[0001] HA-DENDRIMER CORNEA FORMULATION FOR REGENERATION OF CORNEAL TISSUE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The application claims benefit of and priority to U.S. Provisional Application No. 63 / 560,472, filed March 1, 2024, which is hereby incorporated by reference in its entirety.

[0004] STATEMENT OF GOVERNMENT INTEREST

[0005] None.

[0006] FIELD OF INVENTION

[0007] The present invention relates to the field of corneal repair, and more particularly, to corneal wound repair.

[0008] BACKGROUND OF THE INVENTION

[0009] Cornea is a highly transparent tissue which protects the human eye from external intrusion and provides two-thirds of the total refractive power. Pathologies or diseases occurring in this vital tissue diseases are the leading cause of visual impairment and blindness globally, affecting approximately 2.2 billion people as of 2019. Various etiologies contribute to corneal blindness, including various inherited or acquired diseases such as severe dry eye disease, ocular surface disorders such as infections and inflammation, chemical burns, traumatic injuries, and surgical complications. If left untreated, these conditions can lead to significant problems, including corneal stromal fibrosis, scarring, and opacity. Currently, the most effective therapy for comeal blindness is corneal transplantation. Corneal transplantation requires healthy human donor tissue, specialized equipment and advanced surgical skills. However, considerable global donor shortage (available to less than 5% of the affected population), particularly in developing countries, complications related to graft rejections and infections often makes this procedure problematic. In addition, most developed countries do not have eye bank facilities to procure tissue for corneal transplants. Further, multiple sutures are required to secure the transplant into recipient tissue and these suture points act as a nidus for inflammation, infections and additional trauma contributing towards graft failure and astigmatism. The shortages and challenges associated with grafting comeal tissues has led to efforts to create artificial comeal tissue substitutes, which have not been effective so far.

[0010] Comeal fibrosis is one of the leading causes of blindness in ophthalmology. While corneal keratoplasty is a common treatment, its widespread use is limited due to the shortage of donor corneas and the complexity of the surgical procedure. Additionally, there is a lack of suitable materials that can effectively form comeal tissue or support corneal regeneration. Careful selection of material composition for corneal substitutes is important as ocular tissues are highly sensitive. Existing options include artificial corneas and in situ formed comeal substitutes derived from natural and synthetic polymeric materials. In situ formed corneal substitutes present a beneficial option in certain clinical applications due to their cost effectiveness and ease of use. Biomaterials for in situ formed substitutes, include but are not limited to polymethyl methacrylate, gelatin, collagen, silk fibroin, and hyaluronic acid (HA). More effective material compositions are needed to better support comeal regeneration, improve biocompatibility, and promote functional tissue integration.

[0011] Comeal tissue substitutes with sutureless implantation would increase availability of treatment to patients, reduce cost and complexity, and could promote endogenous regeneration of the cornea.

[0012] It is therefore an object of the present invention to produce an alternative material for use in repair and / or regeneration of corneal defects.

[0013] It is another object of the present invention to provide an alternative material for use in keractectomies.

[0014] SUMMARY OF THE INVENTION

[0015] A dendrimer-hyaluronic acid hydrogel which is generally a viscous liquid containing a mixture of two components: a methacrylate-functionalized hyaluronic acid (“HA”) and a methacrylate-functionalized hydroxyl dendrimer [poly(amidoamine), preferably of about 14 kDa, has been developed for use in ocular applications.

[0016] The dendrimer-hyaluronic acid hydrogel can be easily applied over or into a corneal wound or a partially removed cornea (e.g. in preparation for a Superficial Anterior Lamellar Keratoplasty (SALK, corresponding to -40-50% removal of corneal stroma) or a Deep Anterior Lamellar Keratoplasty(DALK, corresponding to -80-90% removal of comeal stroma) to form new comeal tissue. The HA crosslinked hydrogel slowly degrades and facilitates regeneration of the cornea over a 6-10-month period, resulting in a tissue structure with structural and functional elements of the original cornea. The HA promotes corneal tissue regeneration, while the dendrimer component allows the ideal modulus within the hydrogel network, preserving comeal surface integrity.

[0017] Examples demonstrate utility in a rabbit DALK and SALK model used to assess the application of gel as an in situ formed comeal substitute. A 7.0 mm diameter comeal defect, simulating two conditions: a 40-50% removal of corneal stroma to mimic SALK and an 80-90% removal to replicate DALK conditions in human corneal keratoplasty, demonstrated robust corneal regeneration and reconstruction when using the dendrimer-hyaluronic acid gel as an artificial comeal material. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIGs 1A-1D show the synthesis and formulation of the corneal repair hydrogel. FIG 1A is a schematic of the synthesis of methyl acrylated dendrimer (“D-MA”) conjugates by attaching methyl acrylate groups to the surface -OH groups of dendrimers using methacrylic anhydride. FIG IB is a schematic showing the three steps synthesis of methacrylated hyaluronic acid (“HA-MA”). FIG 1C shows D-MA and HA-MA mixed at a ratio of 30:70 (D-MA:HA-MA) with a photo-initiator IRGACURE 2959 and stored at 4°C under dark until use in SALK or DALK surgery. This mixture is referred to herein as “OcuPair”. FIG ID is a schematic representing the application of OcuPair adhesive hydrogel solution in the corneal defect followed by photo-crosslinking to form a transparent corneal substitute.

[0019] FIGs 2A-2D show longitudinal stroma regeneration and hydrogel regression.

[0020] FIGs 2A and 2B are bar graphs showing the stromal thickness (FIG 2A) and the hydrogel thickness (FIG 2B) measured by Imaged to show the regeneration of the stroma and regression of the hydrogel in SALK (left panel in FIGs 2A, 2B) and DALK (right panel in FIGs 2A and 2B) groups. The first column and last column in FIG 2B are epithelium layers, not the hydrogel thickness. FIG 2C is a bar graph of the fluorescein staining area shown in mm2. The vertical dotted line indicates the initial trephine wound area. FIG 2D is a bar graph showing the Kaplan-Meier curve of the time that hydrogel was totally cleared from the cornea in the SALK and DALK groups. The vertical dotted line is median (50%) clearance time of the adhesive hydrogel from the cornea. SALK n=7, DALK: n=9.

[0021] FIGs 3A and 3B show the transmission electron microscopy (“TEM”) ultra- structure of regenerated cornea stroma. FIG 2A is a schematic drawing of the TEM sample preparation to ensure the embedded TEM surface was within the regenerated cornea. The dotted orange line indicates the original cornea wound of 7mm. FIG 3B are TEM images acquired at the epithelium basal member / anterior cornea layer in SALK, DALK and CTRL rabbit cornea. The imaging area (red square in row 1 ) enlarged in row 2, and the blue square in row 2 enlarged in row 3 were located 10-20 pm below the basal member. Yellow arrow in 1st row: basal membrane. In 2nd and 3rd row: red arrow: parallel arranged collagen. Green arrow: orthogonally arranged collagen. White arrows: the stromal cells. E: Epithelium. S: Stroma.

[0022] FIG 4 are in vivo confocal images of the regenerated cornea. The images were acquired at 9 months in SALK, DALK, and control rabbit eyes. The images from the basal epithelium layer, SNP, anterior stroma, and endothelium were selected from the volume acquisition. Yellow triangle in basal epithelium and SNP layer (1st row and 2nd row): microglia cells. Red triangle in SNP layer (2nd row): SNP nerve fibers. Yellow triangle in anterior stroma (3rd row): keratocytes. Red triangle in anterior stroma (3rd row): nerve fibers. Red triangle in endothelium layer: endothelium cells. Scale bar: 100|im.

[0023] FIG 5 are bar graphs showing individual rabbit corneas at 9 months from SALK and DALK wounds. In the SALK group, there were 5 in 7 animals that had no gel left on the cornea; in DALK, there were 7 in 9 animals that had no gel on the cornea. In those animals, tear production was measured at various timepoint as well (left panel: SALK, right panel: DALK), compared with the average reading of the right (R-Operated) side with the left (CTRL) side. *p<0.05, **p<0.01.

[0024] FIG. 6 is a synthesis scheme for an exemplary dendrimer.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] Several cornea replacement strategies are on the market, but none have shown the ability to regenerate the cornea. They are all marketed as a cornea lenticule to replace the cornea transplant. In addition, they all require their cornea substitute to be sutured. Furthermore, the cost of manufacture is relatively inexpensive, so that it can be readily available for most patients. Irregular astigmatism is also an issue with these other corneal substitutes. The hydrogel-assisted cornea regeneration system described herein demonstrates that the cornea can regenerate and maintain normal astigmatism. The dendrimer hydrogel can also be used as a corneal glue to treat cornea perforation, which is not an option for the other available materials. This dendrimer-hyaluronic acid hydrogel has the ability to heal corneal wounds, and act as a corneal tissue, with many applications.

[0027] I. Definitions

[0028] The term "therapeutic agent" refers to an agent that can be administered to prevent or treat one or more symptoms of a disease or disorder. These may be a nucleic acid, a nucleic acid analog, a small molecule, a peptidomimetic, a protein, peptide, carbohydrate or sugar, lipid, or surfactant, or a combination thereof. As used herein, the term “active agent” or “biologically active agent” are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and / or physiological effect, which may be prophylactic, therapeutic or diagnostic. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of active agents, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, and analogs.

[0029] The term “diagnostic agent”, as used herein, generally refers to an agent that can be administered to reveal, pinpoint, and define the localization of a pathological process.

[0030] The term “prophylactic agent”, as used herein, generally refers to an agent that can be administered to prevent disease or to prevent certain conditions like pregnancy. The phrase "pharmaceutically acceptable" refers to compositions, polymers and other materials and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier" refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, solvent or encapsulating material involved in carrying or transporting any subject composition, from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient.

[0031] The phrase "therapeutically effective amount" refers to an amount of the therapeutic agent that produces some desired effect at a reasonable benefit / risk ratio applicable to any medical treatment. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. A prophylactic agent refers to an agent that may prevent a disorder, disease or condition. Examples include vaccines which prevent infection and birth control pills that prevent pregnancy.

[0032] The term "treating" refers to preventing or alleviating one or more symptoms of a disease, disorder or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

[0033] The term “biocompatible” as used herein, generally refers to materials that are, along with any metabolites or degradation products thereof, generally non-toxic to the recipient, and do not cause any significant adverse effects to the recipient. Generally speaking, biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient.

[0034] The term “biodegradable” as used herein, generally refers to a material that will degrade or erode under physiologic conditions to smaller units or chemical species that are capable of being metabolized, eliminated, or excreted by the subject. The degradation time is a function of composition and morphology. Degradation times can be from hours to years.

[0035] The term “seal”, as used herein, means to substantially cover a rough surface, a cavity, or a gap in the substrate (e.g., a tissue) or coat on top of the tissue surface, and optionally to form a covalent or non-covalent bond at the contact surface. “Seal” may also refer to the barrier effect of preventing the migration or transport of certain gas, liquid, solute, macromolecules, or bacteria.

[0036] The term "dendrimer", as used herein, includes, but is not limited to, a molecular architecture with an interior core, interior layers (or "generations") of repeating units regularly attached to this initiator core, and an exterior surface of terminal groups attached to the outermost generation.

[0037] The term “bioadhesive polymer”, as used herein, refers to a natural or synthetic polymer that can adhere to a biological substrate. The adhesion of polymers to tissues may be achieved by (i) physical or mechanical bonds, (ii) primary or covalent chemical bonds, and / or (iii) secondary chemical bonds (i.e., ionic).

[0038] The term “crosslink”, as used herein, means the formation of covalent linkages between a precursor molecule containing nucleophilic groups and a precursor molecules containing electrophilic group resulting in an increase in the molecular weight of the material. “Crosslink” may also refer to the formation of non-covalent linkages, such as ionic bonds, or combinations of covalent and non-covalent bonds.

[0039] The term “photocrosslink”, as used herein, means to cause vinyl or other unsaturated bonds to break and form cross-links by the application of radiant energy.

[0040] The term “external stimulus”, as used herein, evokes a specific functional reaction, which is not intrinsic, such as a physical, chemical, biological, mechanical, and irradiation stimuli.

[0041] “Polymeric network”, as used herein, refers to the product of a process in which substantially all of the monomers, oligomers, or polymers are bound by intermolecular covalent linkages through their available functional groups to form a macromolecule.

[0042] “Physiological”, as used herein, refers to conditions found in living vertebrates. In particular, physiological conditions refer to the conditions in the human body such as temperature, pH, aqueous medium, etc. “Physiological temperatures”, as used herein, refers to a temperature range of between 35°C to 42°C, preferably around 37°C.

[0043] “Crosslink density”, as used herein, refers to the average molecular weight between two crosslinks (Mc) of the respective molecules.

[0044] “Swelling”, as used herein, refers to the increase in volume and mass due to the uptake of water by the biomaterial. The terms” water-uptake” and “swelling” are used synonymously.

[0045] “Gel point” or “gelation” as used herein refers to the point where the viscous modulus and complex modulus cross each other and viscosity increases. Thus the gel point is the stage at which a liquid begins to take on the semisolid characteristics of a gel. “In situ formation” as generally used herein refers to the ability of mixtures of precursor molecules which are substantially not crosslinked prior to and at the time of injection, but form covalent linkages, non-covalent linkages, or a combination, with each other at a physiological condition or upon trigger by external stimuli at the site of injection in the body.

[0046] “Equilibrium state”, as used herein, refers to the state in which a hydrogel undergoes no mass increase or loss when stored under constant conditions in water.

[0047] “Functionalize”, as used herein, means to modify in a manner that results in the attachment of a functional group or moiety. For example, a molecule may be functionalized by the introduction of a molecule which makes the molecule a strong nucleophile or strong electrophile. For example, a molecule, such as hyaluronic acid, may be functionalized to become a thiol, amine, acrylate, or quinone.

[0048] II. Formulations

[0049] Dendrimer Hydrogel Compositions

[0050] A hydrogel or hydrogel precursor composition for sealing tissue and optionally delivering therapeutic, prophylactic, and / or diagnostic agents has been developed and is referred to herein as a “dendrimer-hyaluronic acid”. The dendrimer-hyaluronic acid is formed with one or more dendrimer molecules (or derivatives thereof) and one or more bioadhesive polymers (or derivatives thereof), wherein the dendrimer molecules and the bioadhesive polymers are crosslinked upon application of one or more external stimuli or one or more physiological conditions within the tissue. In preferred embodiments, the dendrimers and the bioadhesive polymers are chemically modified to contain photo-crosslinkable groups (e.g., thiol groups and vinyl groups; thiol groups and maleimide groups; thiol groups and alkene groups; thiol groups and alkyne groups; amine groups and aldehyde groups; amine groups and carboxylate groups; methacrylate groups; and acrylate groups). These dendrimers and bioadhesive polymers with functional groups are cured to form the dendrimer-hyaluronic acid in situ at tissue sites after exposure to external stimuli such as ultraviolet irradiation, cobalt blue light, argon laser or visible light. Optionally, the therapeutic, prophylactic, or diagnostic agent is conjugated to or complexed with a dendrimer molecule, and is released at the administered sites in a sustained and controlled manner.

[0051] In certain embodiments, one or more dendrimer molecules are generation 2-10 poly(amidoamine) (PAMAM) dendrimers optionally terminated with one or more functional groups such as hydroxyl, amino, carboxyl, alkoxysilyl, thiol, pyridyl, vinyl, methacryloyl, alkene, alkyl and / or cyclic unsaturated hydrocarbon. A. Dendrimers

[0052] In preferred embodiments, the dendrimers and non-toxic and have numerous surface groups enabling modification with multiple photo-crosslinkable groups for high crosslinking densities at low concentrations and potential conjugation with active agents.

[0053] Dendrimers suitable for use include, but are not limited to, polyamidoamine (PAMAM), polypropylamine (POPAM), polyethylenimine, polylysine, polyester, iptycene, aliphatic poly(ether), and / or aromatic polyether dendrimers. Each dendrimer of the dendrimer complex may be same or of similar or different chemical nature than the other dendrimers (e.g., the first dendrimer may include a PAMAM dendrimer, while the second dendrimer may be a POPAM dendrimer). In some embodiments, the first or second dendrimer may further include an additional agent such as a multiarm PEG polymer including a polyethylene glycol having at least two branches bearing sulfhydryl or thiopyridine terminal groups. Other PEG polymers bearing other terminal groups such as succinimidyl or maleimide terminations can be used. The PEG polymers in the molecular weight 10 kDa to 80 kDa can be used. Complexes can be formed of one or more dendrimers.

[0054] Examples of dendrimers include, but are not limited to, poly(amidoamine) (PAMAM), polyester, polylysine, and polypropylenimine (PPI). The PAMAM dendrimers may contain different cores, with amidoamine building blocks, and can have carboxylic, amine and hydroxyl terminations of any generation including, but not limited to, generation 1 PAMAM dendrimers, generation 2 PAMAM dendrimers, generation 3 PAMAM dendrimers, generation 4 PAMAM dendrimers, generation 5 PAMAM dendrimers, generation 6 PAMAM dendrimers, generation 7 PAMAM dendrimers, generation 8 PAMAM dendrimers, generation 9 PAMAM dendrimers, or generation 10 PAMAM dendrimers. In the preferred embodiment, the dendrimers are soluble in the formulation and are generation (“G”) 4, 5 or 6 dendrimers.

[0055] As used herein, the term “PAMAM dendrimer” means poly(amidoamine) dendrimer, which may contain different cores, with amidoamine building blocks. The method for making them is known to those of skill in the art and generally involves a two-step iterative reaction sequence that produces concentric shells (generations) of dendritic P-alanine units around a central initiator core. This PAMAM core-shell architecture grows linearly in diameter as a function of added shells (generations) and the surface groups amplify exponentially at each generation according to dendritic-branching mathematics. They are available in generations GO - 10 with 5 different core types and 10 functional surface groups. The dendrimer-branched polymer may consist of poly amidoamine (PAMAM), poly glycerol, polyester, poly ether, poly lysine, or polyethylene glycol (PEG), polypeptide dendrimers. The dendrimers may have hydroxyl groups attached to their functional surface groups.

[0056] In some embodiments, the dendrimers are in nanoparticle form and are described in detail in international patent publication Nos. W02009 / 046446, PCT / US2015 / 028386, PCT / US2015 / 045112, PCT / US2015 / 045104, and U.S. Patent No. 8,889,101.

[0057] In some forms, the dendrimers are polyamidoamine hydroxyl dendrimers. Polyamidoamine hydroxyl dendrimers (D-OH) are hyperbranched polymeric materials that can be used as nanocarriers for targeted drug delivery to both the anterior and posterior segments of the eye. D-OH has a good non-toxic profile, well tolerated and demonstrates favorable clinical outcomes in phase- 2 trials. Dendrimers possess favorable structural properties such as branched architecture and multivalent surface groups that are amenable for covalent attachment of crosslinkable groups. These characteristics are beneficial for synthesis of hydrogels with good crosslinking densities with tailorable mechanical strength particularly for comeal substitute application where long-term hydrogel stability is required for complete wound healing of large comeal defects.

[0058] B. Hyaluronic acid (HA)

[0059] HA is a naturally occurring, immunoneutral glycosaminoglycan of the extracellular matrix that plays an important role in development, wound healing, and inflammation. Its viscoelastic properties and long ocular surface residence time have rendered it suited for use in several tissue repair practices, including ophthalmic use to protect the corneal endothelium. HA is also identified to be a ligand for CD44 (Zhu SN, et al., Br J Ophthalmol., 81 (l):80-4 (1997)), a transmembrane cell surface adhesion molecule.

[0060] As a component of the ocular tissues, HA is abundant in the corneal limbal stem cell niche, and it plays a role in maintaining the pluripotency of limbal epithelial stem cells. HA is unique biomaterial of ocular surface drug delivery for its high-water retention, non-immunogenic, biodegradable, and relatively demonstrated mucoadhesive properties. HA has been incorporated into the fabrication of next-generation comeal substitutes, including the skirt material of T-style keratoprostheses and in situ forming collagen-hyaluronate hydrogels.

[0061] In a preferred embodiment, HA crosslinked by dendrimer molecules forms the hydrogel sealant for tissue repair. HA has a wide range of molecular weights; and in some embodiments, HA of 15-20kDa is selected. To crosslink, HA is first modified chemically at one or more of the three functional groups: the glucuronic acid carboxylic acid, the primary and secondary hydroxyl groups, and the N -acetyl group (following deamidation). Most prominently, carboxylates have been modified by carbodiimide-mediated reactions, esterification, and amidation; hydroxyls have been modified by etherification, divinylsulfone crosslinking, esterification, and bis-epoxide crosslinking. Detailed reviews on chemical modification of HA can be seen in Kuo JW, Prestwich GD, in Materials of Biological Origin - Materials Analysis and Implant Uses, Comprehensive Biomaterials, Elsevier (2010).

[0062] C. Therapeutic, Prophylactic and Diagnostic Agents

[0063] Dendrimer dendrimer-hyaluronic acids have been developed which both seal, support regeneration of corneal tissue and provide for controlled, sustained local release of therapeutic, prophylactic and / or diagnostic agent. The dendrimer-hyaluronic acid has desirable mechanical properties to hold the tissue. It also can be used to deliver agents accelerating wound healing and delivering antibacterial and anti-inflammatory drugs to prevent infection and scar tissue formation. The formulation is surgeon friendly. It is a viscous liquid during application and polymerizes rapidly upon laser illumination, providing a longer time window for surgeons to work with. The strength of the dendrimer-hyaluronic acid can be modulated by the duration of laser application or by altering the percentages of individual components of the dendrimer-hyaluronic acid.

[0064] Other materials can be incorporated into the formulations to increase flexibility, strength or control drug delivery properties. Therapeutic, prophylactic and / or diagnostic agents can be incorporated directly, bound to the dendrimers, or formulated into particles, to facilitate sustained release of therapeutic agents such as steroids and antibacterial drugs (antibiotic drugs).

[0065] D. Therapeutic, Prophylactic and Diagnostic Agents

[0066] The dendrimer-hyaluronic acid may include one or more therapeutic, prophylactic, or diagnostic agents that are encapsulated, conjugated to the components of the hydrogel, or encapsulated in / conjugated to sustained release nanoparticle / microparticle formulations that are dispersed in the hydrogel precursor(s). In some embodiments, the agents can be modified with a succinate group via a reaction with succinic anhydride, which are later conjugated to dendrimers and / or bioadhesive polymers at their hydroxyl groups to permit ester linkage and hydrolysis- mediated, sustained release of the agents in tissue.

[0067] Representative therapeutic agents include, but are not limited to, anti-inflammatory drugs, including immunosuppressant agents and anti- allergenic agents, and anti-infectious agents. Some examples of anti-inflammatory drugs include steroids like triamcinolone acetonide, fluocinolone acetonide, prednisolone, dexamethasone, loteprendol, fluoromethoIone. Immune modulating drugs such as: cyclosporine, tacrolimus and rapamycin. Non-steroidal anti-inflammatory drug include ketorolac, nepafenac, and diclofenac. Anti-infectious agents include antiviral agents, antibacterial agents, antiparasitic agents, and anti-fungal agents. Exemplary antibiotics include moxifloxacin, ciprofloxacin, erythromycin, levofloxacin, cefazolin, vancomycin, tigecycline, gentamycin, tobramycin, ceftazidime, ofloxacin, gatifloxacin; antifungals: amphotericin, voriconazole, natamycin.

[0068] Active agents can include anti-glaucoma agents that lower intraocular pressure (IOP), antiangiogenesis agents, growth factors, and combinations thereof. Examples of anti-glaucoma agents include prostaglandin analogs such as travoprost and latanoprost, prostamides such as bimatoprost; beta-adrenergic receptor antagonists such as timolol, betaxolol, levobetaxolol, and carteolol, alpha- 2 adrenergic receptor agonists such as brimonidine and apraclonidine, carbonic anhydrase inhibitors such as brinzolamide, acetazolamine, and dorzolamide, miotics (i.e., parasympathomimetics) such as pilocarpine and ecothiopate), seretonergics, muscarinics, and dopaminergic agonists.

[0069] Representative anti-angiogenesis agents include, but are not limited to, antibodies to vascular endothelial growth factor (VEGF) such as bevacizumab (AVASTIN®) and rhuFAb V2 (ranibizumab, LUCENTIS®), and other anti- VEGF compounds including aflibercept (EYLEA®); MACUGEN® (pegaptanim sodium, anti- VEGF aptamer or EYE001) (Eyetech Pharmaceuticals); pigment epithelium derived factor(s) (PEDF); COX-2 inhibitors such as celecoxib (CELEBREX®) and rofecoxib (VIOXX®); interferon alpha; interleukin- 12 (IL-12); thalidomide (THALOMID®) and derivatives thereof such as lenalidomide (REVLIMID®); squalamine; endostatin; angiostatin; ribozyme inhibitors such as ANGIOZYME® (Sirna Therapeutics); multifunctional antiangiogenic agents such as NEOVASTAT® (AE-941) (Aetema Laboratories, Quebec City, Canada); receptor tyrosine kinase (RTK) inhibitors such as sunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib (Nexavar®) and erlotinib (Tarceva®); antibodies to the epidermal grown factor receptor such as panitumumab (VECTIBIX®) and cetuximab (ERBITUX®), as well as other antiangiogenesis agents known in the art.

[0070] In some cases, the active agent is a diagnostic agent imaging or otherwise assessing the eye. Examples of diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, and contrast media.

[0071] The active agents may be present in their neutral form, or in the form of a pharmaceutically acceptable salt. In some cases, it may be desirable to prepare a formulation containing a salt of an active agent due to one or more of the salt's advantageous physical properties, such as enhanced stability or a desirable solubility or dissolution profile.

[0072] Generally, pharmaceutically acceptable salts can be prepared by reaction of the free acid or base forms of an active agent with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Pharmaceutically acceptable salts include salts of an active agent derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts as well as salts formed by reaction of the drug with a suitable organic ligand (e.g., quaternary ammonium salts). Lists of suitable salts are found, for example, in Remington’s Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704. Examples of ophthalmic drugs sometimes administered in the form of a pharmaceutically acceptable salt include timolol maleate, brimonidine tartrate, and sodium diclofenac.

[0073] In certain embodiments, the dendrimer-hyaluronic acid contains one or more local anesthetics. Representative local anesthetics include tetracaine, lidocaine, amethocaine, proparacaine, lignocaine, and bupivacaine. In some cases, one or more additional agents, such as a hyaluronidase enzyme, is also added to the dendrimer-hyaluronic acid to accelerate and improves dispersal of the local anesthetic.

[0074] The formulation has multimodal applications and can be modified for administration at different locations or purposes, for example, as a looser gel for subconjunctival administration to increase drug availability and aid sustained drug release for various corneal and anterior segment diseases such as corneal inflammation, corneal neovascularization, corneal graft rejection, and possibly iritis and anterior uveitis. The percentage of individual components is modified with appropriate dilution to form transparent flexible drug depot gels that can be injected intravitreally for sustained release of drugs for a variety of posterior segment diseases such as diabetic retinopathy, choroidal neovascularization (CNV) secondary to age related macular degeneration (AMD) and other retinal pathologies. Using hyaluronic acid as the major component in the modified formulation integrates well with the vitreous gel, causing dendrimer with drugs to release from the gel and delivering drugs to targeted retinal cells for enhanced and long-term efficacy resulting in significant decreased frequency in intravitreal injections.

[0075] The formulation provides advantages for treatment of conditions requiring frequent eye drops that often cause ocular surface irritation and toxicity and may have a better safety profile in terms of intraocular pressure (IOP) and patient discomfort. The subconjunctival gels have reduced side effects as compared to eye drops. The photo-crosslinkable dendrimer-hyaluronic acid based dendrimer-hyaluronic acids are useful in sealing corneal incisions and simultaneously releasing antibiotic / steroids for prevention of infection and inflammation and to accelerate comeal wound healing. In addition to the benefits of being able to be applied in situ and photo-cured in a tailored manner to provide a high crosslink density through the use of the dendrimer; the inclusion of hyaluronic acid in the gel promotes wound healing and integrates into corneal stroma; while simultaneously releasing antibiotics / steroids to address infections / inflammation in a sustained manner. The formulation is also transparent, which is clearly beneficial in ocular applications. In certain embodiments, the therapeutic agents are anti-inflammatory, anti-infectious, or anti-angiogenesis small molecules or biomacromolecules.

[0076] III. Methods of Making the Composition

[0077] The method for making dendrimers is known to those of skill in the art and generally involves a two-step iterative reaction sequence that produces concentric shells (generations) of dendritic P-alanine units around a central initiator core. This PAMAM core-shell architecture grows linearly in diameter as a function of added shells (generations). Meanwhile, the surface groups amplify exponentially at each generation according to dendritic -branching mathematics. They are available in generations GO - 10 with 5 different core types and 10 functional surface groups. The dendrimer-branched polymer may consist of polyamidoamine (PAMAM), polyester, polyether, polylysine, or polyethylene glycol (PEG), polypeptide dendrimers. Alternatively, dendrimers with various termination groups can be purchased from vendors such as Dendritech.

[0078] Modifications to dendrimers and / or bioadhesive polymers to allow for stimuli-triggered gelation are known with various functional groups. Thiolation of hyaluronic acid is described in the literature. Kafedjiiski K, et al., Ini J Pharm, 343(l-2):48-58 (2007) reported the synthesis of thiolated hyaluronic acid by conjugating L-cysteine ethyl ester to the carboxyl (-COOH) groups of the hyaluronic acid using EDC-NHS coupling reaction in aqueous solvent. The resultant product was subjected to oxidation to form disulfide (-S-S-) bridges, which permits HA to form gels for drug delivery. As an alternative, free thiols on hyaluronic acid can be generated to allow for thioene click reactions. In this approach, since a reaction in the aqueous medium may result in disulfide formation, an inert condition for this modification reaction is preferred, which provides better control on the degree of substitution and avoids in-situ disulfide formation. Detailed procedures for this modification are explained in the Examples section.

[0079] Active agents can also be modified to permit covalent attachment and controlled release. Alternatively, active agents can be mixed with one or both of the precursor components, and are dispersed in the formed hydrogel.

[0080] IV. Methods of Using the Composition

[0081] The cornea of the eye serves an important role in refracting and focusing light rays necessary for clear vision. The cornea possesses unique characteristics of an orderly arrangement of stromal collagen fibrils and a lack of blood vessels that result in transparency.

[0082] Corneal wounds arise from surgical procedures (e.g., transplants, incisions for cataract removal and intraocular lens implantation, laser-assisted in situ keratomileusis), infections (e.g., ulcers), and traumatic injury (e.g., lacerations, perforations). The disclosed hydrogel-dendrimer compositions are suitable for aiding corneal regeneration of corneal wounds that occur during surgical procedures. In a preferred embodiment, the hydrogel formed from hyaluronic acid and dendrimers is applied as a sealant for corneal wounds, where the precursors are applied to the comeal wound and which may be photo-cured in situ in a tailored manner.

[0083] The hydrogel-dendrimer compositions are suitable for administering to a patient undergoing optical procedures including but not limited to superficial keratectomy, lamellar keratoplasty, mechanical keratectomy, and photokeratectomy.

[0084] In superficial keratectomy (SK), the surgeon excises the superficial layers of cornea (epithelium, Bowman layer, or superficial stroma) without replacing the tissue. The primary indications are as follows: removal of hyperplastic or necrotic tissue (eg, corneal dermoid, pterygium, Salzmann nodular degeneration, epithelial basement membrane redundancy, degenerative calcification); excision of retained foreign material in the cornea; need for tissue for diagnosis (histology or microbiology); and excision of scar tissue or superficial corneal dystrophic tissue. Mechanical superficial keratectomy (corneal scraping) usually involves removal of pathological epithelial and sub-epithelial tissues.

[0085] SK is a safe procedure as none of the reports and series in the literature reported intraoperative complications. However, patients should be informed that discomfort due to epithelial debridement might persist for several days. The surgeon may choose topical, peri, or retrobulbar anesthesia based on the level of cooperation. The procedure usually is done by a microscope in the operating theater. Performing SK at the slit lamp is also possible but depends on patients' cooperation and the type of technique surgeons choose. The eye is prepared with povidone-iodine, and a lid speculum is inserted. Then, the superficial comeal layers are debrided with a sterile sponge or surgical blade. Some surgeons remove the loose epithelium and then polish Bowman's layer with a diamond burr. Manual or automated diamond burr polishing helps debride persistent abnormalities. If there is dystrophic tissue, the lesion is peeled off from Bowman's layer using a blunt blade or cellulose micro-sponge in one continuous plane. Sharp dissection may be performed if it is not possible to shave off the peripheral membrane from the limbus or if the surgeon finds making a smooth surface under the lesion is not possible without using sharp dissection.

[0086] The surgeon may choose some adjuvant treatments based on the indication for SK. For example, in patients with band keratopathy, applying Ethylene diamine tetra-acetic acid (EDTA) helps dissolve calcific deposits. Another adjunctive medication is the use of a sponge soaked with mitomycin C 0.02% (MMC). MMC is an antimetabolite that decreases activated fibrocytes and keratocytes. It may subsequently prevent the recurrence of pathologies such as Salzmann nodular degeneration and lessens the chance of corneal haze following SK.

[0087] After the procedure, a therapeutic contact lens should be placed, and the patient discharged with artificial tears, topical antibiotics and cycloplegic drops. Some ophthalmologists prescribe corticosteroid eye drops to suppress inflammation. In addition, topical non-steroidal antiinflammatory drugs like ketorolac may alleviate pain. Patients are revisited in the first week postoperatively to ensure healing of the epithelial defect. After healing of the epithelial defects is confirmed, topical antibiotics are ceased, and topical steroids can be tapered.

[0088] SK is an available and effective method for various indications, including tissue diagnosis, excision of comeal degenerations, dystrophies, scarring, recurrent comeal erosions and retained comeal foreign body (Table 1). This part briefly explains the indication and outcomes, complications, and adjunct treatments for each indication separately.

[0089] Recurrent corneal erosion syndrome (RCES) is a relapsing breakdown of the comeal epithelium and the Bowman's layer. Trauma and corneal dystrophies are considered the main etiology. Many patients respond to conservative therapy with a combination of lubrication with drops or gels and bandage contact lenses. However, some people experience symptoms that are refractory to conservative topical therapy and require surgery. Many treatment options are available for the treatment of RCES, but there is no consensus on which is the best. Options include epithelial debridement (ED), SK, anterior stromal puncture (ASP), Nd: YAG laser treatment, alcohol delamination, and PTK. SK is one of the most common interventions in patients with RCES. SK is a procedure that can be easily performed in an office, imposes a lesser cost to the healthcare system, and requires a lower skill level than PTK. A Cochrane review on interventions for RCES showed that performing diamond burr polishing in addition to the epithelial debridement decreases the recurrence significantly. Moreover, PTK may have a higher recurrence rate (373 per 1,000 vs. 294 per 1,000 in mean 24 months follow up) and lower symptom relief (378 per 1,000 vs. 590 per 1,000 in 3 months of follow up) in comparison with epithelial debridement. Most reports prefer SK with diamond burr polishing over simple SK. The recurrence rate ranged from 0 to 24%. Most studies reported that best-corrected visual acuity (BCVA) was unchanged after SK. Table 1 - Indications for superficial keratectomy.

[0090] Lamellar keratoplasty (epikeratophakia) involves suturing a pre-lathed donor cornea onto the surface of the recipient's cornea. Used as a means of correcting adult and pediatric aphakia, Anterior Lamellar Keratoplasty (ALK) is an alternative treatment that selectively replaces the front part of the cornea when it is scarred or distorted. In ALK, the surgeon dissects the cornea into two thin pieces and removes the front, scarred part. If corneal biopsy is performed for histology, preservation of tissue integrity and anatomical orientation is important. A small specimen can be placed on a filter or thin card to maintain the tissue orientation before fixation or cryosection. For microbiology workup, the biopsied specimens can be minced or homogenized before inoculation of the culture media or tissue smear for histochemical staining.

[0091] Mechanical keratectomy may be used if the corneal lesion is superficial, and it is possible to scrape or peel it away from the surface of the cornea without sharp dissection. Often, a smooth anatomical tissue plane anterior to the Bowman layer can be achieved with sweeping strokes parallel to the tissue through the use of a metal spatula blade or a cellulose sponge. In some cases, such as in Salzmann nodular degeneration, it is possible to gently peel the abnormal tissue off using forceps. When deeper dissection is required, the surgeon can either mark the area freehand with an adjustable-depth blade or use a trephine. A 2- to 3-mm disposable dermatologic skin punch trephine blade can be used to create a partial-thickness incision, and forceps and scissors are then used to excise a lamellar flap of cornea.

[0092] In phototherapeutic keratectomy, an excimer laser is used to remove superficial stromal tissue. However, abnormal tissue, like corneal scars or calcium deposits (as in band keratopathy), may ablate at a different rate than normal tissue, so an uneven surface results even if the original surface was smooth. Manual techniques are more likely to respect the Bowman layer and maintain a smooth ocular surface, as the laser does not respect anatomical planes. Frequent application of viscous liquid to the comeal surface during laser ablation can fill in gaps in the surface and help achieve a smooth surface after ablation. Most patients experience a hyperopic shift after phototherapeutic keratectomy (PTK) from the corneal-flattening effect of the procedure. Nevertheless, PTK is an excellent option in selected patients with superficial (less than 100 [im deep) stromal scarring or dystrophies when manual techniques are not feasible. PTK may postpone or eliminate the need for corneal transplantation. Topical application of a formulation such as mitomycin-C (“MMC”) may be applied to the corneal ablation zone for a brief period following PTK to decrease postoperative scar formation. PTK can also be used to treat recurrent corneal erosion.

[0093] The hyaluronic-dendrimer compositions can be administered by any appropriately trained individual. This broadens access to care by allowing clinicians such as general ophthalmologists, optometrists with advanced procedural training, or other healthcare providers with appropriate training to administer the hyaluronic-dendrimer compositions.

[0094] The present invention will be further understood by reference to the following non-limiting examples.

[0095] EXAMPLE 1: Corneal Regeneration using an exemplary In situ Photo-crosslinkable Dendrimer Hyaluronic acid based Bioadhesive Hydrogel “OcuPair”

[0096] MATERIALS AND METHODS

[0097] OcuPair adhesive hydrogel: Synthesis, formulation and characterization

[0098] An OcuPair adhesive hydrogel was synthesized. When applied over the full-thickness comeal wounds followed by photo-crosslinking using UV light, the OcuPair adhesive hydrogel forms a transparent hydrogel bandage. In brief, the individual polymeric components of the adhesive hydrogel, PAMAM hydroxyl dendrimer-methacrylate (D-MA) and methacrylated hyaluronic acid (HA-MA), were synthesized using the following protocols. D-MA conjugates were synthesized by modifying and reacting the surface -OH groups dendrimers with methacrylic anhydride in the presence of 4-Dimethylaminopyridine (DMAP) as a base and anhydrous N, N- dimethylacetamide (DMA) as solvent under nitrogen atmosphere (FIG 1A). HA-MA was synthesized using a 3-step synthesis protocol. In the first step, the sodium salt of HA (~42kDa, Lifecore Biomedical) was converted to DMA / DMSO soluble tetrabutylammonium salt of HA (HA- TB A) using ion exchange. In the second step, the -OH groups of hyaluronic acid were reacted with methacrylic acid by Steglich esterification using methacrylic anhydride in the presence of DMAP as a base to form (TBA-HA-MA). In the third step, the TBA ions were replaced with NA ions to form (HA-MA) using ion exchange with NAC1 (FIG IB). All the intermediates and the final products (D- MA and HA-MA) were purified using tangential flow filtration (TFF) and were characterized by proton nuclear magnetic resonance ('H NMR) and high-performance liquid chromatography (HPLC). The D-MA and HA-MA conjugates were dissolved in sterile lOmM phosphate buffer at a concentration of 300mg / mL and 190mg / mL respectively. Both the solutions were mixed at a ratio of 30:70 (D-MA: HA-MA) with catalytic amount of photo-initiator Irgacure 2959 (FIG 1C).

[0099] The synthesis, purification, and formulation protocols were transferred to CMO and CRO for scale-up manufacturing following good manufacturing practices (GMP) under sterile conditions (in accordance with ISO 14644-1). The final formulations were aseptically filled into final delivery devices (sterile glass syringes with lure-lock connector) and stored in 4°C under dark until use (FIG ID).

[0100] Animals, Surgical procedures (lamellar keratectomy) and OcuPair treatment

[0101] Sixteen New Zealand white rabbits aged 3-6 months, both male and females at equal proportion, from Charles River Laboratories International, Inc (MD, USA) were used. All procedures were performed under sterile conditions with approval from the Johns Hopkins University Animal Care and Use Committee (ACUC) and within the parameters outlined by ARVO for the Use of Animals in Ophthalmic and Vision Research. Survival surgeries and all invasive procedures on rabbits were performed under aseptic conditions under general anesthesia: ketamine 25-50mg / kg (Bio-niche Pharma, Lake Forest, IL, USA) and xylazine 2.5-5mg / kg (Phoenix Pharmaceuticals, St. Joseph, MO, USA) or topical anesthesia proparacaine 0.5% (Sandoz, Holzkirchen, Germany). The right eye was utilized for surgical procedures and OcuPair treatment and the left contralateral eye served as controls.

[0102] In the 17 New Zealand White rabbits (SALK=7, DALK-9, both sex), the corneal wound was created by 0=7mm trephine with a preset depth of 300 m (Coronet, Network Medical Products, UK). A gentle turn of the guarded trephine created a wound with the depth of around 150 pm at the edge of the rabbit cornea. Carefully dissecting from the cornea wound edge toward the central cornea at the same plane, the superficial cornea stroma flip was removed to create the SALK wound. To mimic the DALK wound, further stripping of the cornea stroma was carefully carried out till the smooth pre-Descemet’ s layer were reached. Prior to the start of the surgery, the OcuPair adhesive hydrogel was brought from 4°C to room temperature and then mixed and kept in a dark place just prior to the application. The wound / corneal defect space was over-filled with the OcuPair adhesive hydrogel precursor solution by gently extruding via 25 G anterior chamber cannula fitted to the lure-lock connectors of the final delivery devices. Care was taken to eliminate any void spaces and to match the corneal curvature. In situ cross-linking of the HA-dendrimer was conducted using a handheld cobalt blue UV light (365nm, 4W) (Jaxman Electronics), for 90 seconds. Immediately before and after surgery, AS-OCT was performed to measure the cornea height. Wound depths were calculated by remaining stromal height / original corneal height. Subsequently, the rabbits were awakened from anesthesia under close surveillance. The Elizabeth collars were installed to prevent rabbit from scratching the eyes and removed after 24 hours. Animals were visually inspected twice a week, in addition to the other clinical follow up as described below. In all animals, no contact lens bandage, tarsorrhaphy, or reapplication of hydrogel was used following the surgery.

[0103] Schirmer’s tear test

[0104] Tear production was measured simultaneously in both eyes. Rabbits were sedated with ketamine (25-30 mg / kg) and xylazine (2.5-3.0 mg / kg) to allow for ease of measurement. The test was performed by hooking the small, folded end of a standard commercial tear-test paper (Merck, NJ) under the lower lid margin at approximately a third of the distance from the temporal boundary. Test strips were allowed to wet for 5 minutes, during which the progression of a blue dye down the length of the strip was recorded to quantify tear production. Measurements were taken for each animal are 2 weeks, 4 weeks, 2 months, 3 months, 6 months, and 9 months following surgery for both the SALK and DALK groups. These measurements were conducted in a temperature- and humidity-controlled environment, ensuring minimal discomfort for the animals.

[0105] Anterior-Segment Optical Coherence Tomography (AS-OCT)

[0106] During a single sedation session of the animals, AS-OCT images were acquired immediately after Schirmer's test as described above. An ultra-high-resolution anterior segment SD- OCT (Bioptigen, NC) was utilized for the evaluation of wound healing and tissue regeneration. Images were acquired through Raster scan and the cross corneal apex image was selected for analysis. The thickness of the cornea epithelium, stroma, and OcuPair adhesive hydrogel were measured using Image J software (NIH, USA). Similar to Schirmer’s test, measurements were taken for each subject at 2 w, 4 w, 2 m, 3 m, and 6 m and 9 m following surgery for both SALK and DALK groups. Fluorescein staining

[0107] During a single sedation session for the animals, after performing Schirmer’s test and AS- OCT imaging, one drop of 0.25% fluorescein ophthalmic solution (Altaire, Aquebogue, NY) was applied to each eye. Images were then captured using a cobalt blue light at a fixed distance. The fluoresceine stained area was measured by Image J (NIH, USA) in each animal. Similar to Schirmer’s test and AS-OCT, measurements were taken for each subject at 2 weeks, 4 weeks, 2 months, 3 months, and 6 months and 9 months following surgery for both SALK and DALK groups.

[0108] In vivo confocal microscopy (IVCM)

[0109] The rabbits were anesthetized with ketamine (75mg / kg, KetaVed®, VEDCO, St, Joseph, MO, USA) and xylazine (5mg / kg, XylaMed®; Bimeda-MTC; Cambridge, ON, Canada). GenTeal® (Alcon, Fort Worth, TX, USA) was applied to both eyes to prevent drying. The HRT3-RCM with a modified rodent platform was used to examine all corneal layers. Image acquisition "zero" was uniformly set at the cornea epithelium, while the presence of keratocytes indicated the beginning of the anterior stroma. A series of sequential volume acquisitions, typically four to five, were taken from position 0 pm (ocular surface) to around +400 pm (passing the endothelium layer). Images were taken 9 months after SALK and DALK surgery.

[0110] Histology Analysis

[0111] At post operative month 10, corneas from surgery and control eyes of each rabbit were harvested immediately after euthanasia via pentobarbital overdose. The superior half of the cornea was fixed in 10% neutral buffered formalin for 2 hours and then embedded in the paraffin wax for H&E, trichrome, and TUBB III immunofluorescent staining of the cornea nerve. H&E and Trichrome staining were performed by the Johns Hopkins University histology core facility. For TUBB III staining, the slides were blocked with primary antibody TUBB III (1:200, Biolegend, San Diego, CA, USA), and then secondary antibody Alexa Fluror555 (Molecular Probe A21432), at a dilution of 1 :500 for 2 hours at room temperature. Images were acquired by LSM700 fluorescent microscope (Zeiss, Germany).

[0112] For TEM ultrastructure of the regenerated cornea, a 2 mm strip of the inferior half of the cornea was fixed in glutaraldehyde solution and sent to JHU Wilmer TEM core facility. A series of images were acquired from the epithelium to the endothelium layer by Hitachi H7600 TEM. The images across the epithelium basal membrane were selected for comparison with the unoperated cornea. Data Analysis

[0113] The data were presented as mean ± SEM and were acquired at the following time points: pre-surgery, immediate post-surgery (0), 2 weeks, 4 weeks, 2 months, 3 months, 6 months and 9 months. Student's t-test by Prism9 (GraphPad Software, San Diego, CA, USA) was performed to detect statistical differences between the two groups. Statistical significance was defined as *p<0.05, **p<0.0l.

[0114] RESULTS

[0115] OcuPair adhesive hydrogel synthesis

[0116] The synthesis protocol for the individual compositional components of the adhesive hydrogels (D-MA and HA-MA) were developed and expanded for scale-up synthesis amenable at GLP settings. Methacrylate groups were conjugated to the hydroxyl (-OH) groups that are present on the dendrimer surface and the side groups of the hyaluronic acid backbone allowing for photocrosslinking to form hydrogels. The synthesis of D-MA and HA-MA involves utilizing anhydride of methacrylic acid for esterification in the presence of DMAP as a base and without the need for any coupling agents thereby significantly reducing the side products and impurities thereby enabling easy purification (FIGs 1 A and IB). Further, the 3-step synthesis protocol for HA-MA was chosen where in the first step, The sodium salt of hyaluronic acid (NA-HA) was converted to tetrabutylammonium salt (HA-TBA) using ion exchange with Dowex resin. This allows the solubility of HA in organic solvent (DMSO / DMA mixture) thereby reaction can be held under inert and anhydrous conditions. The advantage for this step is 2-fold: (i) it allows better control over the esterification reaction and has consistent degree of methacrylate substitution thereby reducing batch to batch variability and (ii) usage of appropriate mole equivalents of methacrylic anhydride whereas in aqueous reactions, ~20-fold excess was required to obtain modest degree of substitution.

[0117] In the second step, methacrylic acid formed in the reaction is conjugated to the -OH side groups in the HA backbone to form (TBA-HA-MA), In the third step, the TBA ions are exchanges with NA to obtain (NA-HA-MA or HA-MA). Both the conjugates are thoroughly purified using TFF to remove excess reactants and side products.1H NMR analysis demonstrated that -15-20 molecules and -40-50 molecules of methacrylate were conjugated to each dendrimer and hyaluronic acid molecule respectively resulting in high degree of methacrylation (-50%). The formulation was improved by taking parameters such as viscosity, mechanical strength, and crosslinking time into account. The solution concentrations of D-MA (300mg / mL) and HA-MA (190mg / mL) with a mixing ratio of 30:70 (D-MA: HA-MA) resulted in appropriate viscosity of -5200 cps which allows the solution to stay on the cornea for -15-20 seconds without flowing away thereby allowing ample time for cross-linking with UV light (FIG 1C). Upon 90 seconds of UV light illumination, the solution forms a transparent and flexible hydrogel over the corneal surface (FIG ID). The adhesive hydrogel possesses good mechanical and adhesive properties which are beneficial and provide long-term stability to the hydrogel, thereby facilitating corneal wound healing.

[0118] Clinical outcome of the OcuPair adhesive hydrogel filled SALK or DALK wound after 9 months

[0119] Earlier studies on OcuPair hydrogels focused on the short-term efficacy of sealing and stabilizing full thickness traumatic corneal wounds for 5 days. This study reports on the long-term effects of OcuPair adhesive hydrogel in treating large corneal defects, mimicking the keratectomy procedure performed in humans. In all animals, the clinical follow up was performed at various timepoint until 9 months. The DALK and SALK surgical steps included: (i) cornea wounds were made with a diameter of 7 mm trephine and the cornea sheet was removed by a crescent cornea knife; (ii) the cornea wound was overfilled with the adhesive hydrogel; (iii) the cornea wound was filled with hydrogel under curing with a UV light, (iv) and OCT images were taken before surgery and right after surgery in SALK and DALK wound, respectively. The clinical outcome of the OcuPair adhesive hydrogel filled SALK or DALK wound was evaluated. Corneal repair was evaluated after using adhesive hydrogel in SALK and DALK wound at 9 months. Representative macroscope, fluorescein staining, OCT, keratoscope and cornea clarity images were taken of rabbit cornea 10 months after SALK and DALK surgery filled with OcuPair adhesive hydrogel compared to the CRTL cornea.

[0120] At post-operative surgery 9 months, 5 out of 7 corneas in the SALK group and 7 out of 9 corneas in the DALK group remained transparent, showing no signs of opacity. Fluorescein staining demonstrated the absence of residual hydrogel in the surgical cornea, revealing the fully regenerated corneal epithelium was indistinguishable from the native and control (contralateral) corneal tissues. AS-OCT evaluation shows that the OcuPair adhesive hydrogel-treated cornea preserves a curvature and architecture comparable to a healthy control, with full regeneration of both the corneal epithelium and stromal structure. In both the SALK and DALK groups, we did observe a thin hyper-reflective stroma from OCT images with slight unevenness. Keratoscopy imaging at POM 9 showed no apparent difference in corneal curvature in the SALK or DALK group, compared to control. Postmortem cornea tissue has apparent transduction of the light, and no apparent opacity was observed. Although the majority of the rabbits have cleared all the hydrogel on the cornea, the corneas of 2 animals had residual OcuPair adhesive hydrogel left in each group. Residual hydrogel, stained by fluorescein, covered approximately 10-30% of the original wound area. Otherwise, the corneas were transparent, showing no signs of infection, fibrosis, or corneal neovascularization. Tear production was detected in all animals for the 9 months follow up period. At the end of experiment of 9 months (FIG 5), the tear production of both SALK and DALK groups was comparable with the contralateral eye. In the SALK surgery group, Schirmer’s test demonstrated a decrease of tear production with no statistically significant difference (9.1+ 1.8 vs 17.6 +2.3 mm, operated vs CTRL, p=0.0540) between operated and non-operated eyes at postsurgery 2 months (FIG 2 A, left panel). In the DALK group, the tear production was significantly decreased at post-surgery 2 months and 3 months (post-surgery 2 months: 6.7+ 1.9 vs 15.6 +2.6 mm, operated vs CTRL, P<0.05; post-surgery 3 months: 4.0+ 0.8 vs 15.2 +3.0 mm, operated vs CTRL, P<0.0.01), but it returned to a comparable level at post-surgery 6-9 months (FIG 2A, right panel).

[0121] Evaluation of stroma regeneration and hydrogel regression after anterior keratectomy

[0122] In the long-term recovery of corneal wounds with a 0= 7.00mm, it was important to understand the process of corneal regeneration. This research is essential for gaining insights into the regeneration process and guiding the improvement of gel formulation. To capture the dynamic corneal regeneration and reconstruction process, clinical follow up at the various time points were conducted: post 2 weeks, 4 weeks, 2 months, 3 months, 6 months and 9 months, in addition to the visual inspection of the corneal surface twice a week. Longitudinal evaluation of cornea regeneration was conducted using serial high-resolution AS-OCT post-surgery. Immediately after keratoplasty and OcuPair adhesive hydrogel application, the native cornea stromal thickness was reduced from 303.1+9.0 pm to around 150.1+9.8 pm (SALK, average 50.5% corneal removed), and from 317.5+11.5 pm to 94.8+9.2 pm (DALK, 70. 1% corneal removed) (FIG 2B, pre-, post-), with a hydrogel layer thickness of approximately 146.0+63.5 pm (SALK) or 167.6+91.4 pm (DALK) on (FIG 2C, Hydrogel).

[0123] Surprisingly, the cornea stroma quickly regenerated to the pre-surgery level within 2 weeks post-surgery in both SALK and DALK groups (FIG 2B). Within 2 months, the stromal thickness exceeded the original stromal thickness in the SALK group (FIG 2B) (434.9+42.8 vs 303.1+9.0 pm, POS 2m vs pre-, p=0.045), and the stromal thickness reached the original stromal thickness in the DALK group (363.4+39.1 vs 317.5+9.4 pm, post-surgery 2m vs pre-, p=0.2647). At this timepoint, while the SALK had total regenerated cornea compared to the DALK group animals, the net regenerated cornea (post-surgery 2 months minus pre-, SALK vs DALK: 283.9 +24.5 pm vs 268.7+30.2 pm, p=0.8757) is comparable considering the difference of remaining corneal stroma depth in SALK and DALK surgeries. However, the stromal thickness gradually regressed to presurgery thickness at post-surgery 6-9 months (FIG 2A). At post-surgery 9 months, AS-OCT showed that the restored maintained satisfactory curvature of the cornea. The stromal thickness was 335.6+18. 1 pm (SALK) and 333.9 +19.5 um (DALK) (FIG 2A, left panel-SALK, right panel- DALK). The thin hyper- reflective layer in anterior stroma underneath the corneal epithelium in all animals appeared. The comeal stroma appeared to be hyper-reflective to a certain extent with an uneven surface at 2 months and peaked at 3 months.

[0124] Concurrently, the hydrogel thickness measured by OCT was maintained at a stable level at first 2 months, followed by a noticeable reduction of hydrogel thickness between 2 and 3 months (SALK: 99.8+21.9 vs 57.7 .1+11.2 pm, post-2m vs pre-, p=0.0450, DALK: 137.0+33.0 vs 90.3 . 1+31.0 pm, post-2m vs pre-, p=0.0872,). At 9 months, the hydrogel was cleared from the cornea, and the epithelium layer was completely regenerated over the hydrogel surface in both SALK and DALK groups. The corneal epithelium thickness was 48.8 +3.47 mm (SALK) and 51.3+ 2.9 pm (DALK) respectively (FIG 2C, left panel-SALK, right panel-DALK). OCT imaging also revealed epithelium re-population during the comeal repair process with the OcuPair adhesive hydrogel substitute. Interestingly, comeal epithelium regeneration followed the stroma. A protruding edge from the cornea keratectomy edge, appeared to be higher density than the original transparent cornea epithelium, appeared to be intruding into the total transparent OcuPair adhesive hydrogel starting from 2 months in both the SALK and DALK groups. This process continued and, eventually, all the hydrogel was replaced by nascent epithelium at 6-9 months. At the end of experiment, the OCT imaging of epithelium regained transparent but with a relative uneven basal member.

[0125] The corneal surface covered by hydrogel was also monitored using fluorescein staining. Although not shown, initially the hydrogel was overfilled when applied to the corneal trephine wound. The hydrogel covered area was reduced to less than the initial wound area at post-surgery 2 weeks. The hydrogel covered area continued to decrease thereafter. In 5 / 7 of SALK, and 7 / 9 in DALK groups, there was no residual hydrogel detected after 9 months (data not shown; FIG 2C). At all timepoint, there was no statistical difference in hydrogel covered area between SALK and DALK group. Throughout all weekly visual inspection, no fluorescein staining was observed outside of the hydrogel covered area, indicating there is no additional epithelium damage. The median times to clear the hydrogel, by fluorescein staining, were 5.0 and 7.5 months in the SALK and the DALK eyes, respectively. Although it took more time in the DALK group to clear all the hydrogel, there was no statistical significance between the two groups (FIG 2D).

[0126] Histological Analysis of Regenerated Cornea

[0127] While imaging results and clinical follow-up of the rabbits indicate complete comeal regeneration with OcuPair adhesive hydrogel as the comeal substitute, it was important to examine the microstmcture to confirm the regeneration of the epithelium, stroma, comeal nerves, and 9 / 9 comeal endothelium. H&E staining demonstrated similar histology between the regenerated corneas and the healthy controls. The regenerated epithelium layer shows typical structure with the superficial epithelial cells, wing cells, and columnized-shaped basal epithelial cells. A thin layer of anterior stroma with higher keratocyte density was observed, attributing to newly regenerated cornea stroma. Masson's trichrome staining confirmed absence of stromal fibrosis in both SALK and DALK groups. TUBBIII staining was performed to evaluate corneal nerve regeneration. Cornea nerve terminals were detected within the newly regenerated cornea epithelium and the sub- basal nerve complex (SNP) layer. The anterior stromal nerve was also found in the nascent cornea stroma.

[0128] TEM analysis was performed to evaluate the ultrastructure of the regenerated cornea. The tissue specimen chosen for TEM was a 1 -2 mm strip, taken just beneath the corneal transverse plane and spanning across the apex, as illustrated in FIG 3A. The embedding surface for TEM was the medium sagittal plane of the regenerated cornea structure. A randomly selected small area 10- 20 pm below the epithelium basal membrane was imaged. In both the SALK and DALK groups, a complete basal membrane was present (FIG 2B). Stratified collagen bundles were found in the area imaged, a typical 90-degree orientated collage structure, as well as keratocytes of the anterior stroma. These findings suggested a similar ultrastructure between the regenerated cornea and healthy cornea. In vivo confocal microscopy was performed at the conclusion of the experiment (FIG 4). The basal epithelium of operated corneas appeared to be smooth and with a typical cobblestone shape with microglial cells detected. Thin parallel SNP nerves were detected at the sub-basal layer. The anterior stroma was populated with nerve fibers and keratocytes. The endothelium of all groups demonstrated a similar honeycomb structure to healthy controls.

[0129] The orchestrated corneal regeneration using the OcuPair adhesive hydrogel platform

[0130] The long-term goal of the corneal substitute is to achieve complete biocompatibility, comparable to a corneal transplant. Corneal recovery can take months to years, involving tissue remodeling that includes both temporal and spatial dynamic interactions between tissue regeneration and hydrogel degradation. To further elucidate the mechanism of corneal regeneration using the OcuPair hydrogel platform, the corneal structure at post-surgery 3 months was evaluated. Macro imaging demonstrated a significant reduction in the size of the defect, compared to the initial area. Transparency was maintained in both the regenerated tissue and the OcuPair-covered area. AS-OCT image demonstrated good adherence between the hydrogel and the underlying corneal wound bed, suggesting preserved structural and mechanical integrity of the hydrogel. Mildly hyperplastic tissue, presumably nascent epithelium, was observed to be snuggly connected to the hydrogel, at the periphery of the hydrogel area suggestive of native corneal tissue integration into the hydrogel. This affixation between the hydrogel and cornea tissue was also evident on H&E and trichrome staining of the comeal cross-section, since the hydrogel was partially preserved during formalin tissue fixation. The hyperplastic epithelium formed an incursion front at the comea / hydrogel junction, and a thin layer of epithelium infiltrated toward wound center under the hydrogel, forming tiny epithelium island not evident on the AS-OCT.

[0131] The epithelium incursion was also evident in the cornea tissue for TEM, though the hydrogel was not preserved in the glutaraldehyde fixation. Microscopy shows TEM imaging of this transitional area. The basal membrane appeared to be intact at low magnification, but the interruption of basal membrane was evident at high magnification. Superficial epithelial cells with typical microvillus are situated on the basal epithelium. Tight junctions were evident in all the layers of the epithelium. At this epithelium-stromal junction layer, myelinated sub-basal / anterior stromal nerves and unorganized collagen bundle were identified.

[0132] Discussion

[0133] Comeal fibrosis is a leading reversible cause of blindness worldwide. The technical expertise and human donor corneas, required of traditional corneal transplantation, present a significant barrier to restore sight in underserved areas. A biocompatible corneal substitute, OcuPair adhesive hydrogel, was developed, which requires minimal surgical expertise and clinical followup. It promotes robust corneal regeneration and achieves significantly satisfying long-term outcomes, making it a convenient alternative to human tissue transplants. One of the current hurdles for comeal substitutes is the technical complexity involved in the surgery, along with the intensive clinical follow-up required for patients. Corneal substitutes, such as biosynthetic human collagen- based corneas or bio-engineered porcine constructs, generally requires a surgeon expertise to suture the comeal substitute to host cornea or insert the implant to a stromal pocketFormsUing . . Other in situ formed comeal substitutes required less technical expertise, but may necessitate intensive postoperative clinical follow ups, such as cornea substitute reapplication, bandage contact lens, or amniotic membrane placement. In this study, OcuPair adhesive hydrogel was developed by utilizing the unique properties of linear hyaluronic acid biopolymer and a branched globular architecture of hydroxyl PAMAM dendrimer. The multiple surface hydroxyl groups covalently functionalized with methacrylate groups contribute to high crosslinking densities and the hyaluronic acid offers adhesiveness and flexibility resulting in hydrogels with desirable mechanical properties. Once the viscous OcuPair hydrogel precursor solution applied followed by photo-crosslinking using low powered UV light, it forms a transparent and flexible hydrogel within 90 seconds adhering well to the tissue resulting in a stable dome-shaped bioactive prosthesis over the corneal defect. This unique property of the OcuPair adhesive hydrogel eliminates the need for the surgeon to apply the 9 / 9 precise amount of the hydrogel into the stromal bed as other in situ formed hydrogel, and its domelike shape forms naturally without requiring a specific angle on the ocular surface. The OcuPair bioadhesive formulation was improved with appropriate dendrimer to hyaluronic acid ratio (30:70, D-MA:HA-MA) to have appropriate crosslinking density, mechanical and adhesive properties which may contribute resistance to rapid enzymatic degradation of hyaluronic acid by hyaluronidase in the tear film and allowing slow degradation allowing time for stromal integration and complete regeneration of the corneal tissue. This OcuPair adhesive hydrogel is both strong, elastic and withstands high intraocular pressures beyond physiological range (>50 mm Hg) and stabilizes the injured cornea immediately upon photo-crosslinking with no signs of corneal toxicity over 30-day period. These unique properties of OcuPair adhesive hydrogel allow it to withstand the forces from eye blinking and activities like rabbit grooming. This eliminates the need for hydrogel re-application or a contact lens bandage for up to 9 months. Meanwhile, the excess hydrogel spontaneously delaminates from the intact epithelial surface within 24-48 hours, covering only the wounded stroma area. This is due to the external hydrophobic HA branches of the OcuPair adhesive hydrogel, which facilitate easy removal after the curing process, as shown in FIG 4. Thus, the ease of use, mechanical strength, and biocompatibility of this OcuPair adhesive hydrogel are desired characteristics of an in situ formed cornea substitute.

[0134] The data demonstrates robust corneal stroma regeneration, highlighting the effectiveness of the OcuPair adhesive hydrogel. The regenerated stroma exhibited structural integrity and functionality comparable to natural tissue, indicating the hydrogel’s potential as a reliable alternative to traditional corneal transplants. In just 2-4 weeks, the stroma regeneration reached and exceeded the original corneal height (FIGs 2A-2D). This signific regeneration capacity is rarely reported in other in situ cured comeal substitute animal models or human research. However, cornea stroma keratocytes maintain a strong regenerative capacity and can become highly active upon injury. Additionally, there are other local progenitor keratocytes dormant within the corneal stroma, and mesenchymal-like stroma stem cells at the limbal stem cell niche. Proliferation and activation of these cells is modulated by their microenvironment. As a key component of this OcuPair adhesive hydrogel, hyaluronic acid is a natural constituent of the corneal limbus and has been extensively studied for its ability to promote stem cell proliferation and inhibit cell differentiation. Furthermore, its low immunogenicity compared to collagen, another commonly employed corneal substitute material, is highly advantageous for long-term in vivo application. Therefore, the hyaluronic acid in the hydrogel platform is fundamental for the reconstitution of a pro-regenerative environment for the corneal keratocytes, progenitors, and stem cells. Besides the choice of HA as the bioengineered material, another engineered feature that might improve corneal regeneration is the dendrimer component that contributes to high crosslinking density, resulting in hydrogel with appropriate mechanical strength and rigidness. Research indicates that the rigid hydrogel promotes the growth of pluripotent stem cells, while the softer hydrogel accelerates their differentiation. It is believed that the OcuPair adhesive hydrogel possesses strength and flexibility that balances the appropriate rigidness and softness. For these reasons, it is believed that the powerful regeneration of cornea stroma lays in a stem cell-conducive microenvironment provided by the OcuPair adhesive hydrogel.

[0135] In both SALK and DALK groups, the hydrogel achieved satisfactory corneal regeneration at the end of the experiment of 9 months. Longitudinally, the epithelium recovery as indicated by the FIG 2C showed no difference as calculated by the hydrogel covered area. The net stroma regeneration measured by the OCT was comparable in two group (FIG 2A). The hydrogel clearance time was shorter in SALK, but the difference failed to yield any statistical significance (FIG 2D). The only noticeable difference between the two groups was the significant decrease in the tear production between 2-3 months in DALK group. This clearly demonstrates that DALK surgery could lead to a temporary but profound tear production decrease in rabbit model. In the present animal model, this tear reduction did not lead to any noticeable comeal epithelium damage, and the tear production rebounded back to comparable amount at the end of post-surgery 9 months (FIG 5). But in patients, dry eye is a common side effect of comeal keratoplasty, up to 70-90% penetrating keratoplasty patients, and 30-50% of the corneal lamellar keratoplasty patients, presumably due to the deeper stromal nerve damage during the surgery. So, the present model provides valuable insights into the corneal recovery process and the mechanisms underlying dry eye development in these patients.

[0136] The present study also provides clear evidence of the transition from stromal regeneration to stromal remodeling during that long-term corneal recovery process. After the initial stromal regeneration process, which occurs between 2 weeks and 2 months post-surgery, it appears that the comeal regeneration shifts to corneal remodeling beginning around 2 to 3 months and continues thereafter. Before 2-3 months, the cornea curvature was unsatisfactory, the regenerated stroma height exceeded the original cornea height, and the OCT density was homogeneous from the anterior to the posterior cornea. After 2-3 months, the corneal curvature eventually evolved into a smooth surface by 9 months, the cornea stroma returned to the height comparable to the native height of the cornea, accompanied with a slightly increased density band in OCT imaging. The increased density of OCT imaging was also seen in the other long-term comeal substitute study at 3 and 12 months, and tended to become less intense gradually. Additionally, cornea haze, which was frequently documented in other forms of cornea substitutes, was not observed in the study. Additionally, all the microscopic analysis indicated that there is no corneal fibrotic change, so it was confirmed that the increased OCT intensity strip is with no clinical significance. These findings highlight the dynamic nature of cornea stromal regeneration and reconstruction, and the OcuPair adhesive hydrogel exhibited excellent biocompatibility, evoking no fibrotic response.

[0137] Although tissue integration is a highly desirable property for corneal substitutes, these studies raise doubts about this assumption. In this study, a clean demarcation line segregated the stroma from the OcuPair hydrogel during the corneal regeneration and reconstruction. This suggests integration of the corneal stroma and corneal substitute is not a prerequisite for successful corneal tissue regeneration and remodeling, On the contrary, this finding is consistent with some in situ forming cornea substitutes that reported little evidence of keratocyte infiltration in long-term observation. Even in the cornea substitute with embedded stromal cells, regeneration of the cornea was mainly attributed to exosome-secreting programed activated keratocyte, not stem cells integration into the host cornea. Moreover, data from full-thickness corneal transplants also mitigates the importance of keratocyte integration, since the variable integration rates of host cells into transplanted tissue was not correlated with the success rate of cornea transplant. Accordingly, keratocyte regeneration induced by the OcuPair hydrogel, not the keratocyte incorporation into the OcuPair hydrogel, facilitated successful regeneration and remodeling of the cornea.

[0138] Apart from the robust stroma regeneration observed in the present study, it is believed that the mechanism of action of OcuPair adhesive hydrogel is the orchestration of keratocyte regeneration, epithelium repopulation, and HA-dendrimer dislodgment altogether. In addition to providing a robust protective temporary prosthesis over the wounded area and creating a microenvironment conducive for keratocyte integration and activation, the hydrogel also promotes sufficient stratified columnar epithelium formation and stromal extracellular matrix rearrangement during a period of 6-9 months in the present animal model. Unlike other corneal substitutes, which typically show migration of stratified epithelial cells to cover exposed corneal wounds or artificial corneas within one week, fluorescein staining, histology, and OCT images of this OcuPair adhesive hydrogel revealed no epithelial coverage over the hydrogel surface at any time. Instead, newly generated epithelium formed a hyperplastic edge at the OcuPair hydrogel / comea border and infiltrated under the hydrogel layer to form the epithelium island, allowing a much slower but controlled regeneration of the epithelium tissue. This phenomenon is in line with recently discovery that there are two population of epithelium stem cell located in the cornea rim: a rapid migrating epithelium population that could provide a barrier for underlying stromal cells in days, and a slow- replicating cornea outer rim limbal stem cells eventually regenerate the corneal epithelium in a matter of months. In the present study, the physical barrier provided by the OcuPair hydrogel allowed the corneal epithelium to regenerate and undergo a process mimicking the normal cornea regeneration. This process also contributed to the hydrogel dislodge from the cornea. The unique hydrophobic property of HA component that may have contributed to the self-dislodgement from the healthy corneal epithelium 24 hours after the gel curing (the over filling part during OcuPair application) whereas the majority part of the OcuPair hydrogel filling the corneal defect was stable for long-term suggestive of superior adherence of hydrogel to the stromal tissue. Additionally, the complete dislodgement of the OcuPair adhesive hydrogel required several months in both the SALK and DALK groups. Of note, this OcuPair adhesive hydrogel is still susceptible to hyaluronidase enzymatic degradation, which is a rich component in tears, but at a lower rate as the HA in OcuPair hydrogel is in crosslinker-form with dendrimer. The present data demonstrated that the tear production was reduced in both SALK and DALK between 2-3 months. So, the decreased tear enzymes could also have contributed to the prolonged OcuPair adhesive hydrogel integrity on the comeal surface. In summary, the hydrogel dislodging mechanism is likely due to the convergence of multiple factors: (i) high degree of cross-linking resulting in robust hydrogel with a balance of stiffness and flexibility, (ii) the hydrophobic nature of HA making it incompatible with the newly generated comeal epithelium, and the continuous digestion of HA by tear hyaluronidase.

[0139] The clinical implications of this study were done in healthy cornea with intact stromal and epithelial stem cells from cornea rim limbal region of rabbits. Accordingly, its efficacy remains unexplored in situations where these pre-existing conditions are not met, such as limbal cell deficiency, unresolved chronic infection, or ocular autoimmune diseases. In the present study, hydrogel required an average of 6-7 months to disengage from the cornea. While the hydrogel maintained its clarity throughout this period, it may present clinical issues for patients experiencing comeal foreign body sensation and corneal refractive errors. Also, there were two animals in each group that had OcuPair adhesive hydrogel that remained on the comeal until the end of the experiment of 10 months. To minimize the individual variability of the efficacy of this comeal substitute, further improving the OcuPair adhesive hydrogel formulation by incorporating growth factors or drugs that might accelerate corneal regeneration and shorten the hydrogel clearance time.

[0140] In this study, the feasibility of a HA-dendrimer hydrogel platform was demonstrated through promoting corneal regeneration after anterior lamellar keratectomy. The recovery of the corneal structure appeared to be a result of the dynamic interaction between the OcuPair adhesive hydrogel and the corneal tissues. This interaction facilitated the cornea regeneration process, with the hydrogel providing a supportive scaffold that promoted cellular growth and tissue integration. The interaction between the hydrogel and the corneal stmctures ensured that the regenerated tissue maintained its integrity and functionality, closely mimicking the original corneal architecture. This platform reduces the need for technical expertise and obviates the demand for comeal donor tissues, in specific cases of comeal opacities.

Claims

We claim:

1. A kit to treat corneal defects and wounds comprising a sterile biocompatible hyaluronic acid-dendrimer formulation comprising(a) a methacrylate-functionalized hyaluronic acid (“HA”) and(b) a methacrylate-functionalized hydroxyl dendrimer in a dosage unit for treatment of corneal defects or wounds.

2. The kit of claim 1 wherein the methacryate-functionalized hydroxyl dendrimer is a poly(amidoamine).

3. The kit of claim 1 wherein methyl acrylated dendrimer conjugates are made by attaching methyl acrylate groups to the surface -hydroxyl groups of dendrimers using methacrylic anhydride.

4. The kit of claim 3 wherein the methyl acrylated dendrimers are formed form hyaluronic acid from methacrylated dendrimer (D-MA) and hyaluronic acid dendrimer (HA-MA) mixed at a ratio of 30:70 (D-MA:HA-MA) with a photo-initiator such as IRGACURE 2959.

5. A method of inducing corneal regeneration comprising applying to corneal tissue a formulation comprising(a) a methacrylate-functionalized hyaluronic acid (“HA”) and(b) a methacrylate-functionalized hydroxyl dendrimer.

6. The method of claim 5 for treating a comeal wound or a partially removed cornea comprising applying to the comeal wound or area of the partially removed cornea a biocompatible hyaluronic acid-dendrimer formulation7. The method of claim 5 wherein the formulation is application to an eye following Superficial Anterior Lamellar Keratoplasty (SALK) to form new corneal tissue.

8. The method of claim 5 wherein the formulation is application to an eye following Deep Anterior Lamellar Keratoplasty(DALK, corresponding to approximately 80-90% removal of corneal stroma) to form new corneal tissue.

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