hydrogel

Abstract

A composition comprising a crosslinked polymer and horseradish peroxidase (HRP).

Description

ML Ref. 135156-5001HYDROGELCROSS-REFERENCED TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 728,591, filed on December 5, 2024, which is incorporated herein by reference in their entireties.BACKGROUND

[0002] The present disclosure relates to a composition or hydrogel for delivering a therapeutic agent to an eye, a drug delivery system involving a composition or hydrogels for various medical conditions.

[0003] At present, at least 2.2 billion people around the world have a vision impairment, eventually leading to visual impairment or complete blindness. Conventional treatment involves the use of eye drops, pharmacological or surgical treatments. However, these formulations often confer low ocular bioavailability and frequent dosing is required. Therefore, there is an urgent need to develop more effective drug delivery systems to tackle the current limitations.

[0004] A hydrogel is a gel-like material composed of a network of a crosslinked polymer chains that can hold large amounts of water, allowing it to encapsulate and deliver drugs in a controlled manner to specific target areas within the body. Given that hydrogels exhibit excellent biocompatibility and their mechanical properties can be designed in such a way that they match those of many soft biological tissues, they have been used for delivery of drugs into the anterior and posterior segments of the eye. Injectable hydrogel systems serve as a mimic of the extracellular matrix, a bioactive mediator of cellular proliferation, and a celladhesive domain for cell, surface receptors. See Zhu et al., Design properties of hydrogelML Ref. 135156-5001 tissue-engineering scaffolds, Expert Rev Med Devices 8, 607, 2011; and Lutolf et al., Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol 23, 47, 2005.SUMMARY

[0005] In one aspect, the present disclosure provides a composition comprising a crosslinked polymer and horseradish peroxidase (HRP), and a method of storing the composition thereof. The composition, in one embodiment, may be for delivering a therapeutic agent to a subject in need thereof.

[0006] The present disclosure also provides a method of preparing the composition comprising crosslinking gelatin-hydroxyphenyl propionic acid (Gtn-HPA) and / or hyaluronic acid tyramine (HA-Tyr) with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) in a solution. The present disclosure also provides, in one embodiment, a method of delivering a therapeutic agent to an eye in a subject, the method comprising administering the therapeutic agent in the composition comprising a crosslinked polymer and horseradish peroxidase (HRP). The present disclosure also provides, in one embodiment, a kit comprising a polymer and a crosslinking agent. The present disclosure also provides a composition or hydrogel which is injectable for drug delivery.

[0007] The composition of the present disclosure may mimic animal vitreous and exhibit a biomimetic hydrogel matrix, enabling in vivo delivery of drugs such as retinal neurons via, such as, intravitreal and subretinal injections. Post-injection, the composition of the present disclosure may adjust its stiffness and degradation timeline within the subject’s body. By mimicking animal vitreous, such as human vitreous, the hydrogel may undergo enzymatic degradation through collagenase and hyaluronidase present in the subject’s body, such as eye, ensuring a promising safety profile as the metabolites are continuously cleared by the humanML Ref. 135156-5001 body.

[0008] The embodiments of the present disclosure are further illustrated by the following detailed description and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate exemplary' embodiments and, together with the description, serve to explain the disclosed principles.

[0010] FIGURE 1 A is a graph showing the results of a gelation time (min) with storage temperature ofIGTOOl.

[0011] FIGURE IB is a graph showing the results of a gelation time (min) with storage temperature of a composition having 0.1 U / mL of HRP and 1.5 mM of H2O2.

[0012] FIGURE 1C is a graph showing the results of a gelation time (min) with storage temperature of a composition having 0.1 U / mL of HRP and 2 mM of H2O2.

[0013] FIGURE ID is a graph showing the results of a gelation time (min) with storage temperature of a composition having 0.05 U / mL of HRP and 1 mM of H2O2.

[0014] FIGURE IE is a graph showing the results of a gelation time (min) with storage temperature of a composition having 0.13 U / mL of HRP and 1 mM of H2O2.

[0015] FIGURE 2A is a graph showing the final residual of H2C>2(mM) with initial concentration of Gel-HA (mM).

[0016] FIGURE 2B is a graph showing the final residual of HRP (U / mL) with initial concentration of Gel-HA (mM).DETAILED DESCRIPTION

[0017] The delivery of therapeutic agents, for example, to sensitive areas such as theML Ref. 135156-5001 eye, presents significant challenges in terms of biocompatibility, controlled release, and ease of handling. Traditional drug delivery systems often fail to mimic the natural environment of the target site, leading to suboptimal therapeutic outcomes and potential adverse effects. There is a need for an injectable hydrogel composition that is easy to handle and can effectively deliver therapeutic agents while mimicking the natural vitreous of the eye, ensuring biocompatibility and controlled degradation.

[0018] The present disclosure may address these challenges by providing an injectable hydrogel composition comprising a crosslinked polymer and horseradish peroxidase (HRP), which forms a biomimetic hydrogel matrix. This composition may be designed to mimic the animal vitreous, allowing for safe and effective intravitreal and subretinal drug delivery. The hydrogel’s ability to adjust its stiffness and degradation timeline post-injection, along with its enzymatic degradation through naturally occurring enzymes in the body, ensures a promising safety profile and effective clearance of metabolites.

[0019] Hereinafter, the present disclosure will be described in more detail to help the understanding of the present disclosure.

[0020] As used herein and unless otherwise indicated, “wt %” refers to a weight percent based on a total weight of a reference unless otherwise explained.

[0021] As used herein and unless otherwise indicated, “mM” refers to millimoles per liter (mmol / L).

[0022] As used herein and unless otherwise indicated, “U / mL” refers to units per milliliter.

[0023] When the term "about'’ is used, it is used to mean a certain effect or result can be obtained within a certain tolerance, and the skilled person knows how to obtain the tolerance. When the term “about” is used in describing a value or an end-point of a range, the disclosure should be understood to include the specific value or end-point referred to. In oneML Ref. 135156-5001 aspect, the term “about” means plus or minus 20% of the numerical value of the number with which it is being used.

[0024] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having” or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0025] The transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim, closing the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0026] The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed embodiment. A “consisting essentially of’ claim occupies a middle ground between closed claims that are written in a “consisting of’ format and fully open claims that are drafted in a “comprising” format. Optional additives as defined herein, at a level that is appropriate for such additives, and minor impurities are not excluded from a composition by the term “consisting essentially of’.

[0027] Further, unless expressly stated to the contrary, “or” and “and / or” refers to an inclusive and not to an exclusive. For example, a condition A or B, or A and / or B, is satisfied by any one of the following: A is true (or present) and B is false (or not present). A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0028] The use of “a” or “an” to describe the various elements and components hereinML Ref. 135156-5001 is merely for convenience and to give a general sense of the disclosure. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.

[0029] For convenience, many elements of the present embodiments are discussed separately, lists of options may be provided and numerical values may be in ranges; however, for the purposes of the present disclosure, that should not be considered as a limitation on the scope of the disclosure or support of the present disclosure for any claim of any combination of any such separate components, list items or ranges. Unless stated otherwise, each and every' combination possible with the present disclosure should be considered as explicitly disclosed for all purposes.

[0030] In one aspect, the present disclosure relates to a composition comprising a crosslinked polymer and horseradish peroxidase (HRP). In some embodiments, a composition for delivering a therapeutic agent to an eye may comprise a crosslinked gelatin and horseradish peroxidase (HRP). In some embodiments, a composition for delivering a therapeutic agent to an eye may comprise a crosslinked hyaluronic acid (HA) and horseradish peroxidase (HRP). As used herein and unless otherwise indicated, the term “horseradish peroxidase “ or “HRP” may include an enzyme isolated from the roots of the horseradish plant. HRP can catalyze the oxidation of various substrates using hydrogen peroxide as an electron acceptor, leading to the formation of reactive intermediates. Therefore, HRP can act as a catalyst to crosslink tissue-engineered hydrogels due to its mild reaction conditions and ability to modulate the mechanical properties of the matrix. In some embodiments, the crosslinked polymer included in the composition of the present disclosure is prepared by crosslinking a polymer with HRP as a crosslinking agent.

[0031] In some embodiments, the composition may comprise a crosslinked polymer. As used herein and unless otherwise indicated, the term “crosslinked polymer” may include aML Ref. 135156-5001 polymer in which one polymer chain and another polymer chain are connected via one or more linking groups. In some embodiment, the crosslinked polymer of the present disclosure has a structure including hyaluronic acid (HA) and / or gelatin as a main body or a polymer backbone crosslinked with HRP and H2O2 forming networks via enzy matic oxidation, creating covalent bonds between modified hyaluronic acid or modified gelatin, which often tyramine-conjugated, and phenolic groups, resulting in tunable, fast-gelling, biocompatible structures ideal for tissue engineering and drug delivery.

[0032] As used herein and unless otherwise indicated, the term “polymer” may include a molecule composed of repeating structural units linked by covalent chemical bonds and characterized by a large number of repeating units (e.g., equal to or greater than 10 repeating units, and often equal to or greater than 50 repeating units, and often equal to or greater than 100 repeating units) and a high molecular weight. The polymer is typically the polymerization product of one or more monomer precursors. The term polymer includes homopolymers, or polymers consisting essentially of single repeating monomer subunits. The term polymer also includes copolymers, which are formed when two or more different types of monomers are linked in the same polymer. Copolymers may contain two or more monomeric subunits and include random, block, alternating, segmented, grafted, tapered, and other copolymers.

[0033] In some embodiments, the composition may be for delivering a therapeutic agent to an eye. In some embodiments, the composition may be used for delivering a therapeutic agent to a subject or a part of a subject’s body, such as a human eye.

[0034] In some embodiment, the composition may be a hydrogel. As used herein and unless otherwise indicated, the term “hydrogel” may include a substance formed when a polymer becomes a three dimensional open-lattice structure that entraps solution molecules, typical ly water, to form a gel. The three-dimensional molecular network may be heldML Ref. 135156-5001 together by covalent chemical bonds, or by ionic bonds, or by any combination thereof. A polymer may form a hydrogel by, for example, aggregation, coagulation, hydrophobic interactions, cross-linking, salt bridges, etc. Where a hydrogel is to be used as part of a scaffold onto which cells will be seeded, the hydrogel should be non-toxic to the cells.

[0035] In some embodiment, the crosslinked polymer may have a gelation time from 1 minutes to 5 minutes when a hydrogel composition is stored at a room temperature. As used herein and unless otherwise indicated, the term “gelation” may include formation of a gel from a sol. A gelation is the phase transition that a polymer or a crosslinked polymer undergoes when it increases in viscosity and transforms from a fluid state into a semi-solid material, or gel. The gelation time of the crosslinked polymer of the present disclosure may vary depending on the concentrations of HRP and / or H2O2 used as crosslinking agents. The gelation time of the crosslinked polymer of the present disclosure may vary depending on storage temperature of the crosslinked polymer of the present disclosure. In some embodiments, stiffness and degradation rate of the crosslinked polymer may change along with the gelation time.

[0036] In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time from 1 minutes to 5 minutes when a hydrogel is stored at a room temperature. In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time less than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute when store at a room temperature (20 °C). In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time less than 55, 50, 45, 40, 30, 20, 10. 5, 4, 3, 2, or 1 second when store at a room temperature (20 °C). In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time more than 1, 2, 3. 4, 5, 6. 7, 8, 9. 10, 15, 20, 25. 30, 40, 50, or 55 seconds when store at a room temperature (20 °C). In some embodiments, the crosslinked polymer of the present disclosure may have a gelation timeML Ref. 135156-5001 more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 minutes when store at a room temperature (20 °C). In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time from about 10 seconds to about 20 minutes, from about 10 seconds to about 10 minutes, from about 10 seconds to about 5 minutes, from about 10 seconds to about 4 minutes, from about 10 seconds to about 3 minutes, from about 10 seconds to about 2 minutes, from about 10 seconds to about 1 minute, from about 20 seconds to about 20 minutes, from about 20 seconds to about 10 minutes, from about 20 seconds to about 5 minutes, from about 20 seconds to about 4 minutes, from about 20 seconds to about 3 minutes, from about 20 seconds to about 2 minutes, from about 20 seconds to about 1 minute, from about 30 seconds to about 20 minutes, from about 30 seconds to about 10 minutes, from about 30 seconds to about 5 minutes, from about 30 seconds to about 4 minutes, from about 30 seconds to about 3 minutes, from about 30 seconds to about 2 minutes, from about 30 seconds to about 1 minute, from about 1 minute to about 20 minutes, from about 1 minute to about 10 minutes, from about 1 minute to about 5 minutes, from about 1 minute to about 4 minutes, from about 1 minute to about 3 minutes, or from about 1 minute to about 2 minutes when store at a room temperature (20 °C).

[0037] In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time less than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute when store at a body temperature (37 °C). In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time less than 55, 50, 45, 40, 30, 20, 10, 5, 4, 3, 2, or 1 second when store at a body temperature (37 °C). In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time more than 1, 2, 3. 4, 5, 6, 7, 8, 9, 10, 15, 20, 25. 30, 40, 50, or 55 seconds when store at a body temperature (37 °C). In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time more than 1, 2. 3, 4, 5. 6, 7, 8, 9. 10. 15. or 20 minutes when store at a body temperature (37ML Ref. 135156-5001°C). In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time from about 10 seconds to about 20 minutes, from about 10 seconds to about 10 minutes, from about 10 seconds to about 5 minutes, from about 10 seconds to about 4 minutes, from about 10 seconds to about 3 minutes, from about 10 seconds to about 2 minutes, from about 10 seconds to about 1 minute, from about 20 seconds to about 20 minutes, from about 20 seconds to about 10 minutes, from about 20 seconds to about 5 minutes, from about 20 seconds to about 4 minutes, from about 20 seconds to about 3 minutes, from about 20 seconds to about 2 minutes, from about 20 seconds to about 1 minute, from about 30 seconds to about 20 minutes, from about 30 seconds to about 10 minutes, from about 30 seconds to about 5 minutes, from about 30 seconds to about 4 minutes, from about 30 seconds to about 3 minutes, from about 30 seconds to about 2 minutes, from about 30 seconds to about 1 minute, from about 1 minute to about 20 minutes, from about 1 minute to about 10 minutes, from about 1 minute to about 5 minutes, from about 1 minute to about 4 minutes, from about 1 minute to about 3 minutes, or from about 1 minute to about 2 minutes when store at a room temperature (37 °C).

[0038] In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time less than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute when store at an ice temperature (2-8 °C). In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time less than 55, 50, 45, 40, 30, 20, 10, 5, 4, 3, 2, or 1 second when store at a body temperature (2-8 °C). In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time more than 1, 2, 3. 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 55 seconds when store at a body temperature (2-8 °C). In some embodiments, the crosslinked polymer of the present disclosure may have a gelation time more than 1, 2. 3, 4, 5. 6, 7, 8, 9. 10. 15. or 20 minutes when store at a body temperature (2- 8 °C). In some embodiments, the crosslinked polymer of the present disclosure may have aML Ref. 135156-5001 gelation time from about 10 seconds to about 20 minutes, from about 10 seconds to about 10 minutes, from about 10 seconds to about 5 minutes, from about 10 seconds to about 4 minutes, from about 10 seconds to about 3 minutes, from about 10 seconds to about 2 minutes, from about 10 seconds to about 1 minute, from about 20 seconds to about 20 minutes, from about 20 seconds to about 10 minutes, from about 20 seconds to about 5 minutes, from about 20 seconds to about 4 minutes, from about 20 seconds to about 3 minutes, from about 20 seconds to about 2 minutes, from about 20 seconds to about 1 minute, from about 30 seconds to about 20 minutes, from about 30 seconds to about 10 minutes, from about 30 seconds to about 5 minutes, from about 30 seconds to about 4 minutes, from about 30 seconds to about 3 minutes, from about 30 seconds to about 2 minutes, from about 30 seconds to about 1 minute, from about 1 minute to about 20 minutes, from about 1 minute to about 10 minutes, from about 1 minute to about 5 minutes, from about 1 minute to about 4 minutes, from about 1 minute to about 3 minutes, or from about 1 minute to about 2 minutes when store at a room temperature (37 °C).

[0039] In some embodiments, the composition of the present disclosure may include FbChat a concentration less than about 0.0001 mM. As used herein and unless otherwise indicated, the term “H2O2” refers to hydrogen peroxide. H2O2 is widely used for the gelation of aqueous solutions of gelatin derivatives with phenolic hydroxyl groups (Gelatin-Ph) catalyzed by horseradish peroxidase (HRP). H2O2 is known to cause degradation and / or depolymerization of various polymers. The composition of the present disclosure may include H2O2 as a residual as a crosslinking agent used for preparing the crosslinking network of the crosslinked polymer.

[0040] In some embodiments, the composition of the present disclosure may include tbChat a concentration of 10, 9. 8, 7, 6, 5, 4, 3, 2. 1.5, 1, 0.5, 0.3. 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005. 0.003, 0.002. 0.001, 0.0005, 0.0003. 0.0002. 0.0001,ML Ref. 135156-50010.00005, or 0.00001 mM or less. In some embodiments, the composition of the present disclosure may include H2O2 at a concentration of 0.00001, 0.00002, 0.00003, 0.00004, 0.00005, 0.0001, 0.0002, 0.0003, 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mM or more. In some embodiments, the composition of the present disclosure may include H2O2 at a concentration from about 0.0001 to about 2, from about 0.0001 to about 1.5, from about 0.0001 to about 1, from about 0.0002 to about 2, from about 0.0002 to about 1.5, from about 0.0002 to about 1, from about 0.0002 to about 0.5, from about 0.0002 to about 0.1, from about 0.0002 to about 0.05, from about 0.0002 to about 0.01, from about 0.0004 to about 2, from about 0.0004 to about 1.5, from about 0.0004 to about 1, from about 0.0004 to about 0.5, from about 0.0004 to about 0.1, from about 0.0004 to about 0.05, from about 0.0004 to about 0.01, from about 0.02 to about 0.1, from about 0.02 to about 2, from about 0.02 to about 1.5, from about 0.02 to about 1, from about 0.02 to about 0.1, from about 0.02 to about 0.08, from about 0.02 to about 0.06, from about 0.02 to about 0.05, from about 0.02 to about 0.04, from about 0.04 to about 2, from about 0.04 to about 1 , from about 0.04 to about 0.5, from about 0.04 to about 0.1, from about 0.04 to about 0.08, from about 0.04 to about 0.06, from about 0.04 to about 0.05, from about 0.05 to about 2, from about 0.05 to about 1.5, from about 0.05 to about 1, from about 0.05 to about 0.1, from about 0.05 to about 0.08, from about 0.05 to about 0.06, from about 0.07 to about 2, from about 0.07 to about 1, from about 0.07 to about 0. 14, from about 0.07 to about 0.12. from about 0.07 to about 0.1, from about 0.1 to about 10, from about 0. 1 to about 5, from about 0. 1 to about 3, from about 0. 1 to about 1, from about 0.5 to about 10. from about 0.5 to about 5, from about 0.5 to about 1, from about 0.8 to about 10. from about 0.8 to about 5, from about 0.8 to about 2, from about 0.8 to about 1.5, from about 0.8 to about 1, from about 1 to about 10. from about 1 to about 5. from about 1 to about 3,ML Ref. 135156-5001 from about 1 to about 2, or from about 1 to about 1.5 mM.

[0041] In some embodiment, the composition of the present disclosure may include HRP at a concentration from about 0.0002 to about 1 U / mL. In some embodiments, the composition may include HRP at a concentration from about 0.004 to about 1 U / mL. In some embodiments, the composition of the present disclosure may include HRP at a concentration of 0.0001, 0.0002, 0.0003, 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 U / mL or more. In some embodiments, the composition of the present disclosure may include HRP at a concentration of 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01 U / mL or less. In some embodiments, the composition of the present disclosure may include HRP at a concentration from about 0.0001 to about 2, from about 0.0001 to about 1, from about 0.0002 to about 2, from about 0.0002 to about 1, from about 0.0002 to about 0.5, from about 0.0002 to about 0.1, from about 0.0002 to about 0.05, from about 0.0002 to about 0.01, from about 0.0004 to about 1, from about 0.0004 to about 0.5, from about 0.0004 to about 0.1, from about 0.0004 to about 0.05, from about 0.0004 to about 0.01 , from about 0.02 to about 0.1 , from about 0.02 to about 0.08, from about 0.02 to about 0.06, from about 0.02 to about 0.05, from about 0.02 to about 0.04, from about 0.04 to about 0.1, from about 0.04 to about 0.08, from about 0.04 to about 0.06, from about 0.04 to about 0.05, from about 0.05 to about 0.1, from about 0.05 to about 0.08, from about 0.05 to about 0.06, from about 0.07 to about 0.14, from about 0.07 to about 0. 12, from about 0.07 to about 0. 1 U / mL.

[0042] In some embodiments, the concentration of H2O2 may be a residual amount of H2O2 in the composition after crosslinking a polymer to produce the crosslinked polymer. In some embodiment, the concentration of HRP may be a residual amount of HRP in the composition after crosslinking a polymer to produce the crosslinked polymer. In someML Ref. 135156-5001 embodiments, the term “residual amount” may include an amount of less than 5 U / mL. In some embodiment, the term “residual amount” may include an amount of less than about 0.0001 mM. In some embodiments, the residual may be a substance that remains after a process, like crosslinking reaction, or a leftover part of a reagent not fully used up, often indicating incomplete conversion, contamination, or a necessary' leftover for a function.

[0043] In some embodiments, the crosslinked polymer may be hydrophilic. In some embodiments, the crosslinked polymer may be hydrophobic. As used herein and unless otherwise indicated, the term “hydrophilic” may include a property having a strong affinity for water. As used herein and unless otherwise indicated, the term “hydrophobic” may include a property having a water repellency. As the crosslinked polymer of the present disclosure may have semisolid hydrophilic polymer network like hydrogel, and may absorb large amount of water molecules and swell while maintaining their network structured due to physical or chemical crosslinking of unique polymer chains. Accordingly, water content of the hydrophilic crosslinked polymer may have high water content as high as 90-99% with good biocompatibility and biodegradability and low cytotoxicity. Accordingly, the crosslinked polymer of the present disclosure may provide cells with a suitable microenvironment similar to extracellular matrix to play a positive role in drug delivery, cell migration, adhesion, proliferation and differentiation.

[0044] In some embodiment, the crosslinked polymer of the present disclosure may comprise gelatin. As used herein and unless otherwise indicated, the term “gelatin” means gelatin and derivatives thereof and may include any mixture of peptides and proteins produced by the partial hydrolysis of collagen. In some embodiments, gelatin may include natural gelatin made from naturally derived collagen. In some embodiments, gelatin may include chemically synthesized peptide or chemically synthesized gelatin, which is an artificially synthesized peptide or gelatin. In some embodiment, a molecular weight of theML Ref. 135156-5001 gelatin may be in the approximate range of 5 kDa to 100 kDa. In some embodiment, a molecular weight of the gelatin may be in the approximate range from 4, 5, 6, 7, 8, 9, 10. 20. 30, 40, 50, 60, 70, 80, or more 90 kDa or more and 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3 or 2 kDa or less. In some embodiments, the crosslinked polymer of the present disclosure may comprise gelatin which may be crosslinked and / or not crosslinked. In some embodiments, gelatin may be crosslinked with horseradish peroxidase (HRP) and / or H2O2. In some embodiments, the composition for delivering a therapeutic agent may comprise gelatin which may be crosslinked and / or not crosslinked.

[0045] In some embodiments, the crosslinked polymer may comprise gelatin including a side chain having a substituted or unsubstituted C6 to C20 aryl group, a linear or branched substituted or unsubstituted Cl to C8 alkyd group, a hydroxyl group, amino group and / or a carboxylic acid. As used herein and unless otherwise indicated, the term ‘'side chain” refers a chemical group or structure that is attached to a core part of the molecule called the “main chain” or backbone. As used herein and unless otherwise indicated, the term “substituted” refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent selected from the group consisting of alkyl, alkoxy, cycloalkoxy, alkenyl, alkynyl, aryl, arylalkyl, COOH, CHO, oxyacyl, acyloxy, cycloacyloxy, phenol, phenoxy, pyridinyl, pyrrolidinyL amino, amido, hydroxy, F, CL Br, NO2, cyano, sulfuryl, and the like. As used herein and unless otherwise indicated, the term “unsubstituted” means that for carbon atoms, only hydrogen atoms are present in addition to the valences connecting the atoms to the parent molecular group.

[0046] Examples of the C6 to C20 aryl group may include phenyl, 1 -naphthyl. 2- naphthyl. 1-anthryl, 2-anthryL 9-anthryl, 1 -phenanthryl. 2-phenanthryl. 3 -phenanthryl. 4- phenanthryl, 9-phenanthryl, 1 -naphthacenyl, 2-naphthacenyl, 9-naphthacenyl, 1-pyrenyl. 2- pyrenyl, 4-pyrenyl, 2-biphenylyl, 3-biphenylyl, 4-biphenylyl. p-terphenyl-4-yl. p-terphenyl-3-ML Ref. 135156-5001 yl, p-terphenyl-2-yl, m-terphenyl-4-yl, m-terphenyl-3-yl, m-terphenyl-2-yl, o-tolyl, m-tolyl, p-tolyl, p-t-butylphenyl, p-(2-phenylpropyl)phenyl, 3-methyl-2-naphthyl, 4-methyl-l- naphthyl, 4-methyl-l -anthryl, 4-methylbiphenylyl, 4-t-buty l-p-terphenyl-4-yl, fluoranthenyl, fluorenyl and the like. Examples of the Cl to C8 alkyl group include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-buty l, tert-butyl, n-pentyl, isopenty l, neopentyl, cyclopenty l, n-hexyl, isohexyl, cyclohexyl, heptyl, cy cloheptyd, octyd, cyclooctyd, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl 1, pentadecyd, hexadecyl, heptadecyl, octadecyl, nonadecyd, eicosyl and so on. In the present disclosure, unless otherwise specified, examples of' "substituent" include halogen, hydroxy, cyano, nitro, alkyd, haloalky 1, hydroxyalkyd, alkoxyalkyd, acyl, acyloxy, oxy, carboxy, alkoxycarbonyl, aralkoxycarbonyl, carbamoyl.When the above-mentioned substituents are present, the number thereof is usually one, two or three.

[0047] In some embodiments, the crosslinked polymer may7comprise gelatin including hydroxyphenyl propionic acid as a side group in an amount of about 0. 1 to about 10 % of a monomer unit of the gelatin or about 0. 1 to about 10 wt% of the gelatin. As used herein and unless otherwise indicated, the term “hydroxyphenyl propionic acid” or “HP A” refers to 3-(4-hydroxyphenyl)propionic acid, also known as phloretic acid, and 3-(2- hydroxyphenyl)propionic acid. In some embodiment, gelatin including hydroxyphenyl propionic acid as a side group may have a structure that HPA is attached to gelatin to create gelatin conjugated with hydroxyphenyl propionic acid (Gtn-HPA) hydrogels. In some embodiments, gelatin may include Gtn-HPA.

[0048] In some embodiments, the gelatin may include hydroxyphenyl propionic acid as a side group in an amount of about 0.1. 0.2. 0.3, 0.4, 0.5. 0.6, 0.7, 0.8, 0.9. 1, 2, 3, 4. 5, 6, 7, 8 or 9% or more and / or about 10, 9. 8, 7, 6. 5, 4, 3, 2. 1, 0.9, 0.8. 0.7. 0.6, 0.5, 0.4. 0.3, or 0.2 % or less of a monomer unit of the gelatin. In some embodiments, the gelatin mayML Ref. 135156-5001 include hydroxyphenyl propionic acid as a side group in an amount of about .1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8 or 9% or more and / or about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2 % or less of the gelatin.

[0049] In some embodiments, the crosslinked polymer may comprise gelatin crosslinked with a crosslinking agent. In some embodiments, the crosslinked polymer may comprise crosslinked gelatin crosslinked with a crosslinking agent. As used herein and unless otherwise indicated, the term “crosslinking agent” may include a composition and / or compound used to facilitate forming crosslinks between separate polymer molecules or between separate monomer sites on the same polymer molecule. Some crosslinking agents may, for example, be a catalyst that is not covalently incorporated into the polymer molecule but merely increases a crosslinking rate and / or lowers an energy requirement for a crosslinking reaction. Some crosslinking agents may be directly incorporated, at least in part, within a covalent link between polymer molecules or between separate monomer sites on the same polymer molecule. Some cross-linking agents may have two or more functional groups at their ends that chemically bond carbon atoms from different polymer chains. This process transforms linear segments into a three-dimensional network of connected molecules.

[0050] In some embodiment, the crosslinked polymer of the present disclosure may comprise hyaluronic acid (HA). As used herein and unless otherwise indicated, the term the term “hyaluronic acid” or “HA” means a polymer of hyaluronic acid, where different HA chains are covalently linked by a crosslinker to form a 3-dimensional polymer network. In some embodiment, hyaluronic acid (HA) may be a linear copolymer consisting of (P-1,4) linked D-glucuronate (D) and (|3-l,3)-N-acetyl-D-glucosamine (N). In some embodiments, hyaluronic acid (HA) may have a molecular weight in a range from about 5 kDa to 20.000 kDa. In some embodiments. HA may have a molecular weight about 5, 6, 7. 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1.000, 5,000, 10,000. 11.000, 12,000, 13,000, 14,000,ML Ref. 135156-500115,000. 16,000, 17,000, 18,000, 19,000 kDa or more, and / or about 20,000, 19,000, 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, 10,000, 5,000, 1,000, 500, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7 or 6 kDa or less.

[0051] HA may be derivatives of hyaluronic acid that include esters of hyaluronic acid, salts of hyaluronic acid and also includes the term hyaluronan. Hyaluronic acid may have a molecular weight of less than 10 kDa. HA is a biodegradable, biocompatible, nontoxic, non-immunogenic and non-inflammatorv linear polysaccharide.

[0052] In some embodiment, the crosslinked polymer of the present disclosure may comprise HA, and HA may be crosslinked with a crosslinking agent. In some embodiments, the crosslinked polymer may be hyaluronic acid (HA). In some embodiments, the crosslinked polymer may comprise a crosslinked hyaluronic acid (HA) crosslinked with a crosslinking agent. In some embodiments, the composition for delivering a therapeutic agent may comprise hyaluronic acid (HA) which may be crosslinked and / or not crosslinked. In some embodiments, hyaluronic acid (HA) may include hyaluronic acid tyramine (HA-Tyr). In some embodiments, hyaluronic acid (HA) may be crosslinked with horseradish peroxidase (HRP) and / or H2O2.

[0053] In some embodiment, the crosslinked polymer of the present disclosure may comprise HA including a side chain having a C6 to C20 aryl group, a linear or branched Cl to C8 alkyl group, a hydroxyl group, an amino group and / or a carboxylic acid. In some embodiments, the crosslinked polymer of the present disclosure may comprise HA including tyramine as a side chain in an amount of about 10 to about 90 % of a monomer unit of HA or about 10 to about 90 wt% of HA. HA including tyramine as a side chain may include hyaluronic acid-tyramine (HA-Tyr) conjugates or HA-Tyr hydrogel which may be prepared by a radical crosslinking reaction with H2O2 and HRP. In some embodiments, an amount of HA including tyramine as a side chain in the crosslinked polymer may be about 10, 20, 30,ML Ref. 135156-500140, 50, 60, 70, or 80 wt% or more and / or about 90, 80, 70, 60, 50, 40, 30 or 20 wt% or less.

[0054] In some embodiment, the crosslinked polymer comprises a biopolymer. In some embodiments, the crosslinked polymer may be prepared by crosslinking a biopolymer with a crosslinking agent. As used herein and unless otherwise indicated, the term “biopolymer’ may include a polymeric substance derived from a biological source. The biopolymer may include at least one selected from the group consisting of a starch, a chitosan, a polysaccharide, a protein, a gelatin, a biopolyesters and modifications and mixtures thereof. A biopolymer may be synthetically obtained (e.g., through laboratory synthesis) and / or obtained and / or derived from nature (e.g., from a living or previously living organism). In one embodiment, the biopolymer is the same as a polymer found in nature (i.e., a native biopolymer) or is a derivative thereof. In another embodiment, the biopolymer is a derivative of a polymer produced by a living organism, the derivative caused by the synthetic method used to obtain or isolate the biopolymer from nature. In yet another embodiment, the biopolymer is a polymer produced by bacteria and / or microbes.

[0055] In some embodiments, the crosslinking agent of the present disclosure may comprise at least one selected from the group consisting of horseradish peroxidase (HRP), hydrogen peroxide (H2O2), calcium ions (CaCL), thrombin, tripolyphosphate, transglutaminase, carbodiimide, genipin, epichlorohydrin, glutaraldehyde, sodium periodate, permanganate, tyrosinase, a pH adjusting agent such as sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, and combinations thereof. In some embodiments, an amount of crosslinker / crosslinking agent in the present disclosure may be in a range from about 0. 1 mM to about 30 mM. In some embodiments, an amount of crosslinker / crosslinking agent is at least about 0. 1, 0.5, 1, 2. 3, 4, 5. 6, 7, 8. 9, 10. 15. 20 or 25 or more and / or about 30. 25. 20. 15, 10, 9. 8, 7, 6. 5, 4, 3, 2. 1, 0.5 mM or less. In someML Ref. 135156-5001 embodiments, an amount of crosslinker / crosslinking agent may be in a range from about 0.01 U / mL to about 1 U / mL. In some embodiments, an amount of crosslinker / crosslinking agent may be at least about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4 0.5, 0.6, 0.7, 0.8, 0.9 U / mL or more and / or about 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 or 0.02 U / mL or less.

[0056] In some embodiments, the crosslinked polymer of the present disclosure may be crosslinked via a chemical crosslinking, a UV crosslinking, a photocros slinking, an enzymatic crosslinking, a thermal crosslinking, an ionic crosslinking, a physical crosslinking or combinations thereof. As used herein and unless otherwise indicated, the term “chemical crosslinking” may include forming strong, stable covalent bonds between polymer chains or molecules (like proteins, peptides) using chemical agents (crosslinkers) to create a robust, interconnected 3D network structure, significantly enhancing properties like strength, stability, and resistance to swelling, compared to weaker physical interactions. As used herein and unless otherwise indicated, the term “UV crosslinking” may include the process where ultraviolet (UV) light is used to initiate a chemical reaction that bonds polymers together, creating a more robust and durable material. As used herein and unless otherwise indicated, the term “photocrosslinking” may include chemical crosslinking triggered by a photoinitiator allowing the hydrogel to form in situ at physiological pH and room temperature without using toxic chemical crosslinkers. As used herein and unless otherwise indicated, the term "enzymatic crosslinking” may include a polymerization route that relies on enzymes as reagents to cleave or form covalent bonds. As used herein and unless otherwise indicated, the term “thermal crosslinking” may include a method that utilizes heat to initiate chemical reactions in polymers, leading to the creation of stable microspheres. As used herein and unless otherwise indicated, the term “ionic crosslinking” may include the formation of a 3D polymer network by linking charged polymer chains with oppositely charged ions, creatingML Ref. 135156-5001 physical ‘‘bridges” (ionic bonds) that enhance material properties like strength, stability', and gel formation. As used herein and unless otherw ise indicated, the term “ionic crosslinking” may include the crosslinking of polymers through physical interactions. Physical crosslinking is mainly realized through the interaction between ions and proteins, and physical forces such as hydrogen bonding induce the interpenetration of multiple polymers to form a mixture of macromolecular structures.

[0057] In some embodiments, the crosslinked polymer of the present disclosure maybe prepared by crosslinking at least one polymer selected from the group consisting of polyethylene glycol, alginate, fibrin, chitosan, collagen, poly(vinyl alcohol) (PVA), dextran, methacrylated hyaluronic acid (HAMA), poly(lactic-co-glycolic acid) (PLGA), Gelatin methacrylate (GelMa), a long chain / high molecular w eight hyaluronic acid (HA), a long chain gelatin, poly(N-isopropylacrylamide) (PNIPAAm), heparin, hydroxyethyl methacrylate (HEMA), polycaprolactone (PCL) , poly(ethyleneimine) (PEI), chondroitin sulfate, gelatin- hydroxyphenyl propionic acid (Gtn-HPA) and hyaluronic acid tyramine (HA-Tyr), and a copolymer thereof.

[0058] In some embodiments the crosslinked polymer of the present disclosure may comprise a polyethylene glycol (PEG) crosslinked via Michael addition reaction or thiol-ene reaction. In some embodiments, PEG (polyethylene glycol) can be crosslinked via Michael addition. In some embodiments, PEG (polyethylene glycol) can be crosslinked via thiol-ene chemistry.

[0059] In some embodiment, the crosslinked polymer of the present disclosure may comprise alginate crosslinked with a calcium ion such as CaCb. In some embodiments, alginate can be ionically crosslinked using calcium ions (CaCb). In some embodiment, the crosslinked polymer of the present disclosure may comprise fibrin enzymatically crosslinked with thrombin. In some embodiment, the crosslinked polymer of the present disclosure mayML Ref. 135156-5001 comprise chitosan crosslinked with genipin or ionic crosslinked with tripolyphosphate. In some embodiment, the crosslinked polymer of the present disclosure may comprise collagen physically crosslinked or enzymatically crosslinked with transglutaminase. In some embodiment, the crosslinked polymer of the present disclosure may comprise poly(vinyl alcohol) (PVA) crosslinked with a freeze-thaw cycles or borax. In some embodiment, the crosslinked polymer of the present disclosure may comprise dextran crosslinked with epichlorohydrin or a UV crosslinking. In some embodiment, the crosslinked polymer of the present disclosure may comprise methacrylated hyaluronic acid (HAMA) photocrosslinked with UV light and / or photoinitiator such as Irgacure®. In some embodiment, the crosslinked polymer of the present disclosure may comprise poly(lactic-co-gly colic acid) (PLGA) crosslinked with a emulsion or solvent evaporation method. In some embodiment, the crosslinked polymer of the present disclosure may comprise gelatin methacrylate (GelMA) photocrosslinked with UV light and a photoinitiator. In some embodiment, the crosslinked polymer of the present disclosure may comprises a long chain / high molecular weight hyaluronic acid (HA) physically crosslinked with a hydrogen bonding and / or an entanglement. In some embodiment, the crosslinked polymer of the present disclosure may comprise a long chain gelatin physically crosslinked via self-assembly or gelation.

[0060] In some embodiment, the crosslinked polymer of the present disclosure may comprise poly(N-isopropylacrylamide) (PNIPAAm) thermally crosslinked (temperaturesensitive gelation). In some embodiment, the crosslinked polymer of the present disclosure may comprise heparin (glycosaminoglycan) covalently crosslinked with a carbodiimide. In some embodiment, the crosslinked polymer of the present disclosure may comprise hydroxy ethyl methacrylate (HEMA) photocrosslinked with UV light and / or a photoinitiator. In some embodiment, the crosslinked polymer of the present disclosure may comprises poly caprolactone (PCL) crosslinked via a thermal or UV-initiated polymerization. In someML Ref. 135156-5001 embodiment, the crosslinked polymer of the present disclosure may comprise poly(ethyleneimine) (PEI) crosslinked with glutaraldehyde or covalently crosslinked. In some embodiment, the crosslinked polymer of the present disclosure may comprise chondroitin sulfate photocrosslinked with UV light or covalently crosslinked with carbodiimide.

[0061] In some embodiment, the crosslinked polymer of the present disclosure may be prepared by crosslinking gelatin-hydroxyphenyl propionic acid (Gtn-HPA) and / or hyaluronic acid tyramine (HA-Tyr). In some embodiment, the crosslinked polymer of the present disclosure may be prepared by crosslinking hydroxyphenyl propionic acid (Gtn-HPA) and / or hyaluronic acid tyramine (HA-Tyr) with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). In some embodiment, the crosslinked polymer of the present disclosure may be prepared by crosslinking the polymer with a crosslinking agent in an amount from 0. ImM to about 30 mM.

[0062] In some embodiments, an amount of crosslinker / crosslinking agent used for preparing the crosslinked polymer may be in a range from about 0. 1 mM to about 30 mM. In some embodiments, an amount of crosslinker / crosslinking agent used for preparing the crosslinked polymer may be at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more and / or about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 mM or less. In some embodiments, an amount of crosslinker / crosslinking agent used for preparing the crosslinked polymer may be in a range from about 0.01 U / mL to about 1 U / mL. In some embodiments, an amount of crosslinker / crosslinking agent used for preparing the crosslinked polymer may be at least about 0.01, 0.02, 0.03, 0.04, 0.05. 0.06. 0.07. 0.08. 0.09, 0.1. 0.2, 0.3, 0.4 0.5, 0.6. 0.7, 0.8, 0.9 U / mL or more and / or about 1, 0.9, 0.8, 0.7. 0.6, 0.5, 0.4, 0.3. 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 or 0.02 U / mL or less.

[0063] In some embodiment, the crosslinked polymer of the present disclosure mayML Ref. 135156-5001 be prepared by crosslinking the polymer with H2O2 as a crosslinking agent in an amount from about 0. 1 mM to about 5 mM or about 0.75 mM to about 1.5 mM. In some embodiment, the crosslinked polymer of the present disclosure may be prepared by crosslinking the polymer with HRP as a crosslinking agent in an amount from about 0.05 U / mL to about 1 U / mL, or about 0.07 U / ml to 0.15 U / mL.

[0064] In some embodiment, the crosslinked polymer of the present disclosure may comprise a copolymer of hydroxy phenyl propionic acid (Gtn-HPA) and / or hyaluronic acid tyramine (HA-Tyr). In some embodiment, a weight ratio of the Gtn-HPA to the HA-Tyr may be in a range from about 10:90 to about 90: 10. In some embodiment, a weight ratio of the Gtn-HPA to the HA-Tyr may be 50:50. In some embodiments, a weight ratio of the Gtn- HPA to the HA-Tyr can be 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, or 90: 10. In some embodiments, a weight ratio of the Gtn-HPA to the HA-Tyr is in a range from 10:90 to 90: 10.

[0065] In some embodiment, the composition of the present disclosure may be stored at a temperature above about 0 °C. In some embodiment, the composition of the present disclosure may be stored at a room temperature. In some embodiments, the composition of the present disclosure may be stored at temperature above 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 °C. In some embodiments, the composition can be stored at temperature from about 1 to about 100, from about 1 to about 80, from about 1 to about 60, from about 1 to about 50, from about 1 to about 40, from about 1 to about 30, from about 1 to about 25, from about 1 to about 20, from about 1 to about 10, from about 5 to about 100, from about 5 to about 80, from about 5 to about 60, from about 5 to about 50, from about 5 to about 40. from about 5 to about 30, from about 5 to about 25. from about 5 to about 20, from about 5 to about 10, from about 10 to about 100, from about 10 to about 80, from about 10 to about 60. from about 10 to about 50, from about 10 to about 40, from about 10 to about 30,ML Ref. 135156-5001 from about 10 to about 25, from about 10 to about 20, from about 10 to about 15, from about 15 to about 100, from about 15 to about 80, from about 15 to about 60, from about 15 to about 50, from about 15 to about 40, from about 15 to about 30, from about 15 to about 25, from about 15 to about 20, from about 20 to about 100, from about 20 to about 80, from about 20 to about 60, from about 20 to about 50, from about 20 to about 40, from about 20 to about 30, from about 20 to about 25, from about 25 to about 100, from about 25 to about 80, from about 25 to about 60, from about 25 to about 50, from about 25 to about 40, from about 25 to about 30, from about 30 to about 50, or from about 30 to about 40 °C.

[0066] In some embodiment, the composition of the present disclosure may enhance stability and / or prolongs liquid state. As used herein and unless otherwise indicated, the term “enhance stability’’ may include making a target composition / compound / polymer / hydrogel stronger, more resistant to reakdown (by heat, w ater, enzymes), better at holding their shape / structure (mechanical integrity), and improving their performance over time or under stress, often by adding physical or chemical crosslinks, nanoparticles, or better polymer designs to prevent syneresis (water loss) or degradation, crucial for applications like drug delivery or tissue engineering. As used herein and unless otherwise indicated, the term “prolongs liquid state” may include making a target composition or compound or polymer or hydrogel can exist in liquid or sol state to be injected as liquid and solidify in the body of the subject for drug delivery or tissue engineering.

[0067] In some embodiment, the present disclosure relates to a method of storing the composition described herein, by storing the composition at a temperature above about 0 °C. In some embodiment, the present disclosure relates to a method of storing the composition of the present disclosure by storing the composition at a room temperature.

[0068] In some embodiment, the present disclosure relates to a method of preparing the composition described herein. In some embodiments, the method may compriseML Ref. 135156-5001 crosslinking gelatin-hydroxyphenyl propionic acid (Gtn-HPA) and / or hyaluronic acid t ramine (HA-Tyr) with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) in a solution. In some embodiment, in the method of preparing the composition the present disclosure, a concentration of H2O2 in the solution may be from 0.8 mM to 1.5 mM.

[0069] In some embodiments, the method of preparing the crosslinked polymer of the present disclosure may comprise a concentration of H2O2 in the solution of 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.5, 1, 0.5, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.005, 0.003, 0.002, 0.001, 0.0005, 0.0003, 0.0002, 0.0001, 0.00005, or 0.00001 mM or less. In some embodiments, the method comprises a concentration of FbChin the solution of 0.00001, 0.00002, 0.00003, 0.00004, 0.00005, 0.0001, 0.0002, 0.0003, 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mM or more. In some embodiments, the method comprises a concentration of PLCh in the solution from about 0.0001 to about 2, from about 0.0001 to about 1.5, from about 0.0001 to about 1, from about 0.0002 to about 2, from about 0.0002 to about 1.5, from about 0.0002 to about 1, from about 0.0002 to about 0.5, from about 0.0002 to about 0. 1 , from about 0.0002 to about 0.05, from about 0.0002 to about 0.01, from about 0.0004 to about 2, from about 0.0004 to about 1.5, from about 0.0004 to about 1, from about 0.0004 to about 0.5, from about 0.0004 to about 0.1, from about 0.0004 to about 0.05, from about 0.0004 to about 0.01, from about 0.02 to about 0.1, from about 0.02 to about 2, from about 0.02 to about 1.5, from about 0.02 to about 1, from about 0.02 to about 0.1. from about 0.02 to about 0.08. from about 0.02 to about 0.06, from about 0.02 to about 0.05, from about 0.02 to about 0.04, from about 0.04 to about 2, from about 0.04 to about 1, from about 0.04 to about 0.5, from about 0.04 to about 0. 1, from about 0.04 to about 0.08, from about 0.04 to about 0.06, from about 0.04 to about 0.05, from about 0.05 to about 2, from about 0.05 to about 1.5, from about 0.05 to about 1.ML Ref. 135156-5001 from about 0.05 to about 0.1, from about 0.05 to about 0.08, from about 0.05 to about 0.06, from about 0.07 to about 2, from about 0.07 to about 1, from about 0.07 to about 0. 14, from about 0.07 to about 0.12, from about 0.07 to about 0.1, from about 0.1 to about 10, from about 0. 1 to about 5, from about 0. 1 to about 3, from about 0.1 to about 1, from about 0.5 to about 10, from about 0.5 to about 5, from about 0.5 to about 1, from about 0.8 to about 10, from about 0.8 to about 5, from about 0.8 to about 2, from about 0.8 to about 1.5, from about 0.8 to about 1, from about 1 to about 10, from about 1 to about 5, from about 1 to about 3, from about 1 to about 2, or from about 1 to about 1.5 mM.

[0070] In some embodiment, in the method of preparing the composition the present disclosure, a concentration of HRP in the solution is more than 0.07 U / mL. In some embodiments, the method of preparing the crosslinked polymer of the present disclosure may comprises a concentration of HRP in the solution of 0.0001, 0.0002, 0.0003, 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 U / mL or more. In some embodiments, the method comprises a concentration of HRP in the solution of 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.5, 0.3, 0.2, 0.1 , 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 U / mL or less. In some embodiments, the method comprises a concentration of HRP in the solution from about 0.0001 to about 2, from about 0.0001 to about 1, from about 0.0002 to about 2, from about 0.0002 to about 1, from about 0.0002 to about 0.5, from about 0.0002 to about 0. 1, from about 0.0002 to about 0.05, from about 0.0002 to about 0.01, from about 0.0004 to about 1, from about 0.0004 to about 0.5, from about 0.0004 to about 0.1, from about 0.0004 to about 0.05, from about 0.0004 to about 0.01, from about 0.02 to about 0. 1, from about 0.02 to about 0.08, from about 0.02 to about 0.06, from about 0.02 to about 0.05. from about 0.02 to about 0.04. from about 0.04 to about 0. 1, from about 0.04 to about 0.08. from about 0.04 to about 0.06. from about 0.04 to about 0.05.ML Ref. 135156-5001 from about 0.05 to about 0.1, from about 0.05 to about 0.08, from about 0.05 to about 0.06, from about 0.07 to about 0. 14, from about 0.07 to about 0. 12, or from about 0.07 to about 0. 1 U / mL.

[0071] In some embodiment, in the method of preparing the composition the present disclosure, the crosslinking is performed at a room temperature. In some embodiment, in the method of preparing the composition the present disclosure, the crosslinking is performed at a temperature above 35 °C. In some embodiments, the crosslinking is performed at a room temperature. In some embodiments, the crosslinking is performed at a temperature above 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 °C. In some embodiments, the crosslinking is performed at a temperature below 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 °C. In some embodiments, the crosslinking is performed at a temperature from about 1 to about 100, from about 1 to about 80, from about 1 to about 60, from about 1 to about 50, from about 1 to about 40, from about 1 to about 30, from about 1 to about 25, from about 1 to about 20, from about 1 to about 10, from about 5 to about 100, from about 5 to about 80, from about 5 to about 60, from about 5 to about 50, from about 5 to about 40, from about 5 to about 30, from about 5 to about 25, from about 5 to about 20, from about 5 to about 10, from about 10 to about 100, from about 10 to about 80, from about 10 to about 60, from about 10 to about 50, from about 10 to about 40, from about 10 to about 30, from about 10 to about 25, from about 10 to about 20, from about 10 to about 15, from about 15 to about 100, from about 15 to about 80, from about 15 to about 60, from about 15 to about 50, from about 15 to about 40, from about 15 to about 30, from about 15 to about 25, from about 15 to about 20, from about 20 to about 100, from about 20 to about 80, from about 20 to about 60, from about 20 to about 50. from about 20 to about 40. from about 20 to about 30, from about 20 to about 25. from about 25 to about 100, from about 25 to about 80, from about 25 to about 60. from about 25 to about 50, from about 25 to about 40, from about 25 to about 30,ML Ref. 135156-5001 from about 30 to about 50, or from about 30 to about 40 °C.

[0072] In some embodiments, the crosslinking is performed at least 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 seconds. In some embodiments, the crosslinking is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or 60 minutes. In some embodiments, the crosslinking is performed at least 1, 1.5, 2, 2.5, 3, 2.5, 4, 4.5, or 5 hours. In some embodiments, the crosslinking is performed at most 1, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 seconds. In some embodiments, the crosslinking is performed at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or 60 minutes. In some embodiments, the crosslinking is performed at most 1, 1.5, 2, 2.5, 3, 2.5, 4, 4.5, or 5 hours.

[0073] In some embodiments, the crosslinking is performed from about 1 second to about 3 hours, from about 1 second to about 2 hours, from about 1 second to about 1 hour, from about 1 second to about 30 minutes, from about 1 second to about 20 minutes, from about 1 second to about 10 minutes, from about 1 second to about 5 minutes, from about 1 second to about 3 minutes, from about 1 second to about 1 minute, from about 10 second to about 3 hours, from about 10 second to about 2 hours, from about 10 second to about 1 hour, from about 10 second to about 30 minutes, from about 10 second to about 20 minutes, from about 10 second to about 10 minutes, from about 10 second to about 5 minutes, from about 10 second to about 3 minutes, from about 10 second to about 1 minute, from about 30 second to about 3 hours, from about 30 second to about 2 hours, from about 30 second to about 1 hour, from about 30 second to about 30 minutes, from about 30 second to about 20 minutes, from about 30 second to about 10 minutes, from about 30 second to about 5 minutes, from about 30 second to about 3 minutes, from about 30 second to about 1 minute, from about 1 minute to about 3 hours, from about 1 minute to about 2 hours, from about 1 minute to about 1 hour, from about 1 minute to about 30 minutes, from about 1 minute to about 20 minutes, from about 1 minute to about 10 minutes, from about 1 minute to about 5 minutes, fromML Ref. 135156-5001 about 1 minute to about 3 minutes, from about 3 minutes to about 3 hours, from about 3 minutes to about 2 hours, from about 3 minutes to about 1 hour, from about 3 minutes to about 30 minutes, from about 3 minutes to about 20 minutes, from about 3 minutes to about 10 minutes, from about 3 minutes to about 5 minutes, from about 10 minutes to about 3 hours, from about 10 minutes to about 2 hours, from about 10 minutes to about 1 hour, from about 10 minutes to about 30 minutes, from about 10 minutes to about 20 minutes, from about 30 minutes to about 3 hours, from about 30 minutes to about 2 hours, from about 30 minutes to about 1 hour, from about 30 minutes to about 50 minutes, or from about 30 minutes to about 40 minutes.

[0074] In some embodiment, the present disclosure relates to a method of delivering a therapeutic agent to an eye in a subject by administering the therapeutic agent in the composition of the present disclosure. In some embodiment, the method of delivering the therapeutic agent may include in vivo delivery of the retinal neurons and subretinal injections. In some embodiment, the method of the present disclosure may include delivering the therapeutic agent to knee joints for arthritis lubrication, skin / subcutaneous tissue for dermal filers or drug depots, muscle for sustained drug release, and cardiac / retinal space for tissue repair.

[0075] In some embodiment, the therapeutic agent may comprise at least one selected from the group consisting of human epidermal grow th factor (hEGF), anti-VEGF, blocks VEGFR1, blocks VEGFR2, blocks VEGFR3, anti-PDGF, anti-angiogenesis, Sunitinib, E7080, Takeda-6d, Tivozanib, Regorafenib, Sorafenib, Pazopanib, Axitinib, Nintedanib, Cediramb, Vatalanib, Motesanib, macrolides, sirolimus, everolimus, tyrosine kinase inhibitors (TKIs), Imatinib (GLEEVAC) gefinitib (IRES SA), toceranib (PALLADIA). Erlotinib (TARCEVA), Lapatinib (TYKERB) Nilotinib, Bosutinib Neratinib, lapatinib, Vatalanib, dasatinib. erlotinib, gefitinib. imatinib. lapatinib. lestaurtinib, nilotinib, semaxanib,ML Ref. 135156-5001 toceranib, vandetanib.

[0076] In some embodiment, the present disclosure relates to a method of delivering a therapeutic agent to a subject in need thereof, comprising forming a hydrogel comprising the composition of the present disclosure in situ in the subject in the subject. As used herein and unless otherwise indicated, the term “subject” may include any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. In some embodiments, the subject is a human. “Subject in need thereof’ means a subject identified as in need of a therapy or treatment. In some embodiment, the present disclosure provides a method of delivering a therapeutic agent to the subject’s body, such as eye.

[0077] In some embodiment, the present disclosure relates to a kit comprising a polymer and a crosslinking agent, wherein the polymer comprises at least one selected from the group consisting of alginate, fibrin, chitosan, collagen, Poly(vinyl alcohol) (PVA), dextran, Methacrylated Hyaluronic Acid (HAMA), Poly(lactic-co-gly colic acid) (PLGA), GelMA (Gelatin Methacrylate), long HA (Hyaluronic Acid), long gelatin, Poly(N- isopropylacrylamide) (PNIPAAm), heparin, Hydroxyethyl Methacrylate (HEMA), Poly caprolactone (PCL) , Poly(ethyleneimine) (PEI), chondroitin sulfate, gelatin- hydroxyphenyl propionic acid (Gtn-HPA) and hyaluronic acid tyramine (HA-Tyr), and a copolymer thereof.

[0078] In some embodiments, the crosslinked polymer may comprise at least one selected from the group consisting of alginate, fibrin, chitosan, collagen, poly(vinyl alcohol) (PVA). dextran, methacrylated hyaluronic acid (HAMA). poly(lactic-co-gly colic acid) (PLGA), GelMA (gelatin methacrylate), long HA (hyaluronic acid), long gelatin, poly(N-ML Ref. 135156-5001 isopropylacrylamide) (PNIPAAm), heparin, hydroxy ethyl methacry late (HEMA), Poly caprolactone (PCL) , poly(ethyleneimine) (PEI), and chondroitin sulfate, gelatin- hydroxyphenyl propionic acid (Gtn-HPA), hyaluronic acid tyramine (HA-Tyr), and a copolymer thereof. In some embodiments, the crosslinked polymer may be prepared by crosslinking at least one selected from the group consisting of alginate, fibrin, chitosan, collagen, poly(vinyl alcohol) (PVA), dextran, methacrylated hyaluronic acid (HAMA), poly(lactic-co-gly colic acid) (PLGA), GelMA (gelatin methacrylate), long HA (hyaluronic acid), long gelatin, poly(N-isopropylacrylamide) (PNIPAAm), heparin, Hydroxyethyl methacry late (HEMA), poly caprolactone (PCL) , poly(ethyleneimine) (PEI), and chondroitin sulfate, gelatin-hydroxyphenyl propionic acid (Gtn-HPA), hyaluronic acid tyramine (HA- Tyr), and a copolymer thereof.

[0079] In one aspect, the present disclosure provides a method of delivering a therapeutic agent to an eye. In some embodiments, the method comprises administering the therapeutic agent in the composition described herein.

[0080] Hereinafter, the present disclosure will be described in more detail with reference to the following examples. But the following Examples are intended to illustrate the present embodiments, and the scope of the Examples is not limited thereto only.EXAMPLES

[0081] Example 1: Gelation time of composition depend on storage temperature

[0082] A composition comprising HRP and H2O2 is stored at different conditions (- 200 °C, -80°C, -20°C, 4°C, and 20°C) and measured gelation time at different temperatures (body temperature, room temperature, and ice temperature). The result showed a trend of faster gelation time dependent on storage, specifically for room temperature transition (20°C) (shown in FIG. 1 A). The gelation time was less than 1 minute when the composition is storedML Ref. 135156-5001 at body temperature (37 °C), and the gelation time was less than 5 minutes when the composition is stored at room temperature (20 °C). The gelation time was less than 2 minutes regardless of stored temperatures. Similar trends are shown in different compositions (as shown in FIGs. IB to IE).

[0083] Example 2: Residual of H2O2 (mM) or HRP (U / mL) with initial concentration of Gel-HA (mM)

[0084] Residuals of HRP and H2O2 was tested during hydrogel formation in different concentrations. As shown in FIG. 2A, there was no H2O2 residual from 0.75 mM to 1.5 mM of H2O2 while the hydrogel still form a gel. FIG. 2B shows there is no residual of HRP below 0.07 U / mL and there is no gel below 0.02 U / mL of HRP.

[0085] EXEMPLARY EMBODIMENTSEmbodiment 1 . A composition for delivering a therapeutic agent to an eye, the composition comprising a crosslinked polymer and horseradish peroxidase (HRP). Embodiment 2. The composition according to embodiment 1, wherein the crosslinked polymer has a gelation time from 1 minutes to 5 minutes when a hydrogel composition is stored at a room temperature.Embodiment 3. The composition according to any one of the preceding embodiments, wherein the composition includes H2O2 at a concentration less than about 0.0001 mM.Embodiment 4. The composition according to any one of the preceding embodiments, wherein the composition includes HRP at a concentration from about 0.0002 to about 1 U / mL.Embodiment 5. The composition according to any one of the preceding embodiments, wherein the composition includes HRP at a concentration from about 0.004 to about 1 U / mL. Embodiment 6. The composition according to any one of the preceding embodiments.ML Ref. 135156-5001 wherein the concentration of H2O2 is a residual amount of H2O2 in the composition after crosslinking a polymer to produce the crosslinked polymer.Embodiment 7. The composition according to any one of the preceding embodiments, wherein the concentration of HRP is a residual amount of HRP in the composition after crosslinking a polymer to produce the crosslinked polymer.Embodiment 8. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer is hydrophilic.Embodiment 9. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises gelatin.Embodiment 10. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises gelatin, and the gelatin is crosslinked with a crosslinking agent.Embodiment 1 1. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises gelatin including a side chain having a substituted or unsubstituted C6 to C20 ary l group, a linear or branched substituted or unsubstituted Cl to C8 alkyl group, a hydroxyl group, amino group and / or a carboxylic acid.Embodiment 12. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises gelatin including hydroxyphenyl propionic acid as a side group in an amount of 0. 1 to 10 % of a monomer unit of the gelatin or 0. 1 to 10 wt% of a the gelatin.Embodiment 13. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises gelatin crosslinked with a crosslinking agent. Embodiment 14. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises hyaluronic acid (EIA).Embodiment 15. The composition according to any one of the preceding embodiments.ML Ref. 135156-5001 wherein the crosslinked polymer comprises hyaluronic acid (HA), and the hyaluronic acid (HA) is crosslinked with a crosslinking agent.Embodiment 16. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises hyaluronic acid (HA) including a side chain having a C6 to C20 ar l group, a linear or branched Cl to C8 alkyl group, a hydroxyl group, an amino group and / or a carboxylic acid.Embodiment 17. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises hyaluronic acid (HA) including t ramine as a side chain in an amount of about 10 to about 90 % of a monomer unit of the hyaluronic acid (HA) or about 10 to about 90 wt% of the hyaluronic acid (HA).Embodiment 18. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises a biopolymer.Embodiment 19. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer is prepared by crosslinking a biopolymer with a crosslinking agent.Embodiment 20. The composition according to any one of the preceding embodiments, wherein the crosslinking agent comprises at least one selected from the group consisting of horseradish peroxidase (HRP), hydrogen peroxide (H2O2), calcium ions (CaCL), thrombin, tripolyphosphate, transglutaminase, carbodiimide, genipin, epichlorohydrin, glutaraldehyde, sodium periodate, permanganate, tyrosinase, a pH adjusting agent such as sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, strontium hydroxide, barium hydroxide, and combinations thereof.Embodiment 21. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer is crosslinked via a chemical crosslinking, a UVML Ref. 135156-5001 crosslinking, a photocrosslinking, an enzymatic crosslinking, a thermal crosslinking, an ionic crosslinking, a physical crosslinking or combinations thereof.Embodiment 22. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer is prepared by crosslinking at least one polymer selected from the group consisting of polyethylene glycol, alginate, fibrin, chitosan, collagen, poly (vinyl alcohol) (PVA), dextran, methacrylaled hyaluronic acid (HAMA), poly(lactic-co- gly colic acid) (PLGA), Gelatin Methacrylate (GelMa), a long chain / high molecular weight Hyaluronic Acid (HA), a long chain gelatin, poly(N-isopropylacry lamide) (PNIPAAm), heparin, hydroxyethyl methacrylate (HEMA), poly caprolactone (PCL) , poly(ethyleneimine) (PEI), chondroitin sulfate, gelatin-hydroxyphenyl propionic acid (Gtn-HPA) and hyaluronic acid tyramine (HA-Tyr), and a copolymer thereof.Embodiment 23. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises a polyethylene glycol (PEG) crosslinked via Michael addition reaction or thiol-ene reaction.Embodiment 24. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises alginate crosslinked with a calcium ion such as CaCh.Embodiment 25. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises fibrin enzymatically crosslinked with thrombin.Embodiment 26. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises chitosan crosslinked with genipin or ionic crosslinked with tripolyphosphate.Embodiment 27. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises collagen physically crosslinked or enzymatically- crosslinked with transglutaminase.ML Ref. 135156-5001Embodiment 28. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises poly(vinyl alcohol) (PVA) crosslinked with a freeze-thaw cycles or borax.Embodiment 29. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises dextran crosslinked with epichlorohydrin or a UV crosslinking.Embodiment 30. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises methacrylated hyaluronic acid (EIAMA) photocrosslinked with UV light and / or photoinitiator such as Irgacure®.Embodiment 31. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises poly(lactic-co-gly colic acid) (PLGA) crosslinked with a emulsion or solvent evaporation method.Embodiment 32. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises gelatin methacrylate (GelMA) photocrosslinked with UV light and a photoinitiator.Embodiment 33. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises a long chain / high molecular weight Hyaluronic Acid (HA) physically crosslinked with a hydrogen bonding and / or an entanglement.Embodiment 34. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises a long chain gelatin physically crosslinked via self-assembly or gelation.Embodiment 35. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises heparin (glycosaminoglycan) covalently crosslinked with a carbodiimide.Embodiment 36. The composition according to any one of the preceding embodiments.ML Ref. 135156-5001 wherein the crosslinked polymer comprises poly caprolactone (PCL) crosslinked via a thermal or UV-initiated polymerization.Embodiment 37. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises poly(ethyleneimine) (PEI) crosslinked with glutaraldehyde or covalently crosslinked.Embodiment 38. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprises chondroitin sulfate photocrosslinked with UV light or covalently crosslinked with carbodiimide.Embodiment 39. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer is prepared by crosslinking gelatin-hydroxyphenyl propionic acid (Gtn-EIPA) and / or hyaluronic acid tyramine (HA-Tyr).Embodiment 40. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer is prepared by crosslinking hydroxyphenyl propionic acid (Gtn-HPA) and / or hyaluronic acid tyramine (HA-Tyr) with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2).Embodiment 41 . The composition according to any one of the preceding embodiments, wherein crosslinked polymer is prepared by crosslinking the polymer with a crosslinking agent in an amount from 0. ImM to about 30 mM.Embodiment 42. The composition according to any one of the preceding embodiments, wherein crosslinked polymer is prepared by crosslinking the polymer with H2O2 as a crosslinking agent in an amount from about 0. 1 mM to about 5 mM or about 0.75 mM to about 1.5 mM.Embodiment 43. The composition according to any one of the preceding embodiments, wherein crosslinked polymer is prepared by crosslinking the polymer with HRP as a crosslinking agent in an amount from about 0.05 U / mL to about 1 U / mL. or about 0.07 U / mlML Ref. 135156-5001 to 0.15 U / mLEmbodiment 44. The composition according to any one of the preceding embodiments, wherein the crosslinked polymer comprising a copoly mer of hy droxy pheny l propionic acid (Gtn-HPA) and / or hyaluronic acid tyraminc (HA-Tyr).Embodiment 45. The composition according to any one of the preceding embodiments, wherein a weight ratio of the Gtn-HPA to the HA-Tyr is in a range from about 10:90 to about 90:10.Embodiment 46. The composition according to any one of the preceding embodiments, wherein a weight ratio of the Gtn-HPA to the HA-Tyr is 50:50.Embodiment 47. The composition according to any one of the preceding embodiments, wherein the composition is stored at a temperature above about 0 °C.Embodiment 48. The composition according to any one of the preceding embodiments, wherein the composition is stored at a room temperature.Embodiment 49. The composition according to any one of the preceding embodiments, wherein the composition enhances stability and / or prolongs liquid state.Embodiment 50. A method of storing the composition of any one of the preceding embodiments, the method comprising storing the composition at a temperature above about 0 °C.Embodiment 51. A method of storing the composition of any one of the preceding embodiments 1-49, the method comprising storing the composition at a room temperature. Embodiment 52. A method of preparing the composition of any one of embodiments 1- 49, comprising: crosslinking gelatin-hydroxyphenyl propionic acid (Gtn-HPA) and / or hyaluronic acid tyramine (HA-Tyr) with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) in a solution.Embodiment 53. The method according to embodiment 52, wherein a concentration ofML Ref. 135156-5001FbChin the solution is from 0.8 mM to 1.5 mM.Embodiment 54. The method according to embodiment 52 or 53, wherein a concentration of HRP in the solution is more than 0.07 U / mL.Embodiment 55. The method according to any one of embodiments 52-54, wherein the crosslinking is performed at a room temperature.Embodiment 56. The method according to any one of embodiments 52-55, wherein the crosslinking is performed at a temperature above 35 °C.Embodiment 57. A method of delivering a therapeutic agent to an eye in a subject, the method comprising administering the therapeutic agent in the composition of any one of embodiments 1-49.Embodiment 58. The method according to embodiment 57, wherein the therapeutic agent comprises at least one selected from the group consisting of human epidermal growth factor (hEGF), anti-VEGF, blocks VEGFR1, blocks VEGFR2, blocks VEGFR3, anti-PDGF, anti-angiogenesis, Sunitinib, E7080, Takeda-6d, Tivozanib, Regorafenib, Sorafenib, Pazopanib, Axitinib, Nintedanib, Cediranib, Vatalanib, Motesanib, macrolides, sirolimus, everolimus, tyrosine kinase inhibitors (TKIs), Imatinib (GLEEVAC) gefmitib (IRESSA), toceranib (PALLADIA), Erlotinib (TARCEVA), Lapatinib (TYKERB) Nilotinib, Bosutinib Neratinib, lapatinib, Vatalanib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib, toceranib, vandetanib.Embodiment 59. A method of delivering a therapeutic agent to an eye in a subj ect, comprising forming a hydrogel comprising the composition of any one of embodiments 1-49 in situ in the eye in the subject.Embodiment 60. A kit comprising a polymer and a crosslinking agent, wherein the polymer comprises at least one selected from the group consisting of alginate, fibrin, chitosan, collagen. Poly(vinyl alcohol) (PVA), dextran, Methacrylated Hyaluronic AcidML Ref. 135156-5001(HAMA), Poly(lactic-co-glycolic acid) (PLGA), GelMA (Gelatin Methacry late), long HA (Hyaluronic Acid), long gelatin, Poly(N-isopropylacrylamide) (PNIPAAm), heparin, Hydroxyethyl Methacrylate (HEMA), Polycaprolactone (PCL) , Poly(ethyleneimine) (PEI), chondroitin sulfate, gelatin-hydroxyphenyl propionic acid (Gtn-HPA) and hy aluronic acid t ramine (HA-Tyr), and a copolymer thereof.

Claims

ML Ref. 135156-5001CLAIMS1. A composition for delivering a therapeutic agent to an eye, the composition comprising a crosslinked polymer and horseradish peroxidase (HRP).

2. The composition according to claim 1, wherein the crosslinked polymer has a gelation time from 1 minutes to 5 minutes when the composition is stored at a room temperature.

3. The composition according to any one of the preceding claims, wherein the composition includes the H2O2 at a concentration less than about 0.0001 mM.

4. The composition according to any one of the preceding claims, wherein the composition includes the HRP at a concentration from about 0.0002 to about 1 U / mL.

5. The composition according to any one of the preceding claims, wherein the crosslinked polymer comprises gelatin or hyaluronic acid (HA), and the gelatin or the hyaluronic acid (HA) is crosslinked with a crosslinking agent.

6. The composition according to any one of the preceding claims, wherein the crosslinked polymer comprises a copolymer of hydroxyphenyl propionic acid (Gtn-HPA) and / or hyaluronic acid tyramine (HA-Tyr).

7. The composition according to any one of the preceding claims, wherein the composition is stored at a temperature above about 0 °C or a room temperature.

8. A method of storing the composition of any one of the claims 1 -7, the method comprising storing the composition at a temperature above about 0 °C.

9. A method of preparing the composition of any one of claims 1-7, comprising: crosslinking gelatin-hydroxyphenyl propionic acid (Gtn-HPA) and / or hyaluronic acid tyramine (HA-Tyr) with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) in a solution.

10. A method of delivering a therapeutic agent to an eye in a subject, comprising forming a hydrogel comprising the composition of any one of claims 1-7 in situ in the eye in theML Ref. 135156-5001 subject.

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