Biopolymer Formulations

Abstract

The present disclosure provides a biopolymer formulation comprising thiolated gelatin and methacrylated hyaluronic acid.The formulation is useful for corneal application, for example, for treating corneal disease or injury in a subject.Also provided is the method of preparing the biopolymer formulation and its application.

Description

[Background technology]

[0001] Corneal blindness is the fourth leading cause of blindness in the world, with an estimated 1.5 million new cases reported worldwide each year. Approximately 10 million people worldwide suffer from bilateral corneal blindness, and an additional 23 million suffer from unilateral corneal blindness. Major causes of corneal dysfunction include trachoma (including corneal scarring and neovascularization), ocular trauma, corneal ulcers, and infection with herpes simplex virus. One of the primary medical treatments for corneal disease is keratoplasty (corneal transplantation). However, there are various complications associated with corneal transplants, including: (i) corneal transplant patients experiencing tissue (corneal) rejection; (ii) scarring due to infections such as ocular herpes or fungal keratitis; (iii) glaucoma (elevated intraocular pressure); (iv) vision problems (visual acuity) caused by irregular curves in the corneal shape; (v) corneal graft delamination; and (vi) the high cost and inconvenience surrounding the safe extraction, storage, and transportation of the biopsy tissue.

[0002] In view of the limitations associated with corneal transplantation, various efforts have been made to minimize or eliminate the need to resort to keratoplasty. For example, biopolymer-based ocular adhesives that, once applied to a diseased, degenerated, damaged, or partially resected cornea, mimic the properties of the natural cornea. Such ocular adhesives may be used, for example, by methods to close ocular wounds after injury or during corneal surgery. In corneal surgery, biopolymers may be employed as sutureless substitutes to close post-operative perforations. Various biomaterials have been reported in the literature to treat corneal injuries resulting from ocular diseases.

[0003] However, conventional ocular adhesives still have suboptimal properties, such as rapid polymerization, heat generation, poor biocompatibility, poor transparency and rough surfaces, difficult handling, short residence time, and poor integration with host ocular tissue.

[0004] Thus, there remains a long-standing need in the art to develop effective, biocompatible and biodegradable crosslinked hydrogel formulations that match the characteristics of the native cornea to aid in the treatment of corneal diseases while avoiding side effects. Generating hydrogels that are compatible with and match the physical properties of the native cornea has remained a challenging task due to (1) the remarkable physical properties of the cornea, which combine a high degree of transparency with durability, and (2) the unpredictable effects of polymer chemistries and combinations on the physical properties of the resulting hydrogels. Summary of the Invention

[0005] Provided herein are embodiments of biopolymer formulations that include thiolated gelatin and methacrylated hyaluronic acid.

[0006] In some variations, the thiolated gelatin can have an average molecular weight of about 50 kDa to about 200 kDa and an average degree of substitution of about 0.2 mmol / g to about 1.2 mmol / g; the methacrylated hyaluronic acid can have an average molecular weight of 20 kDa to 80 kDa and an average degree of substitution of 0.5 mmol / g to 1 mmol / g.

[0007] In some variations, the biopolymer formulation may include thiolated gelatin having a Bloom value of about 200 Bloom to about 275 Bloom and an average degree of substitution of about 0.5 mmol / g to about 0.9 mmol / g, and methacrylated hyaluronic acid having an average molecular weight of 25 kDa to 40 kDa and an average degree of substitution of about 0.7 mmol / g to about 0.9 mmol / g, the thiolated gelatin and the methacrylated hyaluronic acid being in a weight ratio of about 100:30 to about 100:70. Optionally, the thiolated gelatin may have a Bloom value of about 220 Bloom to about 230 Bloom and an average degree of substitution of about 0.5 mmol / g to about 0.7 mmol / g, the thiolated gelatin and the methacrylated hyaluronic acid being in a weight ratio of about 100:55 to about 100:65. In some cases, the thiolated gelatin can have a Bloom value of about 240 Bloom to about 260 Bloom and an average degree of substitution of about 0.7 mmol / g to about 0.9 mmol / g, and the thiolated gelatin and the methacrylated hyaluronic acid can be in a weight ratio of about 100:35 to about 100:45.

[0008] In some variations, the biopolymer formulation may be in the form of a dry premix, where the methacrylated hyaluronic acid and the thiolated gelatin are in the form of a dry powder.

[0009] In some variations, the biopolymer formulation may be in the form of a liquid or hydrogel and may include a saline solution and at least one photoinitiator compound.

[0010] Also provided herein are embodiments of methods for treating a disease or injury of the cornea of ​​a subject. In some variations, the methods may include mixing a dry biopolymer premix comprising thiolated gelatin and methacrylated hyaluronic acid with a photoinitiator solution to produce a liquid biopolymer mixture; applying the liquid biopolymer mixture to the cornea of ​​the subject; and exposing the liquid biopolymer mixture applied onto the cornea to light having a wavelength within the excitation range of at least one photoinitiator compound. In some variations, the dry biopolymer premix may include thiolated gelatin, optionally having an average molecular weight of 50 kDa to 200 kDa and an average degree of substitution of 0.2 mmol / g to 1.2 mmol / g, and methacrylated hyaluronic acid, optionally having an average molecular weight of 20 kDa to 80 kDa and an average degree of substitution of 0.5 mmol / g to 1 mmol / g. [Brief description of the drawings]

[0011] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain aspects of the subject matter disclosed herein and, together with the description, serve to explain some of the principles associated with the disclosed implementations.

[0012] [Figure 1A] Figures 1A-1C show hCSSCs grown on cover glass cultures and stained with calcein-AM, ethidium homodimer and DAPI to assess cell viability after 1 day (Figure 1A), 2 weeks (Figure 1B) and 4 weeks (Figure 1C) of culture.

[0013] [Figure 1B] Figures 1A-1C show hCSSCs grown on cover glass cultures and stained with calcein-AM, ethidium homodimer, and DAPI to assess cell viability after 1 day (Figure 1A), 2 weeks (Figure 1B), and 4 weeks (Figure 1C) of culture.

[0014] [Figure 1C]Figures 1A-1C show hCSSCs grown on cover glass cultures and stained with calcein-AM, ethidium homodimer, and DAPI to assess cell viability after 1 day (Figure 1A), 2 weeks (Figure 1B), and 4 weeks (Figure 1C) of culture.

[0015] [Figure 1D] Figures 1D-1F show hCSSCs grown within gel-SH hydrogels and stained with calcein-AM, ethidium homodimer, and DAPI to assess cell viability after 1 day (Figure 1D), 2 weeks (Figure 1E), and 4 weeks (Figure 1F) of culture.

[0016] [Figure 1E] Figures 1D-1F show hCSSCs grown within gel-SH hydrogels and stained with calcein-AM, ethidium homodimer, and DAPI to assess cell viability after 1 day (Figure 1D), 2 weeks (Figure 1E), and 4 weeks (Figure 1F) of culture.

[0017] [Figure 1F] Figures 1D-1F show hCSSCs grown within gel-SH hydrogels and stained with calcein-AM, ethidium homodimer, and DAPI to assess cell viability after 1 day (Figure 1D), 2 weeks (Figure 1E), and 4 weeks (Figure 1F) of culture.

[0018] [Figure 2A] Figures 2A-2C show hCSSCs grown on cover glass cultures and stained with CD90, αSMA and DAPI to assess cell differentiation after 1 day (Figure 2A), 2 weeks (Figure 2B) and 4 weeks (Figure 2C) of culture.

[0019] [Figure 2B] Figures 2A-2C show hCSSCs grown on cover glass cultures and stained with CD90, αSMA and DAPI to assess cell differentiation after 1 day (Figure 2A), 2 weeks (Figure 2B) and 4 weeks (Figure 2C) of culture.

[0020] [Figure 2C] Figures 2A-2C show hCSSCs grown on cover glass cultures and stained with CD90, αSMA and DAPI to assess cell differentiation after 1 day (Figure 2A), 2 weeks (Figure 2B) and 4 weeks (Figure 2C) of culture.

[0021] [Figure 2D] Figures 2D-2F show hCSSCs grown within gel-SH hydrogels and stained with CD90, αSMA, and DAPI to assess cell differentiation after 1 day (Figure 2D), 2 weeks (Figure 2E), and 4 weeks (Figure 2F) of culture.

[0022] [Figure 2E] Figures 2D-2F show hCSSCs grown within gel-SH hydrogels and stained with CD90, αSMA, and DAPI to assess cell differentiation after 1 day (Figure 2D), 2 weeks (Figure 2E), and 4 weeks (Figure 2F) of culture.

[0023] [Figure 2F] Figures 2D-2F show hCSSCs grown within gel-SH hydrogels and stained with CD90, αSMA, and DAPI to assess cell differentiation after 1 day (Figure 2D), 2 weeks (Figure 2E), and 4 weeks (Figure 2F) of culture.

[0024] [Figure 3A] Figures 3A-3B show phase contrast images of hCEC cells after 4 days of growth in cover slip culture, comparing hCECs grown on a cover slip surface (Figure 3A) and on a hydrogel surface (Figure 3B).

[0025] [Figure 3B] Figures 3A-3B show phase contrast images of hCEC cells grown on cover slip cultures after 4 days, comparing hCECs grown on cover slip surfaces (Figure 3A) and on hydrogel surfaces (Figure 3B).

[0026] [Figure 4A]Figures 4A-4C show hCEC cells grown on a coverslip surface and stained with DAPI (Figure 4A), ZO1 (Figure 4B) and rhodamine-phalloidin (Figure 4C).

[0027] [Figure 4B] Figures 4A-4C show hCEC cells grown on a coverslip surface and stained with DAPI (Figure 4A), ZO1 (Figure 4B) and rhodamine-phalloidin (Figure 4C).

[0028] [Figure 4C] Figures 4A-4C show hCEC cells grown on a coverslip surface and stained with DAPI (Figure 4A), ZO1 (Figure 4B) and rhodamine-phalloidin (Figure 4C).

[0029] [Figure 4D] Figures 4D-4F show hCEC cells grown on hydrogel surfaces and stained with DAPI (Figure 4D), ZO1 (Figure 4E), and rhodamine-phalloidin (Figure 4F).

[0030] [Figure 4E] Figures 4D-4F show hCEC cells grown on hydrogel surfaces and stained with DAPI (Figure 4D), ZO1 (Figure 4E), and rhodamine-phalloidin (Figure 4F).

[0031] [Figure 4F] Figures 4D-4F show hCEC cells grown on hydrogel surfaces and stained with DAPI (Figure 4D), ZO1 (Figure 4E), and rhodamine-phalloidin (Figure 4F).

[0032] Detailed Description Non-limiting examples of various embodiments and variations of the present invention are described herein.

[0033] Provided herein are embodiments of biopolymer formulations that, in some variations, can be used to produce hydrogels that are compatible with and match the physical properties of the natural cornea.

[0034] In some variations, the biopolymer formulation comprises thiolated gelatin (gel-SH) and methacrylated hyaluronic acid (HA-MA). gel-SH is gelatin modified with thiol groups. HA-MA is hyaluronic acid modified with methacrylate. Without being bound by theory or mechanism, modifying a polymer such as hyaluronic acid with methacrylate can crosslink the polymer. The crosslinks can be dimethacrylate crosslinks between two methacrylate groups (e.g., between two HA-MA molecules), thiol-ene crosslinks between methacrylate groups and thiol groups (e.g., between a gel-SH molecule and a HA-MA molecule), or a combination thereof. Crosslinks can be induced via a photoinitiator compound in the presence of light. Examples of photoinitiator compounds for use in methacrylate crosslinking include eosin, e.g., eosin Y, and / or triethanolamine.

[0035] Properties of Gel-SH In some variations, the gel-SH of the biopolymer formulation of the present disclosure may have an average molecular weight of about 50 kDa to about 200 kDa, about 50 kDa to about 100 kDa, about 40 kDa to about 120 kDa, about 60 kDa to about 90 kDa, about 60 kDa to about 150 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 120 kDa, about 140 kDa, about 160 kDa, about 180 kDa, and about 200 kDa. The average molecular weight of the gel-SH may be determined by size exclusion chromatography (SEC) or based on the bloom value, which may be determined using a gel meter.

[0036] In some variations, the gel-SH in the formulation is from about 0.2 mmol / g to about 1.2 mmol / g, from about 0.2 mmol / g to about 0.9 mmol / g, from about 0.3 mmol / g to about 0.9 mmol / g, from about 0.5 mmol / g to about 0.9 mmol / g, from about 0.3 mmol / g to about 0.8 mmol / g, from about 0.3 mmol / g to about 0.7 mmol / g, from about 0.65 mmol / g to about 0.75 mmol / g, from about 0.2 mmol / g, from about 0.3 mmol / g, mol / g, about 0.3 mmol / g, about 0.4 mmol / g, about 0.5 mmol / g, about 0.6 mmol / g, about 0.62 mmol / g, about 0.64 mmol / g, about 0.66 mmol / g, about 0.68 mmol / g, about 0.7 mmol / g, about 0.72 mmol / g, about 0.75 mmol / g, about 0.8 mmol / g, about 0.9 mmol / g, about 1 mmol / g, about 1.1 mmol / g, or about 1.2 mmol / g.

[0037] In some variations, the gel-SH may have a bloom value of about 100 bloom to about 300 bloom, about 200 bloom to about 300 bloom, about 100 bloom to about 150 bloom, about 150 bloom to about 250 bloom, about 200 bloom to about 250 bloom, about 200 bloom to about 275 bloom, about 220 bloom to about 260 bloom, about 220 bloom to about 280 bloom, about 210 bloom to about 240 bloom, about 100 bloom, about 120 bloom, about 125 bloom, about 150 bloom, about 175 bloom, about 200 bloom, about 215 bloom, about 225 bloom, about 240 bloom, about 250 bloom, about 260 bloom, about 270 bloom, about 280 bloom, about 290 bloom, or about 300 bloom.

[0038] Properties of HA-MA In some variations, the HA-MA of the biopolymer formulations disclosed herein has an average molecular weight of about 20 kDa to about 80 kDa, about 30 kDa to about 60 kDa, about 20 kDa to about 50 kDa, about 30 kDa to about 40 kDa, about 30 kDa to about 35 kDa, about 25 kDa, about 28 kDa, about 30 kDa, about 33 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 60 kDa, about 70 kDa, and about 80 kDa. The average molecular weight of HA-MA can be determined by size exclusion chromatography (SEC).

[0039] In some variations, the HA-MA of the biopolymer formulations of the present disclosure can have an average degree of substitution of about 0.5 mmol / g to about 1 mmol / g, about 0.6 mmol / g to about 1 mmol / g, about 0.7 mmol / g to about 0.9 mmol / g, about 0.5 mmol / g, about 0.6 mmol / g, about 0.7 mmol / g, about 0.75 mmol / g, about 0.8 mmol / g, 0.81 mmol / g, about 0.85 mmol / g, and about 0.9 mmol / g.

[0040] Dry Premix In some variations, the biopolymer formulation of the present disclosure may be in the form of a dry premix preparation, where gel-SH and HA-MA are in the form of a dry powder, optionally lyophilized. In some variations, gel-SH and HA-MA are mixed homogeneously and stored in a sterile container for reconstitution with water, physiologically compatible saline, or optionally buffered saline. Such reconstitution can be performed at the corneal treatment site, for example, at the bedside. Optionally, gel-SH and HA-MA are premixed in the form of a solution. The desired ratio of gel-SH and HA-MA are presolubilized together in water or saline, and after solubilization, are then dried (lyophilized) to obtain a thoroughly mixed powder of gel-SH and HA-MA. Presolubilization may be performed in a dark environment or in a light-proof container and / or without a photoinitiator, so that the gel-SH and HA-MA in the premix remain uncrosslinked after presolubilization and drying.

[0041] In some variations, gel-SH and HA-MA can be mixed in the dry premix in a weight ratio (gel-SH:HA-MA) of about 100:30 to about 100:90, about 100:30 to about 100:70, about 100:30 to about 100:60, about 100:40 to about 100:70; about 100:50 to about 100:70, about 100:40, about 100:50, about 100:60, about 100:70, or about 100:80.

[0042] In some variations, the premix may further comprise a therapeutic agent in dried or lyophilized form. Exemplary therapeutic agents include, but are not limited to, exosomes and liposomes.

[0043] Hydrogels In some variations, the biopolymer formulation of the present disclosure may be in the form of a liquid biopolymer mixture in which gel-SH and HA-MA are in an aqueous solution, in which gel-SH and HA-MA are not cross-linked (or are minimally cross-linked), or in the form of a hydrogel in which gel-SH and HA-MA are generally cross-linked. In the form of a liquid biopolymer mixture or hydrogel, the biopolymer formulation may include an aqueous solvent. In some variations, the aqueous solvent may be water or saline. The saline may be a physiological saline solution with an osmolality or osmolality compatible with tissue, for example, the cornea. The saline may be a buffered saline solution, such as phosphate buffered saline (PBS).

[0044] In some variations, the gel-SH and HA-MA may be crosslinked to form a hydrogel. The crosslinks may include dimethacrylate crosslinks between HA-MA molecules and / or thiol-ene crosslinks between gel-SH and HA-MA molecules. The HA-MA in the liquid biopolymer liquid or hydrogel may be about 0% to about 100% crosslinked, about 10% to about 90% crosslinked, about 20% to about 80% crosslinked, not crosslinked, about 5% crosslinked, about 10% crosslinked, about 15% crosslinked, about 20% crosslinked, about 25% crosslinked, about 30% crosslinked, about 40% crosslinked, about 50% crosslinked, about 60% crosslinked, about 70% crosslinked, about 80% crosslinked, about 90% crosslinked, or about 100% crosslinked.

[0045] In some variations, the biopolymer formulation in the form of a liquid biopolymer mixture or hydrogel may contain gel-SH at a concentration of about 50 mg / ml to about 140 mg / ml, about 70 mg / ml to about 140 mg / ml, about 75 mg / ml to about 125 mg / ml, about 90 mg / ml to about 110 mg / ml, about 90 mg / ml to about 130 mg / ml, about 100 mg / ml to about 120 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 110 mg / ml, about 120 mg / ml, about 125 mg / ml, about 130 mg / ml, or about 140 mg / ml.

[0046] In some variations, the biopolymer formulation in the form of a liquid biopolymer mixture or hydrogel may contain HA-MA at a concentration of about 40 mg / ml to about 90 mg / ml, about 40 mg / ml to about 70 mg / ml, about 45 mg / ml to about 65 mg / ml, about 50 mg / ml to about 60 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml, about 55 mg / ml, about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, or about 80 mg / ml.

[0047] In some variations, the biopolymer formulation in the form of a liquid biopolymer mixture or hydrogel may include at least one photoinitiator. Photoinitiators are generally compounds or combinations of compounds that catalyze the crosslinking of functionalized polymers such as HA-MA and / or gel-SH. In some variations, the at least one photoinitiator includes eosin, triethanolamine, or a combination thereof. In some variations, the eosin is eosin Y. In some variations, the biopolymer formulation may include eosin Y at a concentration of about 0.02 mM to about 0.2 mM, about 0.05 mM to about 0.2 mM, about 0.05 mM to about 0.1 mM, about 0.05 mM, about 0.08 mM, about 0.1 mM, or about 1.5 mM. In some variations, the biopolymer formulation may contain Eosin Y at a concentration of about 0.02% v / v to about 0.2% v / v, about 0.05% v / v to about 0.2% v / v, about 0.02% v / v to about 0.1% v / v, about 0.05% v / v to about 0.1% v / v, about 0.07% v / v to about 0.09% v / v, about 0.6% v / v, about 0.7% v / v, about 0.76% v / v, about 0.8% v / v, about 0.9% v / v, or about 1% v / v.

[0048] In some variations, the biopolymer formulation in the form of a liquid biopolymer mixture or a hydrogel may include a therapeutic agent, which may be a therapeutic exosome or liposome.

[0049] Method for producing biopolymer hydrogels In some variations, biopolymer hydrogels according to embodiments of the present disclosure may be produced by mixing a dry biopolymer premix with a photoinitiator solution to produce a liquid biopolymer mixture, applying the liquid biopolymer mixture to a surface, and exposing the liquid biopolymer mixture applied to the surface to light having a wavelength within the excitation (absorption) range of the photoinitiator. As an example, the excitation range of an exemplary photoinitiator, Eosin Y, is from about 440 nm to about 575 nm, with an absorption peak at about 525 nm. The light may be white light that includes a mix of wavelengths that include wavelengths within the excitation range of the photoinitiator.

[0050] In some variations, the liquid biopolymer mixture may include a therapeutic agent mixed with the dry biopolymer premix and the photoinitiator solution. The therapeutic agent is mixed in a dry or lyophilized form, optionally included in the premix, included in a second solution, or included in the photoinitiator solution. In some variations, the therapeutic agent may be a therapeutic exosome or liposome.

[0051] The surface to which the liquid biopolymer mixture is applied and then crosslinked by exposure to light to form a crosslinked hydrogel can be a biological surface. The biological surface is, for example, a tissue such as the cornea. The cornea can be a diseased, degenerated, damaged, or partially resected cornea.

[0052] Treatment The biopolymer formulations of the present disclosure in the form of hydrogels exhibit one or more of the following improvements compared to conventional hydrogels used as ocular adhesives: improved transparency, improved adhesive strength, low swelling (measured by weight and / or volume), reduced degradation over time, a compressive modulus that is compatible with tissues such as the cornea, and improved biocompatibility with cells such as corneal cells. Thus, it may be advantageous to use the biopolymer formulations of the present disclosure as ocular adhesives or temporary corneal substitutes to treat corneal injuries or diseases.

[0053] In some variations, a method of treating a corneal disease or injury in a subject may include providing a liquid biopolymer mixture of the present disclosure that includes a photoinitiator, applying the liquid biopolymer mixture to a corneal wound site of a subject, and exposing the liquid biopolymer mixture applied onto the cornea to light having a wavelength within the excitation range of the photoinitiator, thereby crosslinking the liquid biopolymer mixture within the corneal wound site and forming a hydrogel replacement cornea (HRC). The corneal wound site may be a wound site surgically excavated, for example, by keratectomy, to remove diseased, scarred, or damaged corneal tissue. The HRC may provide an environment that stimulates corneal regeneration, such that the HRC is replaced over time by regenerative corneal tissue formed at the corneal wound site.

[0054] In some variations, the liquid biopolymer mixture may be applied to a subject's eye as follows: (1) surgically excavate and remove the diseased, damaged, or scarred cornea, for example, by corneal abrasion; (2) apply an appropriate amount of the liquid biopolymer mixture to fill the gaps in the corneal tissue; (3) irradiate the application site with white light of appropriate intensity to crosslink the liquid biopolymer and convert the mixture into HRC. In some variations, after the HRC is formed, the site may be washed with saline and treated with an antibiotic, such as moxifloxacin. In some variations, the cornea may be covered with a bandage contact lens to further protect the surgical site.

[0055] In some variations, the white light for crosslinking the liquid biopolymer mixture is 1 mW / cm 2 ~100mW / cm 2 , 10mW / cm 2 ~100mW / cm 2 , 5mW / cm 2 ~50mW / cm 2 , 5mW / cm 2 ~20mW / cm 2 , 8mW / cm 2 ~12mW / cm 2 , 80mW / cm 2 ~100mW / cm 2, about 5mW / cm 2 , about 8mW / cm 2 , about 10mW / cm 2 , about 12mW / cm 2 , about 15mW / cm 2 , about 20mW / cm 2 , about 30mW / cm 2 , about 40mW / cm 2 , about 60mW / cm 2 , or about 80 mW / cm 2 , or about 100 mW / cm 2 The duration of light application can be 5 minutes to 20 minutes, 5 minutes to 10 minutes, 6 minutes to 8 minutes, about 5 minutes, about 6 minutes, about 8 minutes, about 10 minutes, and about 12 minutes. At lower intensities, longer durations are sufficient, and at higher intensities, shorter durations are sufficient.

[0056] In some variations, the liquid biopolymer mixture can be produced by mixing a dry biopolymer premix of the present disclosure with a photoinitiator of the present disclosure.

[0057] In some variations, the liquid biopolymer mixture may include one or more therapeutic agents suitable for treating corneal disease or injury. Optionally, the one or more therapeutic agents may include therapeutic exosomes or liposomes.

[0058] Corneal diseases include anterior corneal scarring with epithelial and stromal damage / infection (active inflammation), stage 1 neurotrophic keratitis (NK) (persistent corneal epithelial defect), stage 2 NK (large persistent epithelial defect characterized by smooth rounded edges), stage 3 NK (deep corneal ulcer, stromal melting, sterile hypopyon), corneal ulcer such as Mooren's ulcer, keratoconus, corneal perforation, or corneal damage due to dry eye. Corneal diseases can include corneal limbal damage and corneal dystrophies (CD), such as lattice CD type 1, granular CD type 1, and congenital stromal CD, in which the corneal stroma is damaged in the subject. Corneal diseases can include Schneiderian CD or lattice CD type 2, in which both the epithelium and stroma are compromised. EXAMPLES

[0059] Example 1 - Preparation of liquid biopolymer formulations and hydrogels A first exemplary liquid biopolymer formulation ("Formulation 1") was prepared as follows: A dry premix was prepared containing gel-SH (research grade bovine gelatin from Sigma-Aldrich; functionalized by Blafar Ltd.) and HA-MA (HA from Stanford Chemicals; functionalized by Blafar Ltd.) in a 100:60 weight ratio (gel-SH:HA-MA). The gel-SH had an average molecular weight of 50 kDa-100 kDa (about 225 bloom) and an average degree of substitution of about 0.6 mmol / g. The HA-MA had an average molecular weight of about 33 kDa and an average degree of substitution of about 0.81 mmol / g. The gel-SH / HA-MA dry premix was combined with a photoinitiator solution containing eosin Y and triethanolamine in a saline solution. The resulting liquid biopolymer mixture consisted of eosin Y at a concentration of 0.05-0.1 mM, triethanolamine at a concentration of approximately 0.076% w / v, gel-SH at a concentration of approximately 100 mg / ml, and HA-MA at a concentration of approximately 60 mg / ml.

[0060] A second exemplary liquid biopolymer formulation ("Formulation 2") was prepared as follows: A dry premix was prepared containing gel-SH (clinical grade gelatin from Nitta beMatrix; functionalized by Blafar Ltd.) and HA-MA (HA from Stanford Chemicals; functionalized by Blafar Ltd.) in a gel-SH:HA-MA weight ratio of 120:50 (100:60 for gel-SH hydrogel 1). The gel-SH had an average molecular weight of about 100 kDa (250 bloom) and an average degree of substitution of about 0.8 mmol / g. The HA-MA (HA from Stanford Chemicals; functionalized by Blafar Ltd.) had an average molecular weight of about 33 kDa and an average degree of substitution of about 0.81 mmol / g. The gel-SH / HA-MA dry premix was combined with a photoinitiator solution containing eosin Y and triethanolamine in saline. The resulting liquid biopolymer mixture consisted of eosin Y at a concentration of 0.05 mM, triethanolamine at a concentration of approximately 0.076% w / v, gel-SH at a concentration of approximately 120 mg / ml, and HA-MA at a concentration of approximately 50 mg / ml.

[0061] The procedure for preparing the liquid biopolymer mixture is as follows: i. Weigh out the dry (lyophilized) biopolymers (HA-MA and gel-SH) and mix them in the desired weight ratio in a vial. ii. Dissolve the lyophilized biopolymer in saline, spin (2000g, 10 s) within 5 min of adding the saline, and incubate in the dark at 37 °C until the biopolymer is completely dissolved and the solution is clear (approximately 10-15 min). iii. Add the photoinitiator solution to the biopolymer solution.

[0062] Each liquid biopolymer mixture was transformed into a crosslinked hydrogel by exposure to white light. Formulation 1 was approximately 100 mW / cm 2 for about 6 to 8 minutes, and prescription 2 was about 10 mW / cm 2 The cells were exposed to light at an intensity of 100 nm for approximately 10 minutes.

[0063] For a hydrogel to function as an HRC, it is desirable for the gel-SH hydrogel to have physical properties similar to native corneal tissue. The formulation described above has been shown to have physical properties similar to and compatible with native human cornea, as shown in the examples below.

[0064] Example 2 - Testing of Hydrogels for Physical Properties The hydrogels prepared according to Example 1 were then tested for various physical properties, including the following:

[0065] Transparency - Hydrogels were prepared in triplicate in a 96-well plate and their transmittance to visible light was assessed by recording the spectra on a plate reader (Enspire, Perkin Elmer). The absorbance values ​​obtained were converted to transmittance by Beer Lambert's law and plotted against saline as a reference. The average transmittance was determined by averaging the values ​​obtained over the entire spectrum (400-700 nm).

[0066] Compressive modulus - Cylindrical hydrogels with a diameter of 5 mm and height of 1 mm were prepared and tested in a UTS apparatus (BiSS mechanical testing machine) with a parallel plate fixture to evaluate the compressive modulus. Using a 44 N load cell, the hydrogels were compressed up to 50% at a rate of 1 mm / min. The strain and load values ​​were recorded, and the compressive modulus was calculated from the slope of the stress vs. strain curve using the linear region of strain 0.1-0.2 mm / mm.

[0067] Adhesion strength - The adhesion strength of hydrogels to biological tissues was evaluated according to the ASTM F2458-05 standard. Pig skin tissue purchased from a local butcher was thoroughly washed to remove oil and cut into pieces of 2.5 cm × 1.5 cm. The skin tissue pieces were attached to glass slides. An incision was made between two adjacent glass slides, leaving the skin tissue 1 mm apart, and the gap was filled with 75 μl of pregel solution before photocrosslinking. The hydrogels were incubated in saline for equilibration and then adhesion tests were performed on a BiSS UTS instrument. The glass slides were fixed in wedge grips (without disturbing the alignment). The assembly was stretched at a rate of 1 mm / min until adhesive or cohesive failure was observed. The adhesion strength was calculated from the relationship (maximum load / cross-sectional area) × 1000.

[0068] Gravimetric and volumetric swelling - Hydrogel discs were molded into shapes (5 mm diameter, 1 mm height) and incubated in normal saline to assess the weight and volumetric swelling over time. Gravimetric measurements were performed by removing excess saline from the hydrogels and recording the weight at different time points. Gravimetric swelling % was measured using the following relationship: (Wt-Wi) / Wi]×100, where Wt is the weight recorded at different time points and Wi is the initial weight. Volumetric swelling was calculated by measuring the percentage increase in volume when the dimensions were measured using a caliper. Volumetric swelling % was measured using the following relationship: (Vt-Vi) / Vi]×100, where Vt is the volume recorded at different time points and Vi is the initial volume.

[0069] Degradation - The degradation rate of hydrogels was determined by incubating them in 1X phosphate buffered saline (PBS) at ambient temperature with constant shaking (350 rpm) and measuring the change in weight over time. The degradation medium was replenished every other day to avoid saturation, and hydrogel samples were collected at pre-determined time points and their weights recorded after lyophilization at -110 °C for 24 h. The degradation rate was calculated using the following relationship: (Wi-Wt) / Wt] × 100, where Wt is the dry weight at different time points and Wi is the initial dry weight recorded on the day of sample preparation.

[0070] Burst Pressure - The burst pressure of hydrogels, specifically indicative of their ability to withstand intraocular pressure, was measured using graft-rejected cadaveric corneal tissue. The tissue was cleaned and a 2 mm full-thickness punch was drilled in the center of the tissue. The punch was filled with 8 μl of pregel solution and photocrosslinked. The hydrogel-filled cornea was placed in the anterior chamber with a syringe pump. 1X PBS was flowed through the assembly at a constant rate of 0.5 ml / min until the hydrogel either detached from the site or began to leak. Pressure was monitored with a wireless pressure sensor and the burst pressure was determined by calculating the difference between the initial pressure and the final pressure (maximum recorded just before removal).

[0071] Refractive Index - The refractive index was measured using a digital refractometer (Hanna Instruments) where light was passed through a prism in contact with the sample. An image sensor determined the critical angle at which light refracted from the sample.

[0072] The resulting hydrogel exhibited the following physical properties: [Table 1]

[0073] The physical properties above indicate that hydrogels produced according to the above formulations performed as well or better than conventional biopolymers to mimic the properties of the human cornea. In particular, both gel-SH hydrogel formulations 1 and 2 exhibited lower swelling, measured by both weight and volume, had compressive moduli closer to the native cornea, and exhibited less degradation compared to conventional formulations. In addition, unexpectedly, the refractive index of gel-SH hydrogel formulations 1 and 2 was virtually identical to that of the native cornea (1.34 for both formulations compared to 1.37 for the native cornea).

[0074] For aqueous solutions with gel-SH, HA-MA, and photoinitiator, some spontaneous crosslinking is expected even under low light conditions. However, the slow rate of spontaneous crosslinking is advantageous because it allows the mixture to be kept in an uncrosslinked form until application to a desired location, such as a corneal injury site. Surprisingly, Formulation 2 was found to have an advantageously slow rate of spontaneous crosslinking and to be unstable enough for easy application to a corneal injury site until about 15 minutes after the mixture was prepared. In contrast, Formulation 1 spontaneously crosslinked at a faster rate and was found to be unstable enough for easy application to a corneal injury site until about 5 minutes after the mixture was prepared.

[0075] Example 3 - Biocompatibility of hydrogels for hCSSCs For the gel-SH hydrogel to function as HRC and promote scarless tissue regeneration at the corneal wound site, it is important that the hydrogel maintains the viability of corneal stromal stem cells, maintains their phenotype, and gradually acquires a differentiated state while aiding in scarless healing of the wound. Therefore, we tested the biocompatibility of the hydrogel by encapsulating human corneal stromal stem cells (hCSSCs) in the hydrogel and culturing them for 4 weeks. hCSSCs were passaged three times and then cultured on the cover glass surface or within the hydrogel matrix. For cover glass culture, hCSSCs were cultured at 5000 cells / cm. 2 The hCSSCs were mixed in culture medium at a seeding density of 3x10 and added to the wells containing the coverslips. For hydrogel culture, hCSSCs were cultured at a density of 3x10 6 The cells were mixed into a liquid biopolymer formulation at a seeding density of 1000 cells / ml, and the mixture was crosslinked by exposure to light to form a hydrogel. The crosslinked hydrogel served as a culture medium for the hCSSCs encapsulated within it. The hydrogels were incubated at 37°C and 5% CO. 2 The cells were maintained in sterile conditions at 4 °C for 4 weeks. The cells on the coverslips or the cell-injected hydrogels were cultured in culture medium recommended for CSSCs, and the culture medium was changed every 3 days.

[0076] Cell viability was assessed using calcein-AM / ethidium homodimer / DAPI staining for 4 weeks. Figures 1A-1C: Figures 1A-1C show cells grown on cover slip cultures after 1 day (Figure 1A), 2 weeks (Figure 1B), and 4 weeks (Figure 1C). Figures 1D-1F show overhead and stereo views of cells grown in hydrogel cultures based on Formulation 2 of Example 1 after 1 day (Figure 1D), 2 weeks (Figure 1E), and 4 weeks (Figure 1F). As shown in Figures 1D-1F, the cell distribution within the hydrogel was uniform, and more than 80% of the cell population appeared viable after 4 weeks of culture, thereby indicating that the hydrogel is compatible with human corneal stromal cells. Furthermore, compared to hCSSCs grown on cover slips,

[0077] The phenotype of the cells was assessed using CD90 / αSMA / DAPI staining. CD90 is a biomarker for stromal stem cells, while expression of αSMA by the cells reflects their differentiation state into keratocytes or myofibroblasts. Figures 2A-2C are overhead views of cells grown in cover slip cultures after 1 day (Figure 2A), 2 weeks (Figure 2B), and 4 weeks (Figure 2C) of culture. Figures 2D-2F are overhead and 3D views of cells grown in hydrogel cultures based on Formulation 2 of Example 1 after 1 day (Figure 2D), 2 weeks (Figure 2E), and 4 weeks (Figure 2F) of culture. The staining shows that stromal stem cells initially maintained the phenotype of stromal stem cells, as indicated by CD90 staining, and gradually transitioned to a differentiated state, as indicated by increased αSMA staining over 4 weeks.

[0078] Example 4 - Biocompatibility of hydrogels for hCECs For the gel-SH hydrogel to function as an HRC and provide scar-free tissue regeneration at the corneal wound site, it is also important that the hydrogel allows for the growth of human corneal epithelial cells (hCECs). We evaluated whether the gel-SH hydrogel (formulation 2) provides a supportive surface for epithelial cell growth by seeding primary hCECs on the hydrogel and allowing monolayer formation. The hCEC suspension was then incubated for 10 min at 4°C for 1 h. 5 cells / cm 2The cells were seeded directly onto the hydrogel surface or onto the cover slip control surface at a seeding density of 100 μg / mL. The cells were cultured at 37°C and 5% CO 2 The cells were maintained in sterile conditions at 4 °C for 1 h and monitored daily for 1 week, during which time cell confluency was assessed using phase contrast imaging and staining for a tight junction marker (Zona occludens - ZO1) along with DAPI and rhodamine-conjugated phalloidin.

[0079] Figures 3A-3B are phase contrast images of hCEC cells grown on cover slip cultures after 4 days, comparing hCECs grown on cover slip (Figure 3A) and hydrogel (Figure 3B) surfaces. This comparison shows that hCEC growth was more robust on the hydrogel surface, reaching full confluence by 4 days on the hydrogel surface but not on the cover slip surface.

[0080] Figures 4A-4C show hCEC cells grown on cover slip surfaces and stained with DAPI (Figure 4A), ZO1 (Figure 4B), and rhodamine-phalloidin (Figure 4C). Figures 4D-4F show hCEC cells grown on hydrogel surfaces based on Formulation 2 of Example 1 and stained with DAPI (Figure 4D), ZO1 (Figure 4E), and rhodamine-phalloidin (Figure 4F). Comparison of ZO1 staining indicates that growth on hydrogel surfaces promoted hCEC tight junction formation by day 4 of culture, whereas growth on cover slips did not.

Claims

[Claim 1] The inventions described herein or in the drawings.

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