Compositions and methods for controlled release of targeted agents

Abstract

Compositions and methods for the controlled release of macromolecules (such as proteins and polypeptides) are provided. The compositions include at least one first hydrogel-forming polymer and at least one second hydrogel-forming polymer. Methods for preparing and using the compositions are also provided.

Description

[Technical Field]

[0001] Compositions and methods for the controlled release of macromolecules (such as proteins and polypeptides) are provided. The compositions include at least one first hydrogel-forming polymer and at least one second hydrogel-forming polymer. Methods for preparing and using the compositions are also provided. [Background technology]

[0002] Hydrogels are three-dimensional networks of polymers in which water or other materials (e.g., polymers) are entrapped within the polymer network. The size of the three-dimensional voids formed by the polymer matrix is ​​called the "mesh size," or ξ. In theory, controlling the mesh size in the same way as controlling polymers (e.g., proteins, polypeptides, aptamers, etc.) allows for control of the polymers.

[0003] However, precise control of mesh size and its distribution is difficult due to factors such as the polydispersity of the precursor polymer, the random nature of the crosslinking process, and the presence of cargo proteins that inhibit the formation of the polymer network. Therefore, mesh-size-controlled depot systems do not always achieve satisfactory release characteristics. While polymers in loose meshes are releasable, polymers in tight meshes are virtually impermeable to diffusion and can be considered physically immobilized. If the crosslinks in the polymer matrix were degradable, the tighter mesh size would expand, liberating a portion of the trapped polymer. Therefore, coupling the release of the loading molecules to the degradation of the depot meshwork could be an effective strategy for better controlling drug release behavior.

[0004] A problem with encapsulating macromolecules in hydrogels has been that the macromolecules are often covalently bound to the polymer network and are not free proteins.

[0005] Therefore, there is a strong need for versatile, effective and / or customizable approaches to achieve sustained release of macromolecules such as proteins, polypeptides and aptamers. DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0006] The present disclosure provides compositions and methods for the controlled release of macromolecules (such as proteins and polypeptides). The disclosed systems and methods (e.g., the mass ratio between the first modification and the second modification is less than about 1) can eliminate undesired covalent bonds between the macromolecules and the polymer. For example, at least about 20% of the macromolecules (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 96%, at least about 98%, at least about 99%, or more) are free in the hydrogel network. Furthermore, the disclosed compositions and methods can tailor the hydrogel environment (e.g., firmness, gelation time, swelling rate, etc.) to suit the needs of the hydrogel. The macromolecules may be retained within a structure (e.g., a hydrogel) formed by the polymer, which may degrade (e.g., hydrolytic cleavage) over an extended period of time (e.g., over days, weeks, or months). Degradation occurs under physiological conditions. The polymer and its degradation products may be biocompatible. The polymer structure (e.g., hydrogel) may be formed in situ, for example, by introducing (injecting) a composition (e.g., a liquid formulation) capable of forming a polymer structure (e.g., hydrogel) into tissue, and the polymer structure (e.g., hydrogel) may be formed in situ within the tissue upon introduction. Release of the target molecule from the hydrogel may be controlled. [Means for solving the problem]

[0007] In one aspect, the present disclosure provides a composition comprising at least one first hydrogel-forming polymer and at least one second hydrogel-forming polymer, the first hydrogel-forming polymer capable of reacting with the hydrogel-forming second polymer to form the hydrogel, the hydrogel being degradable and allowing for sustained release of a targeted agent, wherein the first hydrogel-forming polymer comprises a first hydrogel-forming polymer derivative, the first hydrogel-forming polymer derivative comprising a first modification, the first hydrogel-forming polymer derivative being electrophilic, and the second hydrogel-forming polymer comprises a second hydrogel-forming polymer derivative, the second hydrogel-forming polymer derivative comprising a second modification, the second hydrogel-forming polymer derivative being nucleophilic, and the mass ratio of the first hydrogel-forming polymer to the second hydrogel-forming polymer is less than 1.

[0008] In some embodiments, the first modification is selected from the group consisting of vinyl, acryloyl, thiol, alkene, thiol ester, isocyanate, isothiocyanate, alkyl halide, sulfonyl halide, epoxide, imidoester, fluorophenyl ester, carbonate, carbodiimide, disulfide, aziridine, and any combination thereof. In some embodiments, the first modification is selected from vinyl sulfone, maleimide, acrylate, methacrylic acid, epoxide, and any combination thereof. For example, the first modification is maleimide or vinyl sulfone.

[0009] In some embodiments, the second modification is selected from the group consisting of a thiol, an amine, an azide, a hydrazide, a diene, a hydrazine, a hydroxylamine, and any combination thereof.

[0010] In some embodiments, the first hydrogel-forming polymer and / or the second hydrogel-forming polymer is selected from the group consisting of polysaccharides, derivatives thereof, and any combination thereof.

[0011] In some embodiments, the first hydrogel-forming polymer and / or the second hydrogel-forming polymer is selected from the group consisting of hyaluronic acid, chitosan, chondroitin sulfate, alginic acid, carboxymethylcellulose, dextran, derivatives thereof, and any combination thereof.

[0012] In some embodiments, the first hydrogel-forming polymer and / or the second hydrogel-forming polymer is selected from the group consisting of dextran, hyaluronic acid, derivatives thereof, and any combination thereof.

[0013] In some embodiments, the hydrogel is hydrolyzable without the involvement of degradative enzymes.

[0014] In some embodiments, at least one of the first hydrogel-forming polymer and / or the second hydrogel-forming polymer comprises a degradable linker.

[0015] In some embodiments, the degradable linker comprises a hydrolyzable functional group.

[0016] In some embodiments, the hydrolyzable functional groups are selected from ester groups, anhydride groups, and amide groups.

[0017] In some embodiments, it is selected from ester groups.

[0018] In some embodiments, the first hydrogel-forming polymer derivative has a first average degree of modification (first DM) of less than about 40% and the second hydrogel-forming polymer derivative has a second average degree of modification (second DM) of less than about 40%.

[0019] In some embodiments, the ratio of the first DM to the second DM is from about 3:1 to about 1:3.

[0020] In some embodiments, the molar ratio between the first hydrogel-forming polymer derivative and the second hydrogel-forming polymer derivative in the composition is from about 3:1 to about 1:3.

[0021] In some embodiments, the first hydrogel-forming polymer derivative is a dextran derivative modified with one or more vinyl sulfone groups, a hyaluronic acid derivative modified with one or more vinyl sulfone groups, or a combination thereof, and the second hydrogel-forming polymer derivative is a dextran derivative modified with one or more thiol groups, a hyaluronic acid derivative modified with one or more thiol groups, or a combination thereof.

[0022] In some embodiments, the first hydrogel-forming polymer and the second hydrogel-forming polymer have a weight average molecular weight of from about 1 kDa to about 500 kDa.

[0023] In some embodiments, the composition is a powder.

[0024] In some embodiments, the composition is a liquid composition, and the concentration of the first hydrogel-forming polymer and / or the second hydrogel-forming polymer in the liquid composition is from about 1% w / v to about 50% w / v.

[0025] In another aspect, the present disclosure provides a hydrogel for the sustained release of a targeted agent, wherein the hydrogel is formed with a composition.

[0026] In some embodiments, the hydrogel further comprises a targeting agent.

[0027] In some embodiments, the targeting agent comprises a macromolecule.

[0028] In some embodiments, the targeting agent comprises a macromolecule having a molecular weight of at least 80 kDa.

[0029] In some embodiments, the targeting agent comprises a protein or polypeptide.

[0030] In some embodiments, at least about 20% of the targeting agent is free targeting agent that is not bound to the hydrogel.

[0031] In some embodiments, less than about 50% of the targeting agent is cumulatively released from the hydrogel within the first 24 hours, and the remaining portion of the targeting agent is cumulatively released from the hydrogel within about 1 to about 36 months.

[0032] In some embodiments, the hydrogel comprises a macroscopic hydrogel and a micronized hydrogel.

[0033] In some embodiments, the hydrogel further comprises a micronized hydrogel, for example, the hydrogel further comprises a micronized hydrogel within a macroscopic hydrogel.

[0034] In another aspect, the present disclosure provides a method of making a hydrogel, comprising: a) providing a composition; b) mixing the composition with a buffer to form a polymer solution; and c) subjecting the polymer solution to conditions that allow for the formation of a hydrogel.

[0035] In some embodiments, the method comprises injecting the polymer solution into a subject in need thereof.

[0036] In some embodiments, the subject incubates the composition at about 1°C to about 45°C.

[0037] In some embodiments, the polymer solution further comprises the targeting agent. In another aspect, the present disclosure provides a method of making a composition, comprising: a) crosslinking precursor polymers with a degradable linker to obtain a first hydrogel-forming polymer and / or a second hydrogel-forming polymer; and b) mixing the first hydrogel-forming polymer and / or the second hydrogel-forming polymer with an additional polymer, wherein the additional polymer is capable of reacting with the first hydrogel-forming polymer and / or the second hydrogel-forming polymer under conditions that allow the formation of the hydrogel.

[0038] In another aspect, the present disclosure provides a method for sustained release of a targeted agent, comprising mixing the targeted agent with a composition to obtain a mixture, and subjecting the mixture to conditions that allow for the formation of a hydrogel capable of sustained release of the targeted agent.

[0039] In another aspect, the present disclosure provides a method for the sustained release of a targeted agent, comprising encapsulating the targeted agent in a hydrogel.

[0040] In another aspect, the present disclosure provides a kit comprising: a) a composition; and b) a targeting agent to be sustained released by a hydrogel formed using the composition of a).

[0041] In another aspect, the present disclosure provides a use of the composition for preparing a hydrogel.

[0042] In another aspect, the present disclosure provides the use of the composition or hydrogel for the sustained release of a targeted agent.

[0043] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present disclosure are shown and described herein. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description should be regarded as illustrative in nature, and not restrictive. INCORPORATION BY REFERENCE

[0044] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0045] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also referred to herein as "FIG" and "FIG.").

[0046] [Figure 1] The synthesis schemes of vinyl sulfone-grafted dextran (DX-VS) and thiol-grafted dextran (DX-DTT and PDT) are shown in Figure 1. [Figure 2] The synthesis scheme of modified functionalized dextrans with ester bonds (DX-O-SH and DX-O(Me)-SH) is shown in Figure 1. [Figure 3] Figure 1 shows the synthesis scheme of modified functionalized dextrans with degradable linkers (DX-SH-VA-SH and DX-SH-VMA-SH). [Figure 4] Three forms of hydrogels are shown in Figure 1. [Figure 5] Figure 1 shows the swelling ratio (Wt / W0) profiles of selected hydrolytic hydrogel formulations varied with ester linkers. [Figure 6] Figures 6A-6B show non-reducing SDS-PAGE showing the size of F-IgG (FITC-IgG, i.e., IgG labeled with fluorescein FITC) released from the hydrolytic hydrogel under bright field (A) and UV (B). [Figure 7] 1 shows a non-reducing SDS-PAGE showing the molecular weight of bevacizumab released from the hydrolytic hydrogel. [Figure 8]Figure 1 shows the cumulative fractional release of IgG from non-degradable dextran-based hydrogel formulations with varying initial polymer concentrations. [Figure 9] The effect of hydrogel degradation rate on the cumulative release profile of F-IgG is shown in Figure 1. (A) Change in swelling ratio due to bulk erosion. (B) Cumulative release of F-IgG. (C) F-IgG release and hydrogel swelling for formulation 1 (C-1) and formulation 2 (C-2). [Figure 10] Figure 1 shows the cumulative release of F-IgG (A) and the corresponding hydrogel swelling (B). [Figure 11] Figure 11A shows the in vivo pharmacokinetics of the protein bevacizumab and bevacizumab-encapsulated hydrogels, and Figure 11B shows the in vitro release of bevacizumab from the hydrogels. [Figure 12] Figure 1 shows the in vivo biocompatibility of protein-encapsulated hydrogels in rabbit eyes. [Figure 13] Schematic diagrams showing protein release from hydrogels without and during crosslink degradation are shown in FIG. [Figure 14] Figure 1 shows a schematic diagram of a micronized hydrogel in a macroscopic hydrogel. [Figure 15] The format of the degradable linker is shown in Figure 1. [Figure 16] The NMR results of HA-MI are shown in Figure 1. [Figure 17] Figure 1 shows the swelling ratio of hydrogels formed by HA-MI with different DMs. [Figure 18] Figure 1 shows the cumulative release of hydrogels formed by HA-MI. DETAILED DESCRIPTION OF THE INVENTION

[0047] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be used.

[0048] definition

[0049] As used herein, the term "polymer" generally refers to a compound or mixture of compounds formed by polymerization and consisting essentially of repeating structural units.

[0050] As used herein, the term "hydrogel" generally refers to a gel or gel-like structure comprising one or more polymers suspended in an aqueous solution (e.g., water). All hydrogels have some degree of physical attraction between macromers as a result of hydrogen bonding and entanglement with each other. Typically, hydrogels intended for biomedical applications can be strengthened by additional electrostatic interactions or chemical crosslinking.

[0051] As used herein, the term "sustained release" generally refers to a process for relatively slowly releasing a targeted agent over an extended period of time (eg, days, weeks, or months).

[0052] As used herein, the term "degradable" generally refers to the property of a polymer structure (e.g., polymer chains) that can be degraded under physiological conditions (e.g., about 37°C and a pH of about 6.5-8). Degradation can be chemical (e.g., hydrolytic cleavage), physical (e.g., photonic cleavage), or biological (e.g., enzymatic cleavage). In some cases, the degradation can be hydrolytic, and in some cases, the hydrolysis can occur at crosslinks.

[0053] As used herein, the term "hydrolyzable" hydrogel generally refers to a polymeric structure (e.g., polymer chain) that can be at least partially hydrolyzed. For example, a hydrolyzable structure may be formed by crosslinking a linear or branched non-hydrolyzable precursor polymer with a crosslinker containing hydrolyzable groups and / or esters. The linear or branched precursor polymer may be modified with one or more modifications. For example, the hydrolyzable functional groups may be selected from ester groups, anhydride groups, and amide groups. For example, a hydrolyzable structure may be different from a polymer in which the bonds between monomers are hydrolyzable, such as polylactic acid (PLA) or poly(lactic-co-glycolic acid) (PLGA).

[0054] As used herein, the term "hydrogel-forming polymer" generally refers to a natural or synthetic polymer capable of forming a hydrogel. Hydrogel-forming polymers can be classified according to their synthetic origin, composition, electrostatic properties, and gel formation mechanism. In some cases, non-degradable hydrogel-forming polymers may have degradable regions incorporated into their structure to impart finely controlled degradability. Hydrogel-forming polymers include at least a first hydrogel-forming polymer and at least a second hydrogel-forming polymer, where the first hydrogel-forming polymer may be different from the second hydrogel-forming polymer. The first hydrogel-forming polymer may interact with the second hydrogel-forming polymer to form a hydrogel.

[0055] As used herein, the term "hydrolyzable" generally refers to the property of being able to be hydrolyzed, for example, at physiological temperatures (30°C to 40°C) and pH (6.5 to 7.5) without the use of a catalyst such as an enzyme. Hydrolysis is typically a chemical reaction in which one water molecule breaks down one or more chemical bonds.

[0056] As used herein, the term "electrophilic" generally means having an affinity for electron pairs. An electrophile (e.g., a molecule or portion of a molecule) may be an electron pair acceptor. In some embodiments, the electrophilic molecule or group may be selected from the group consisting of vinyl, acryloyl, thiol, alkene, thiol ester, isocyanate, isothiocyanate, alkyl halide, sulfonyl halide, epoxide, imidoester, fluorophenyl ester, carbonate, carbodiimide, disulfide, aziridine, and any combination thereof. In some embodiments, the electrophilic molecule or group may include vinyl sulfone, maleimide, acrylate, methacrylic acid, epoxide, and any combination thereof.

[0057] The term "nucleophilic" as used herein generally refers to having the property of being able to donate an electron pair to form a chemical bond for reaction with an electrophilic substance. In some embodiments, the term may refer to the nucleophilicity of a substance and its affinity for an electrolyte. In some embodiments, the nucleophile (e.g., molecule or portion of a molecule) may be selected from the group consisting of thiols, amines, azides, hydrazides, amines, dienes, hydrazines, hydroxylamines, and any combination thereof. A nucleophilic molecule or group acts

[0058] As used herein, the term "hydrophilic" generally refers to having an affinity for water and being able to absorb or be wetted by water. A hydrophilic molecule or portion of a molecule is one in which interactions with water and other polar substances are thermodynamically more favorable than interactions with oil or other hydrophobic solvents.

[0059] The term "ester group" as used herein generally refers to a chemical group derived from an acid (organic or inorganic) in which at least one -OH (hydroxyl) group is replaced with an -O-alkyl (alkoxy) group. For example, the ester group can be selected from an oxyester group and a thiol ester group.

[0060] As used herein, the term "average degree of modification (DM)" generally refers to the number of pendant groups per 100 repeating units in a polymer. DM may reflect the degree of modification of the hydrogel-forming polymer derivative.

[0061] The term "polydispersity" as used herein generally refers to the property of a polymer in terms of dispersity or heterogeneity when the chain lengths of the polymer vary over a wide range of molecular masses. TIFF2023510380000001.tif6170 can be calculated according to the degree of polymerization. where Mw is the weight-average degree of polymerization and Mn is the number-average molecular weight. For example, hydrogel-forming polymers containing degradable backbones have a polydispersity of 4 or less.

[0062] As used herein, the term "crosslink" generally refers to a bond that connects one polymer chain to another. It can be a covalent or ionic bond. A "polymer chain" can refer to a synthetic polymer or a natural polymer (such as hyaluronic acid). In polymer chemistry, when a polymer is said to be "crosslinked," it usually means that the entire bulk of the polymer has been subjected to a crosslinking method.

[0063] As used herein, the term "precursor polymer" generally refers to a polymer that is used to form another polymer structure or that is further modified, where the reactive groups allow the material to be further polymerized to form higher molecular weight structures.

[0064] As used herein, the term "composition" generally refers to a product (liquid or solid) of various elements or components.

[0065] As used herein, the term "biocompatible" or "biocompatibility" generally refers to the state of being compatible with living tissue or systems by being free from toxicity, injury, or physiological reactivity and / or not causing immunological rejection.

[0066] The term "about," when used in the context of a numerical value, generally refers to a value that is 1% to less than 15% above or below the indicated value (e.g., less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%).

[0067] Where a range of values ​​(e.g., a numerical range) is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range and any other stated or intervening value within that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with the cited publications.

[0069] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "particle" includes a plurality of such particles, a reference to an "array" includes a reference to one or more of such sequences and equivalents thereof known to those skilled in the art, and so forth.

[0070] Those skilled in the art, having read this disclosure, will understand that each of the individual embodiments described and illustrated herein has distinct components and features that may be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Methods described may be carried out in the order of events described or in any other order that is logically possible. All such combinations are intended to be covered.

[0071] The present disclosure provides compositions comprising one or more hydrogel-forming polymers, and methods of making and using the same. The present disclosure also provides hydrogels, and methods of making and using the same.

[0072] In one aspect, the present disclosure provides a composition that can include at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) first hydrogel-forming polymer and at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) second hydrogel-forming polymer, wherein the first hydrogel-forming polymer is capable of reacting with the hydrogel-forming second polymer to form the hydrogel, and the hydrogel is degradable (e.g., hydrolytically, enzymatically degradable, or otherwise cleavable) to allow for sustained release of a targeted agent.

[0073] In the present disclosure, the first hydrogel-forming polymer comprises a first hydrogel-forming polymer derivative, said first hydrogel-forming polymer derivative comprising a first modification, and said first hydrogel-forming polymer derivative can be electrophilic.

[0074] In some embodiments, the first modification can be selected from the group consisting of vinyl, acryloyl, thiol, alkene, thiol ester, isocyanate, isothiocyanate, alkyl halide, sulfonyl halide, epoxide, imidoester, fluorophenyl ester, carbonate, carbodiimide, disulfide, aziridine, and any combination thereof. In some embodiments, the first modification can be selected from the group consisting of vinyl, thiol, alkene, thiol ester, isocyanate, isothiocyanate, alkyl halide, sulfonyl halide, epoxide, imidoester, fluorophenyl ester, carbonate, carbodiimide, disulfide, aziridine, and any combination thereof.

[0075] In some embodiments, the first modification is selected from vinyl sulfone, maleimide, acrylate, methacrylic acid, epoxide, and any combination thereof. In some embodiments, the first modification is selected from maleimide, acrylate, methacrylic acid, epoxide, and any combination thereof. For example, the first modification is maleimide or vinyl sulfone.

[0076] In some embodiments, the first modification can be selected from the group consisting of vinyl, maleimide, acrylate, methacrylic acid, epoxide, thiol, alkene, thiol ester, isocyanate, isothiocyanate, alkyl halide, sulfonyl halide, epoxide, imidoester, fluorophenyl ester, carbonate, carbodiimide, disulfide, aziridine, and any combination thereof. For example, the first modification is maleimide or vinyl sulfone.

[0077] In the present disclosure, the second hydrogel-forming polymer comprises a second hydrogel-forming polymer derivative, said second hydrogel-forming polymer derivative comprising a second modification, and said second hydrogel-forming polymer derivative can be nucleophilic.

[0078] In some embodiments, the second modification can be selected from the group consisting of thiols, amines, azides, hydrazides, amines, dienes, hydrazines, hydroxylamines, and any combination thereof. In some embodiments, the second modification can be selected from the group consisting of amines, azides, hydrazides, amines, dienes, hydrazines, hydroxylamines, and any combination thereof.

[0079] In some embodiments, the first modification may be selected from the group consisting of vinyl, acryloyl (e.g., maleimide, acrylate, methacrylic acid, epoxide, and any combination thereof), thiol, alkene, thiol ester, isocyanate, isothiocyanate, alkyl halide, sulfonyl halide, epoxide, imido ester, fluorophenyl ester, carbonate, carbodiimide, disulfide, aziridine, and any combination thereof. The second modification may be selected from the group consisting of thiol, amine, azide, hydrazide, amine, diene, hydrazine, hydroxylamine, and any combination thereof.

[0080] In some embodiments, the first modification may be selected from the group consisting of vinyl, acryloyl (e.g., vinyl sulfone, maleimide, acrylate, methacrylate, epoxide, and any combination thereof), thiol, alkene, thiol ester, isocyanate, isothiocyanate, alkyl halide, sulfonyl halide, epoxide, imidoester, fluorophenyl ester, carbonate, carbodiimide, disulfide, aziridine, and any combination thereof. The second modification may be selected from the group consisting of amine, azide, hydrazide, amine, diene, hydrazine, hydroxylamine, and any combination thereof.

[0081] In some embodiments, in the composition, the first modification may include one or more vinyl sulfones and the second modification may include one or more thiols.

[0082] In some embodiments, a first polymer derivative can react with a second polymer derivative to form a hydrogel.

[0083] In the present disclosure, the weight ratio between the first hydrogel-forming polymer and the second hydrogel-forming polymer in the composition can be less than about 1 (e.g., less than about 0.95, less than about 0.9, less than about 0.85, less than about 0.8, less than about 0.75, less than about 0.7, less than about 0.65, less than about 0.6, less than about 0.55, less than about 0.5, less than about 0.45, less than about 0.4, less than about 0.35, less than about 0.3, less than about 0.25, less than about 0.2, less than about 0.15, less than about 0.1, less than about 0.05, or less).

[0084] In some embodiments, the weight ratio of the first hydrogel-forming polymer to the second hydrogel-forming polymer in the composition is from about 0 to about 1, e.g., from about 0 to about 0.99, from about 0 to about 0.95, from about 0 to about 0.9, from about 0 to about 0.8, from about 0 to about 0.7, from about 0 to about 0.6, from about 0 to about 0.5, from about 0 to about 0.49, from about 0 to about 0.45, from about 0 to about 0.4, from about 0 to about 0.3, from about 0 to about 0.2, from about 0 to about 0.1, from about 0.1 to about 1, or from about 0. 0.2 to about 1, about 0.3 to about 1, about 0.4 to about 1, about 0.5 to about 1, about 0.51 to about 1, about 0.55 to about 1, about 0.6 to about 1, about 0.7 to about 1, about 0.8 to about 1, about 0.9 to about 1, about 0.1 to about 0.5, about 0.1 to about 0.49, about 0.1 to about 0.45, about 0.1 to about 0.4, about 0.2 to about 0.3, about 0.5 to about 0.99, about 0.51 to about 0.99, about 0.6 to about 0.9, and about 0.7 to about 0.8, etc.

[0085] In some embodiments, the weight ratio between the first hydrogel-forming polymer and the second hydrogel-forming polymer in the composition can be about 0.95, about 0.9, about 0.85, about 0.8, about 0.75, about 0.7, about 0.67, about 0.65, about 0.6, about 0.55, about 0.5, about 0.45, about 0.4, about 0.35, about 0.3, about 0.25, about 0.2, about 0.15, about 0.1, or about 0.05, etc.

[0086] In the present disclosure, a first hydrogel-forming polymer derivative may react with a second hydrogel-forming polymer derivative to form a hydrogel.

[0087] In the present disclosure, the first hydrogel-forming polymer may be selected from the group consisting of polysaccharides, derivatives thereof, and any combination thereof.

[0088] In the present disclosure, the second hydrogel-forming polymer may be selected from the group consisting of polysaccharides, derivatives thereof, and any combination thereof.

[0089] In some cases, the polysaccharide may be a homoglycan, i.e., a polysaccharide with a backbone consisting of one monosaccharide, e.g., colominic acid, or a heteroglycan, i.e., a polysaccharide with one or more sugar residues in the backbone, either in an alternating or less regular arrangement, such as gellan, succinoglycan, arabinogalactan, gum tragacanth or gum tragacanth from Astragalus, Gum Karaya from Sterculia urens, Gum ghatti from Anogeissus latifolia, and derivatives thereof.

[0090] In the present disclosure, the first hydrogel-forming polymer may be selected from the group consisting of hyaluronic acid, chitosan, chondroitin sulfate, alginic acid, carboxymethylcellulose, dextran, derivatives thereof, and any combination thereof. In some cases, the first hydrogel-forming polymer may be selected from the group consisting of hyaluronic acid, chitosan, chondroitin sulfate, alginic acid, carboxymethylcellulose, dextran, derivatives thereof, and any combination thereof.

[0091] In some cases, the first hydrogel-forming polymer in the composition may be selected from the group consisting of dextran, hyaluronic acid, derivatives thereof, and any combination thereof. In some cases, the first hydrogel-forming polymer may be selected from the group consisting of hyaluronic acid, derivatives thereof, and any combination thereof. In some cases, the first hydrogel-forming polymer may be hyaluronic acid.

[0092] In the present disclosure, the second hydrogel-forming polymer may be selected from the group consisting of dextran, hyaluronic acid, chitosan, chondroitin sulfate, alginic acid, carboxymethylcellulose, dextran, derivatives thereof, and any combination thereof. In some cases, the second hydrogel-forming polymer may be selected from the group consisting of hyaluronic acid, chitosan, chondroitin sulfate, alginic acid, carboxymethylcellulose, dextran, derivatives thereof, and any combination thereof.

[0093] In some cases, the second hydrogel-forming polymer in the composition may be selected from the group consisting of dextran, hyaluronic acid, derivatives thereof, and any combination thereof. In some cases, the second hydrogel-forming polymer may be selected from the group consisting of hyaluronic acid, dextran, derivatives thereof, and any combination thereof. In some cases, the first hydrogel-forming polymer may be hyaluronic acid.

[0094] In the present disclosure, the first hydrogel-forming polymer in the composition can be selected from the group consisting of dextran, hyaluronic acid, derivatives thereof, and any combination thereof. The second hydrogel-forming polymer in the composition can be selected from the group consisting of dextran, hyaluronic acid, derivatives thereof, and any combination thereof.

[0095] In some cases, the first hydrogel-forming polymer may be selected from the group consisting of hyaluronic acid and its derivatives, and the second hydrogel-forming polymer in the composition may be selected from the group consisting of dextran, hyaluronic acid, its derivatives, and any combination thereof.In some cases, the first hydrogel-forming polymer may be selected from the group consisting of dextran, hyaluronic acid, and its derivatives, and the second hydrogel-forming polymer in the composition may be selected from the group consisting of hyaluronic acid, its derivatives, and any combination thereof.

[0096] In the present disclosure, the first hydrogel-forming polymer derivative may have a first average degree of modification (first DM) of less than about 40% (e.g., less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 2%, less than about 0.5% or less).

[0097] In some cases, the first hydrogel-forming polymer derivative may have an average DM of about 0% to about 40% (e.g., about 0.001% to about 19.5%, about 0.001% to about 4.9%, about 0.5% to about 5%, about 5.5% to about 19.5%, about 8% to about 19%, about 9% to about 20.0%, about 8.5% to about 18%, or about 8.5% to about 17.5%, about 0.001% to about 39.5%, about 0.001% to about 35%, about 0.001% to about 30%, about 0.001% to about 7.5%, about 9.5% to about 20%, about 20% to about 30%, or about 20% to about 40%, about 10% to about 40%, etc.).

[0098] In the present disclosure, the second hydrogel-forming polymer derivative may have a second average degree of modification (second DM) of less than about 40% (e.g., less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 2%, less than about 0.5% or less).

[0099] In some cases, the second hydrogel-forming polymer derivative may have an average DM of about 0% to about 40% (e.g., about 0.001% to about 19.5%, about 0.001% to about 4.5%, about 0.001% to about 4.9%, about 0.5% to about 5%, about 5% to about 8%, about 5.1% to about 7.9%, about 5.5% to about 19.9%, about 8% to about 19.9%, about 8.1% to about 19.9%, about 8.5% to about 18%, or about 8.5% to about 17.5%, about 20% to about 25%, about 20% to about 30%, about 20% to about 35%, about 20% to about 40%, about 10% to about 40%, etc.).

[0100] In the present disclosure, the ratio between the first DM and the second DM can be about 3:1 to about 1:3 (e.g., about 3:1 to about 1:3, about 3:1.5 to about 1:3, about 3:2 to about 1:3, about 3:2.5 to about 1:3, about 3:1 to about 1:2.5, about 3:1 to about 1:2, about 3:1 to about 1:1.5, about 2.5:1 to about 1:3, about 2:1 to about 1:3, about 1.5:1 to about 1:3, etc.).

[0101] In the present disclosure, the molar ratio between the first hydrogel-forming polymer derivative and the second hydrogel-forming polymer derivative in the composition can be from about 3:1 to about 1:3 (e.g., from about 3:1 to about 1:3, from about 3:1.5 to about 1:3, from about 3:2 to about 1:3, from about 3:2.5 to about 1:3, from about 3:1 to about 1:2.5, from about 3:1 to about 1:2, from about 3:1 to about 1:1.5, from about 2.5:1 to about 1:3, from about 2:1 to about 1:3, from about 1.5:1 to about 1:3, etc.).

[0102] In the present disclosure, the volume ratio between the first hydrogel-forming polymer derivative and the second hydrogel-forming polymer derivative in the composition is from about 10:1 to about 1:10 (e.g., from about 10:1 to about 1:10, from about 8:1 to about 1:10, from about 6:1 to about 1:10, from about 5:1 to about 1:10, from about 4:1 to about 1:10, from about 3:1 to about 1:10, from about 2:1 to about 1:10, from about 1.75:1 to about 1:10, from about 1.5:1 to about 1:10, from about 1.25:1 to about 1:10, from about 1:1 to about 1:10, from about 1:1.25 to about 1:10, from about 1:1.5 to about 1:10, from about 1:1.75 to about The ratio may be about 1:10, about 1:2 to about 1:10, about 1:3 to about 1:10, about 1:4 to about 1:10, about 1:5 to about 1:10, about 6:1 to about 1:6, about 5:1 to about 1:5, about 4:1 to about 1:4, about 3:1 to about 1:3, about 2:1 to about 1:2, about 1.75:1 to about 1:1.75, about 1.5:1 to about 1:1.5, about 1.25:1 to about 1:1.25, or about 1.1:1 to 1:1.1, etc.

[0103] In some cases, the first hydrogel-forming polymer derivative may be modified with one or more vinyl sulfone groups and the second hydrogel-forming polymer derivative may be modified with one or more thiol groups. In some cases, the first hydrogel-forming polymer derivative may be modified with one or more maleimide groups and the second hydrogel-forming polymer derivative may be modified with one or more thiol groups. In some cases, the first hydrogel-forming polymer derivative may be modified with one or more acrylate groups and the second hydrogel-forming polymer derivative may be modified with one or more amine groups. In some cases, the first hydrogel-forming polymer derivative may be modified with one or more methacrylic acid groups and the second hydrogel-forming polymer derivative may be modified with one or more amine groups.

[0104] In the present disclosure, the first hydrogel-forming polymer derivative can be a dextran derivative modified with one or more vinyl sulfone groups, a hyaluronic acid derivative modified with one or more vinyl sulfone groups, a dextran derivative modified with one or more maleimide groups, a hyaluronic acid derivative modified with one or more maleimide groups, a dextran derivative modified with one or more acrylate groups, a hyaluronic acid derivative modified with one or more acrylate groups, a dextran derivative modified with one or more methacrylate groups, a hyaluronic acid derivative modified with one or more methacrylic acid groups, or a combination thereof.

[0105] In the present disclosure, the second hydrogel-forming polymer derivative can be a dextran derivative modified with one or more thiol groups, a hyaluronic acid derivative modified with one or more thiol groups, a dextran derivative modified with one or more amine groups, a hyaluronic acid derivative modified with one or more amine groups, or a combination thereof.

[0106] In the present disclosure, the first hydrogel-forming polymer derivative may be a dextran derivative modified with one or more vinyl sulfone groups, a hyaluronic acid derivative modified with one or more vinyl sulfone groups, a dextran derivative modified with one or more maleimide groups, a hyaluronic acid derivative modified with one or more maleimide groups, a dextran derivative modified with one or more acrylate groups, a hyaluronic acid derivative modified with one or more acrylate groups, a dextran derivative modified with one or more methacrylic acids, a hyaluronic acid derivative modified with one or more methacrylic acids, or a combination thereof, and the second hydrogel-forming polymer derivative may be a dextran derivative modified with one or more thiol groups, a hyaluronic acid derivative modified with one or more thiol groups, a dextran derivative modified with one or more amine groups, a hyaluronic acid derivative modified with one or more amine groups, or a combination thereof.

[0107] In the present disclosure, the first hydrogel-forming polymer derivative may be a dextran derivative modified with one or more vinyl sulfone groups, a hyaluronic acid derivative modified with one or more vinyl sulfone groups, a dextran derivative modified with one or more maleimide groups, a hyaluronic acid derivative modified with one or more maleimide groups, a dextran derivative modified with one or more acrylate groups, a dextran derivative modified with one or more methacrylic acids, a hyaluronic acid derivative modified with one or more methacrylic acids, or a combination thereof, and the second hydrogel-forming polymer derivative may be a hyaluronic acid derivative modified with one or more thiol groups, a dextran derivative modified with one or more thiol groups, a dextran derivative modified with one or more amine groups, a hyaluronic acid derivative modified with one or more amine groups, or a combination thereof.

[0108] In the present disclosure, the first hydrogel-forming polymer derivative may be a hyaluronic acid derivative modified with one or more vinyl sulfone groups, a dextran derivative modified with one or more maleimide groups, a dextran derivative modified with one or more acrylate groups, a hyaluronic acid derivative modified with one or more acrylate groups, a dextran derivative modified with one or more methacrylic acids, a hyaluronic acid derivative modified with one or more methacrylic acids, or a combination thereof, and the second hydrogel-forming polymer derivative may be a dextran derivative modified with one or more thiol groups, a hyaluronic acid derivative modified with one or more thiol groups, a dextran derivative modified with one or more amine groups, a hyaluronic acid derivative modified with one or more amine groups, or a combination thereof.

[0109] For example, the first hydrogel-forming polymer derivative may be a hyaluronic acid derivative modified with one or more maleimide groups, and the second hydrogel-forming polymer derivative may be a dextran derivative modified with one or more thiol groups.

[0110] For example, the first hydrogel-forming polymer derivative may be a dextran derivative modified with one or more maleimide groups, and the second hydrogel-forming polymer derivative may be a hyaluronic acid derivative modified with one or more thiol groups.

[0111] In the present disclosure, the hydrogel is hydrolyzable without the involvement of degradative enzymes.

[0112] In the present disclosure, at least one of the first hydrogel-forming polymer and / or the second hydrogel-forming polymer comprises a degradable linker. In some embodiments, the degradable linker can be hydrolyzable. In other embodiments, hydrolysis can occur at the crosslink.

[0113] In the present disclosure, the degradable linker can include a hydrolyzable functional group, for example, the hydrolyzable functional group can be selected from an ester group, an anhydride group, and an amide group.

[0114] In the present disclosure, the ester group may be selected from an oxyester group and a thiol ester group. For example, an oxyester group may have a functional group of -COOR, and a thiol ester group may have a functional group of R-S-CO-R', which may be the product of esterification of a carboxylic acid with a thiol.

[0115] In the present disclosure, the first hydrogel-forming polymer may be from about 1 kDa to about 500 kDa (e.g., from about 1 kDa to about 500 kDa, from about 3 kDa to about 500 kDa, from about 5 kDa to about 500 kDa, from about 7 kDa to about 500 kDa, from about 10 kDa to about 500 kDa, from about 50 kDa to about 500 kDa, from about 100 kDa to about 500 kDa, from about 150 kDa to about 500 kDa, from about 200 kDa to about 500 kDa, from about 250 kDa to about 500 kDa, from about 300 kDa to about 500 kDa, from about 350 kDa to about 500 kDa, from about 400 kDa to about 500 kDa, from about 450 kDa to about 500 kDa, from about 1 The polymer may have a weight average molecular weight of from about 100 kDa to about 39 kDa, from about 41 kDa to about 200 kDa, or from about 41 kDa to about 500 kDa.

[0116] In some cases, the first hydrogel-forming polymer may have a weight average molecular weight of less than 500 kDa (e.g., less than 490 kDa, less than 480 kDa, less than 450 kDa, less than 400 kDa, less than 300 kDa, less than 200 kDa, less than 150 kDa, less than 100 kDa, less than 50 kDa, less than 40 kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa or less). In some cases, the first hydrogel-forming polymer may have a weight average molecular weight of 1 kDa or greater (e.g., 1 kDa or greater, 5 kDa or greater, 10 kDa or greater, 20 kDa or greater, 30 kDa or greater, 40 kDa or greater, 41 kDa or greater, 45 kDa or greater, 50 kDa or greater, 100 kDa or greater, 200 kDa or greater, 300 kDa or greater, 400 kDa or greater, or greater).

[0117] In the present disclosure, the second hydrogel-forming polymer may be from about 1 kDa to about 500 kDa (e.g., from about 1 kDa to about 500 kDa, from about 3 kDa to about 500 kDa, from about 5 kDa to about 500 kDa, from about 7 kDa to about 500 kDa, from about 10 kDa to about 500 kDa, from about 50 kDa to about 500 kDa, from about 100 kDa to about 500 kDa, from about 150 kDa to about 500 kDa, from about 200 kDa to about 500 kDa, from about 250 kDa to about 500 kDa, from about 300 kDa to about 500 kDa, from about 350 kDa to about 500 kDa, from about 400 kDa to about 500 kDa, from about 450 kDa to about 500 kDa, from about 1 The polymer may have a weight average molecular weight of from about 100 kDa to about 39 kDa, from about 41 kDa to about 200 kDa, or from about 41 kDa to about 500 kDa.

[0118] In some cases, the second hydrogel-forming polymer may have a weight average molecular weight of less than 500 kDa (e.g., less than 490 kDa, less than 480 kDa, less than 450 kDa, less than 400 kDa, less than 300 kDa, less than 200 kDa, less than 150 kDa, less than 100 kDa, less than 50 kDa, less than 40 kDa, less than 30 kDa, less than 20 kDa, less than 10 kDa or less). In some cases, the first hydrogel-forming polymer may have a weight average molecular weight of 1 kDa (e.g., 1 kDa or more, 5 kDa or more, 10 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 41 kDa or more, 45 kDa or more, 50 kDa or more, 100 kDa or more, 200 kDa or more, 300 kDa or more, 400 kDa or more, or more).

[0119] In some cases, the composition may be a powder.

[0120] In some cases, the composition may be a liquid composition, and the concentration of the one or more hydrogel-forming polymers in the liquid composition may be from about 1% w / v to about 30% w / v (e.g., from about 1% w / v to about 50% w / v, from about 5% w / v to about 50% w / v, from about 10% w / v to about 50% w / v, from about 15% w / v to about 50% w / v, from about 20% w / v to about 50% w / v, from about 25% w / v to about 50% w / v, from about 30% w / v to about 50% w / v, from about 35% w / v to about 50% w / v, from about 40% w / v to about 50% w / v, from about 50% w / v to about 50% w / v, from about 1% w / v to about 45% w / v, from about 1% w / v to about 40% w / v, from about 1% w / v to about 35% w / v, or from about 1% The range is from about 30% w / v to about 25% w / v, from about 1% w / v to about 20% w / v, from about 11% w / v to about 15% w / v, from about 1% w / v to about 10% w / v, from about 1% w / v to about 5% w / v, etc.

[0121] In the present disclosure, hydrogel-forming polymers comprising a degradable backbone may be formed by grafting precursor polymers with degradable linkers, which may enable the formation of degradable bonds between the precursor polymers.

[0122] In some cases, the precursor polymer may be hydrophilic and / or water-soluble.

[0123] In some cases, the precursor polymer may be non-hydrolytic, non-enzymatically degradable, or otherwise non-cleavable, e.g., when the degradable linker is degraded by hydrolysis, enzymatically, and other well-defined pathways, the precursor polymer may remain unaffected and maintain the structure of the degradable backbone.

[0124] In the present disclosure, the precursor polymer may be selected from the group consisting of polysaccharides, derivatives thereof, and any combination thereof.

[0125] In some cases, the precursor polymer may be selected from the group consisting of dextran, hyaluronic acid, derivatives thereof, and any combination thereof.

[0126] In the present disclosure, the precursor polymer can be a derivative that includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modifications, and the degree of modification of the precursor polymer is less than about 40% (e.g., less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 18%, less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 4%, less than about 2%, less than about 1% or less).

[0127] In the present disclosure, the modification of the precursor polymer may be selected from the group consisting of acrylate, methacrylic acid, maleimide, vinyl sulfone, thiol, amine, and any combination thereof.

[0128] In the present disclosure, a degradable linker can include two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modifications, and the degree of modification of the degradable linker is less than about 40% (e.g., less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, about 1% or less).

[0129] In the present disclosure, the degradable linker modification may be selected from the group consisting of acrylate, methacrylic acid, maleimide, vinyl sulfone, thiol, amine, and any combination thereof.

[0130] In the present disclosure, the precursor polymer can be a dextran derivative modified with one or more vinyl sulfone groups, a hyaluronic acid derivative modified with one or more vinyl sulfone groups, a derivative modified with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) vinyl sulfone groups, or a combination thereof, and the degradable linker includes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) thiol group modifications. For example, the vinyl sulfone group may have the following functional groups: [ka] .

[0131] In some cases, the precursor polymer may be a hyaluronic acid derivative modified with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) thiol groups, a dextran derivative modified with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) thiol groups, or a combination thereof, and the degradable linker includes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) vinyl sulfone group modifications.

[0132] In the present disclosure, the degradable linker may be selected from divinyl methacrylate, divinyl acrylate, and derivatives thereof.

[0133] In some cases, the degradable linker may be selected from the following group: [ka]

[0134] In the present disclosure, a degradable linker may include a modulator, an ester. In some cases, the degradable linker may further include a linker. In some cases, the ester may be modified by the modulator. For example, one side of the ester may be modified with the modulator, or both sides of the ester may be modified with the modulator. In some cases, a degradable linker having an ester modified on both sides with the modulator may be significantly more stable than a degradable linker having an ester modified on one side with the modulator. In some cases, a degradable linker having an ester modified on both sides with the modulator may exhibit a slower ester hydrolysis rate than a degradable linker having an ester modified on one side with the modulator.

[0135] In some cases, the degradable linker may comprise a modulator, an ester, and a linker. For example, the degradable linker may comprise the format shown in Figure 15.

[0136] In some cases, the two modulators may be the same or different. In some cases, the two modulators may be the same.

[0137] Optionally, the ester may be selected from the following group: [ka]

[0138] In some cases, the modulator may be hydrophobic or hydrophilic. In some cases, a hydrophobic modulator may increase the stability of the degradable linker more than a hydrophilic modulator. In some cases, a hydrophobic modulator may decrease the solubility of the degradable linker in an aqueous environment.

[0139] In some cases, the modulator may be electron withdrawing or electron donating.

[0140] In some cases, the modulator may be selected from the group consisting of: [ka]

[0141] Optionally, the linker may be selected from the following group: [ka]

[0142] In some cases, the hydrogel-forming polymer derivative may include modifications of formula (1), (2), (3), (4), or combinations thereof. [ka]

[0143] wherein P is a polymer, A is a linker or a modifier or a combination of both, and B is a linker or a modifier or a combination of both, which may be the same or different from A; [ka] It is an ester, N is a nucleophile and E is an electrophile.

[0144] In some cases, the concentration of the precursor polymer can affect the hydrolytic degradation of the hydrogel-forming polymer.

[0145] In some cases, the average degree of modification (DM) of the hydrogel-forming polymer (eg, precursor polymer) can affect the hydrolytic degradation of the hydrogel-forming polymer.

[0146] In some cases, the average molecular weight (Mw) of the hydrogel-forming polymer (eg, precursor polymer) can affect the hydrolytic degradation of the hydrogel-forming polymer.

[0147] In another aspect, the present disclosure provides a hydrogel for the sustained release of a targeted agent, wherein the hydrogel can be formed with a composition.

[0148] In the present disclosure, hydrogels can dissociate when the precursor polymers or crosslinkers are degraded. In some cases, the degradation products of the hydrogel can have a wide range of molecular weights.

[0149] In the present disclosure, the release of proteins from hydrogel networks before and after crosslink degradation can be shown in Figure 13, where the lines represent the polymer network, the dotted lines represent the polymer after crosslink degradation, the light background represents water, the triangular objects represent proteins, and the filled circles represent crosslinks.

[0150] In the present disclosure, the hydrogel may further comprise a targeting agent.

[0151] In some cases, the targeting agent comprises a macromolecule of at least about 80 kDa molecular weight, e.g., at least about 80 kDa molecular weight, at least about 90 kDa molecular weight, at least about 100 kDa molecular weight, at least about 120 kDa molecular weight, at least about 150 kDa molecular weight, at least about 180 kDa molecular weight, at least about 200 kDa molecular weight, at least about 250 kDa molecular weight, at least about 300 kDa molecular weight, or more.

[0152] In some cases, the targeting agent comprises a macromolecule. For example, the targeting agent may comprise a protein or polypeptide.

[0153] In the present disclosure, at least about 20% (e.g., at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 96%, at least about 98%, at least about 99%, or more) of the targeting agent can be free targeting agent (e.g., protein) that is not bound to the hydrogel. In some embodiments, at least about 80% (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 96%, at least about 98%, at least about 99%, or more) of the targeting agent can be free targeting agent (e.g., protein) that is not bound to the hydrogel.

[0154] In the present disclosure, less than about 50% (e.g., less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less) of the targeting agent may be cumulatively released from the hydrogel within the first 24 hours (e.g., within the first 24 hours, within 22 hours, within 20 hours, within 18 hours, within 16 hours, within 14 hours, within 12 hours, within 10 hours, within 8 hours, within 6 hours, within 4 hours, within 2 hours), and the remainder of the targeting agent may be cumulatively released from the hydrogel within about 1 to about 36 months (e.g., about 1 to about 36 months, about 1 to about The compound may be cumulatively released from the hydrogel in about 30 months, about 1 to about 24 months, about 1 to about 18 months, about 1 to about 12 months, about 1 to about 10 months, about 1 to about 9 months, about 1 to about 8 months, about 1 to about 7 months, about 1 to about 6 months, about 1 to about 5 months, about 1 to about 4 months, about 1 to about 2 months, about 1 to about 3 months, about 4 to about 36 months, about 5 to about 36 months, about 6 to about 36 months, about 7 to about 36 months, about 8 to about 36 months, about 9 to about 36 months, about 10 to about 36 months, about 12 to about 36 months, about 14 to about 36 months, about 16 to about 36 months, about 18 to about 36 months, about 18 to about 24 months, about 20 to about 24 months, or about 22 to about 24 months.

[0155] In the present disclosure, the targeting agent may be cumulatively released from the hydrogel in more than 1 day, more than 1 week, more than 1 month, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 7 months, more than 8 months, more than 9 months, more than 10 months, more than 11 months, more than 12 months, more than 24 months, or more than 36 months.

[0156] In the present disclosure, the first 24 hours (e.g., within the first 24 hours, within 22 hours, within 20 hours, within 18 hours, within 16 hours, within 14 hours, within 12 hours, within 10 hours, within 8 hours, within 6 hours, within 4 hours, within 2 hours) can start from the time the hydrogel containing the targeting agent is formed.

[0157] In the present disclosure, the hydrogel may be a preformed hydrogel or a composition of polymers that form a hydrogel in vivo upon mixing and injection. In some cases, the hydrogel may be a micron-sized hydrogel (micronized hydrogel) or a regular hydrogel about centimeters or larger in size (macroscopic hydrogel). In other cases, the hydrogel or polymer solvent may include a micronized hydrogel (micronized hydrogel in a macroscopic hydrogel).

[0158] In some cases, the solvent in the hydrogel microspheres described above may contain the protein or may contain a micronized hydrogel that encapsulates the protein.

[0159] In some cases, the macroscopic hydrogel may entrap the micronized hydrogel. In some cases, the micronized hydrogel may physically entrap the macromolecule.

[0160] For example, the hydrogel may include an in situ-formed macroscopic hydrogel and a preformed micronized hydrogel (FIG. 14). The in situ-formed macroscopic hydrogel may encapsulate the preformed micronized hydrogel, which may physically encapsulate macromolecules.

[0161] In another aspect, the present disclosure provides methods for producing a hydrogel, the methods may include: a) providing a composition of the present disclosure; b) mixing the composition with a buffer to form a polymer solution; and c) subjecting the polymer solution to conditions that allow for the formation of a hydrogel.

[0162] In the present disclosure, the subject may include injecting a polymer solution into a subject in need thereof.

[0163] In some cases, the subject may administer the composition at a temperature between about 1°C and about 45°C (e.g., between about 1°C and about 10°C, between about 1°C and about 8°C, between about 1°C and about 6°C, between about 2°C and about 6°C, between about 3°C ​​and about 5°C, between about 1°C and about 45°C, between about 2°C and about 45°C, between about 3°C ​​and about 45°C, between about 4°C and about 45°C, between about 6°C and about 45°C, between about 8°C and about 45°C, between about 10°C and about 45°C, between about 15°C and about 45°C, between about 15°C and about 40°C, between about 20°C and about 37°C, between about 20°C and about 45°C, between about 25°C and about 45°C, between about 30°C and about 45°C). °C, about 31°C to about 45°C, about 32°C to about 45°C, about 33°C to about 45°C, about 34°C to about 45°C, about 35°C to about 45°C, about 36°C to about 45°C, about 37°C to about 45°C, about 38°C to about 45°C, about 39°C to about 45°C, about 40°C to about 45°C, about 41°C to about 45°C, about 42°C to about 45°C, about 43°C to about 45°C, or about 44°C to about 45°C, etc.)

[0164] In the present disclosure, the polymer solution may further include a targeting agent.

[0165] In some embodiments, the second hydrogel-forming polymer may be free of DX-O(Me)-DTT.

[0166] In another aspect, the present disclosure provides methods for making a composition, which may include: a) grafting precursor polymers with a degradable linker to obtain a first hydrogel-forming polymer and / or a second hydrogel-forming polymer; and b) combining the first hydrogel-forming polymer and / or the second hydrogel-forming polymer with an additional polymer (e.g., the second hydrogel-forming polymer or the first hydrogel-forming polymer) under conditions that allow for the formation of a hydrogel.

[0167] In some embodiments, steps a), b), and c) may be performed one or more times (e.g., one, two, three or more times). For example, steps a), b), and c) may be performed once to produce a macroscopic hydrogel or a micronized hydrogel. In another example, steps a), b), and c) may be performed three times to produce a micronized hydrogel in a macroscopic hydrogel.

[0168] In another aspect, the present disclosure provides a method for sustained release of a targeted agent, the method may include mixing the targeted agent with a composition to obtain a mixture, and subjecting the mixture to conditions that allow for the formation of a hydrogel capable of sustained release of the targeted agent.

[0169] In another aspect, the present disclosure provides a method for the sustained release of a targeted agent, which may include entrapment of the targeted agent in a hydrogel.

[0170] In some cases, the method comprises heating the composition to a temperature of from about 1° C. to about 45° C. (e.g., from about 1° C. to about 10° C., from about 1° C. to about 8° C., from about 1° C. to about 6° C., from about 2° C. to about 6° C., from about 3° C. to about 5° C., from about 1° C. to about 45° C., from about 2° C. to about 45° C., from about 3° C. to about 45° C., from about 4° C. to about 45° C., from about 6° C. to about 45° C., from about 8° C. to about 45° C., from about 10° C. to about 45° C., from about 15° C. to about 45° C., from about 15° C. to about 40° C., from about 20° C. to about 37° C., from about 20° C. to about 45° C., from about 25° C. to about 45° C., from about 30° C. to about 45° C. 31°C to about 45°C, about 32°C to about 45°C, about 33°C to about 45°C, about 34°C to about 45°C, about 35°C to about 45°C, about 36°C to about 45°C, about 37°C to about 45°C, about 38°C to about 45°C, about 39°C to about 45°C, about 40°C to about 45°C, about 41°C to about 45°C, about 42°C to about 45°C, about 43°C to about 45°C, or about 44°C to about 45°C, etc.

[0171] In some embodiments, the method may include incubating the composition at about 1°C to about 45°C (e.g., about 1°C to about 10°C, about 1°C to about 8°C, about 1°C to about 6°C, about 2°C to about 6°C, about 3°C ​​to about 5°C, about 1°C to about 15°C, about 1°C to about 20°C, about 1°C to about 30°C, about 1°C to about 40°C, about 32°C to about 40°C, about 35°C to about 40°C, e.g., about 37°C).

[0172] In another aspect, the present disclosure provides a kit, which may include: a) a composition; and b) a targeting agent that is sustained-released by a hydrogel formed using the composition of a).

[0173] In some cases, the kit may further comprise one or more of a stabilizer, bulking agent, filler, diluent, anti-adherent agent, binder, coating agent, colorant, disintegrant, flavor, fragrance, lubricant, and / or antioxidant.

[0174] In another aspect, the present disclosure provides a use of the composition for preparing a hydrogel.

[0175] In another aspect, the present disclosure provides the use of the composition or hydrogel for the sustained release of a targeted agent.

[0176] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present disclosure are shown and described herein. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description should be regarded as illustrative in nature and not restrictive.

[0177] Example

[0178] The following examples are provided to provide those of skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent the following experiments as complete or exclusive. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric. Standard abbreviations may be used, such as bp, base pairs; kb, kilobases; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; nt, nucleotides; im, intramuscular; ip, intraperitoneal; sc, subcutaneous, etc.

[0179] Example 1: Conjugation of vinyl sulfone (VS) and thiol (SH) groups to dextran or hyaluronic acid via a non-hydrolyzable linker

[0180] Dextran (DX) and hyaluronic acid (HA) were functionalized with vinyl sulfone (VS) and thiol (SH) using a previously reported method (see Y. Yu and Y. Chau, "One-step 'click' method for generating vinyl sulfone groups on hydroxyl-containing water-soluble polymers," Biomacromolecules, vol. 13, pp. 937-942, 2012). Briefly, three dextrans with molecular weights of 150 kDa (Wako), 40 kDa (Sigma), and 6 kDa (Sigma), or hyaluronic acids with molecular weights of 29 kDa and 150 kDa, were added to the hydroxyl groups in 0.02 M sodium hydroxide solution (DX) and 0.1 M sodium hydroxide solution (HA) by stirring (Figure 1). VS pendant groups were grafted onto the dextran by reacting with divinyl sulfone (DVS, 97% of which contained less than 650 ppm of hydroquinone as an inhibitor, Aldrich) (1.2–1.5 equivalents relative to the hydroxyl). The reaction was stopped by adding concentrated HCl to lower the reaction pH below 5, and the degree of VS modification was controlled by the reaction time. The product was purified by dialysis (Spectra / Por™ cellulose membrane, 7 kD MWCO, Spectra) against deionized water at ambient temperature to remove excess DVS, followed by lyophilization. The lyophilized product was stored below −20°C until use. The degree of modification (DM) was calculated as the number of VS groups per pyranose unit of dextran or per disaccharide unit of HA. The DM of the VS group was determined using the residual internal HDO (δ 4.75, 300 MHz). 1The relative amounts of vinyl protons were estimated from H NMR spectroscopy: δ 6.27–6.44 (q, 2H, =CH2), δ 6.82–6.97 (m, 1H, -CH=), relative amounts of pyranose: δ 4.87–5.29 (m, 1H of C1), and relative amounts of disaccharides: δ 2.0 (m, 3H, -CH3).

[0181] Nonhydrolyzable DX-SH was synthesized by reacting DX-VS with hydrophobic thiol donors, namely, dithiothreitol (DTT, 99%, J&K) and 1,3-propanedithiol (PDT, 99%, Sigma-Aldrich). For DTT conjugation, DX-VS was dissolved in 0.1 M phosphate buffer (pH 7.4) and purged with nitrogen gas to remove dissolved oxygen. DTT was dissolved in water and then added to the DX-VS solution in excess (6 equivalents) relative to the VS group. The reaction was allowed to proceed under stirring at ambient temperature for 2 hours. The reaction was terminated by lowering the pH to 3 with (1 M) dilute hydrochloric acid. Excess DTT was removed by dialysis (7 kD MWCO) against a dilute HCl solution in deionized water (pH = 3) and then lyophilized. For PDT conjugation, DX-VS was dissolved in dimethylformamide with 2% lithium chloride (DMF / 2% LiCl) in an oil bath at 90 °C, then cooled to ambient temperature and purged with nitrogen. PDT was added in excess (6 equiv.) to VS, and triethylamine (TEA, 99%, Sigma-Aldrich) was added as a catalyst (0.5 equiv.). The mixture was reacted for 1 h, and the product was precipitated in isopropanol. The pellet was resuspended in water and further purified by dialysis as described for DX-DTT.

[0182] 1Complete reaction of VS was confirmed by the disappearance of VS-related peaks in the H NMR spectrum. The actual DM of the thiol groups on DX-SH was determined by Ellman's assay (see Yu Y, Chau Y. "Formulation of in situ chemically cross-linked hydrogel depots for protein release: from the blob model perspective" Biomacromolecules. 2015;16 (1) :56-65).

[0183] Example 2 Conjugation of thiols (SH) to dextran using hydrolyzable ester linkers

[0184] 2.1 Synthesis of Acrylic Acid-Functionalized Dextran (DX-O-CA) via Ester Linkages. Dextran functionalized with chloroacetyl groups (DX-O-CA) was synthesized according to the method of Ramirez (Figure 2; see Ramirez JC, Sanchez-Chaves M, Arranz F., "Functionalization of dextran with chloroacetate groups: immobilization of bioactive carboxylic acids". Polymer (Guildf). 1994;35(12):2651-2655. doi:10.1016 / 0032-3861(94)90394-8). Briefly, dextran (40 kDa) was dissolved in DMF / 2% LiCl in an oil bath at 90 °C. The dextran solution was cooled to ambient temperature, and then pyridine (99%, VWR Chemicals BDH) was added to the solution (1 equivalent relative to the OH group of dextran). Chloroacetyl chloride (99%, Sigma) was added (0.1–0.5 equivalents relative to the OH group of dextran) and the reaction was allowed to proceed for 2–6 hours. The DM could be controlled by the amount of chloroacetyl chloride and the reaction time. The product, DX-O-CA, was purified by reprecipitation in isopropanol and dried in vacuo. The DM of the acrylate was quantified using 1H NMR: chloroacetic acid: δ 4.29–4.37 (m, 2H, –CH2–).

[0185] 2.2 Binding of thiol donor to DX-O-CA

[0186] Dried DX-O-CA was dissolved in 0.5 M phosphate buffer (pH 7.4) and then purged with nitrogen gas. Aqueous DTT solution (6–10 equivalents relative to CA) was added to DX-O-CA and reacted at ambient temperature for two hours (Figure 2). The reaction was stopped by adding dilute hydrochloric acid, and the reaction pH was lowered to 4. Excess DTT was removed by dialysis (7 kD MWCO) with deionized water and lyophilized. The DM of thiols was quantified using the Ellman assay.

[0187] 2.3 Synthesis of methacrylate-functionalized dextran (DX-O-MeA) via ester linkage

[0188] Methacrylate esters were conjugated to dextran via an oxyester bond according to the protocol of Kim and Chu (Figure 2). Briefly, dextran (150 kDa or 40 kDa) was dissolved in DMF / 2% LiCl (5 w / v%) in a 90 °C oil bath and cooled to ambient temperature. Methacrylic anhydride (MA, 94%, Aldrich) was added (0.3–0.5× the pyranose), followed by the addition of catalytic TEA (0.01–0.1 equivalents to MA). The reaction proceeded overnight at ambient temperature with stirring. The intermediate dextran-methacrylic acid (DX-O-MeA) was precipitated three times with isopropanol, and the pellet was vacuum-dried. The dried pellet was resuspended in water and further purified by dialysis (7 kD MWCO) against deionized water and lyophilized. DMSO of methacrylic acid was added. 1 Quantification was performed using H NMR spectroscopy: vinyl protons: δ 5.71–6.20 (d, 2 H, ═CH 2 ), methyl protons: δ 1.9 (m, 3 H, −CH 3 ).

[0189] 2.4 Binding of thiol donor to DX-O-MeA

[0190] Lyophilized DX-O-MeA was dissolved in DMSO at 2–5% w / v and purged with nitrogen gas. Four thiol donors with different hydrophobicities: 1,2-ethanedithiol (EDT, 98%, Sigma-Aldrich); 1,3-propanedithiol (PDT, 99%, Sigma-Aldrich); and 2,3-dimercapto-1-propanol (DMP, 98%, Sigma-Aldrich); and DTT were conjugated to DX-O-MeA via TEA (0.5 eq to MA)-catalyzed Michael addition. The thiol donors were added in excess (6–10 equivalents relative to MA) and reacted for 1 h at ambient temperature with stirring (Figure 2). The thiolated dextran was collected and purified using the same method as for DX-O-MeA. Complete consumption of MA was confirmed by the Michael addition of 0.5 eq to MA. 1 The DM of the thiol group was determined by Ellman's assay.

[0191] 2.5 Conjugation of vinyl acrylate (VA) and vinyl methacrylate (VMA) to dextran

[0192] Vinyl acrylate (VA) and vinyl methacrylate (VMA) were conjugated to dextran as shown in Figure 3. Nonhydrolyzable DX-DTT or DX-PDT (obtained from Example 1) was dissolved in dimethyl sulfoxide (DMSO, 99%, Sigma-Aldrich) at 2–5% w / v and purged with nitrogen. Vinyl acrylate (VA, 98%, Sigma-Aldrich) or vinyl methacrylate (VMA, 98%, Sigma-Aldrich) was added in excess (10–20 equivalents relative to SH). TEA was added as a catalyst at a final concentration of 0.5% v / v. The reaction was carried out at ambient temperature for 1 h with stirring. The polymer was precipitated in isopropanol, and the pellet was briefly dried under vacuum, redissolved in deionized water, and further purified by dialysis (7 kD MWCO) against deionized water, followed by lyophilization. The vinyl DM was determined using 1H NMR spectroscopy: vinyl protons: δ 7.10-7.22 (day, 1H). These two polymers were named DX-SH-VA and DX-SH-VMA.

[0193] 2.6 Binding of thiol groups to DX-SH-VA and DX-SH-VMA

[0194] Dried DX-SH-VA or DX-SH-VMA was dissolved in DMOS at 2–5% w / v and purged with nitrogen gas. The radical initiator I-2959 (Irgacure-2959, 98% Sigma-Aldrich) was added to a final concentration of 0.5% w / v. A thiol donor (PDT or DTT) was added in excess (10 equivalents relative to the vinyl) and conjugated to the vinyl group via radical thiol-ene addition. The reaction proceeded in a quartz tube under UV-A (354 nm) irradiation at ambient temperature for 3 h with stirring. The final product was purified by precipitation and dialysis and lyophilized as in the previous example. The DM of the thiol was quantified using the Ellman assay.

[0195] The resulting modified dextrans with various hydrolyzable ester linkers are shown in Table 1. [Table 1]

[0196] Polymers were abbreviated as [polymer, molecular weight, functional group, DM]. For example, VS-modified dextran with 40 kDa and 5% DM was designated as DX 40 k-VS_5 and DX 40 k-DTT_5. -SH-functionalized dextran with Easter linker was abbreviated as "DX-O-SH."

[0197] Example 3 Synthesis of hydrolyzable maleimide-modified hyaluronic acid (HA-MI)

[0198] Hyaluronic acid (HA) with a molecular weight of 27 kDa was obtained from Contipro as (Dolny Dobruk). The molecule contains a maleimide group (MI molecule) (structure: [ka] ) was provided by the Southern University of Science and Technology of China, a contract research organization. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) was obtained from Aladdin Biotechnology. Non-hydrolyzable thiol-modified dextran (DX-SH) was synthesized as in Example 1.

[0199] 27 kDa HA was dissolved in 1 mM PB at a concentration of 24 mg / ml. MI molecules were dissolved in 1 mM PB at a concentration of 9.72 mg / ml. After dissolution was complete, equal volumes of HA and MI solutions were mixed in a 20 ml glass scintillation vial by stirring, with 2 ml of each solution added. The pH was then adjusted by adding 400 μl or 800 μL of 0.1 M NaOH solution dropwise before adding 66.4 mg of DMTMM. The molar ratio of -COOH and -NH2 from HA to MI to DMTMM was 1:0.5:2. The reaction was stopped at 72 h by adding 160 μL of 25% NaCl and precipitation in 20 mL of ethanol in a 50 mL conical tube. The precipitate was isolated by centrifugation at 8000 rpm for 5 minutes and decanting the supernatant. The residual pellet was redissolved in 10 mL of DI water and purified by dialysis in 4 L of 0.6 mM HCl solution (pH=4) for an additional 3 days. The dialysis buffer was changed twice daily. After lyophilization for 2 days, a white, cotton-like solid was obtained. The structure of the product was 1 It was characterized by H NMR, and the results are shown in Figure 16. HA-MI was successfully synthesized.

[0200] Example 4 Preparation of Hydrogel

[0201] Hydrogels can be in three forms: macroscopic hydrogels, micronized hydrogels, or micronized hydrogels in macroscopic hydrogels (Figure 4).

[0202] 4.1 Preparation of blank and protein-containing hydrogels

[0203] Blank hydrogels were formed by mixing different -VS and -SH functionalized polymers at a 1:1 volume ratio. The -VS functionalized hydrogel precursor (DX-VS) was dissolved in pH 7 PBS. The thiol-functionalized polymers (DX-DTT and hydrolyzable DX-O-SH) were dissolved in water to minimize disulfide cross-linking during dissolution. The precursor polymers were thoroughly mixed at 4°C and pipetted onto a hydrophobic surface as approximately 30–50 μL hemispherical droplets, then incubated overnight in a humid chamber at ambient temperature. The wet weight of the hemispherical hydrogel in the relaxed state was defined as the initial weight, W0. Two IgG proteins were used: bevacizumab (Avastin™, Roche Ltd., Basel, Switzerland) and IgG-FITC (from human serum, Sigma-Aldrich). Protein-loaded hydrogels were formed using the same method as the blank hydrogels, except that the VS polymer was dissolved in a pH-adjusted protein solution (pH = 7). Unless otherwise specified, the -VS polymer was mixed with the -SH polymer in a mass ratio of 1:2 to minimize undesired reactions between the loaded protein and remaining VS groups. For the formulations used in vivo, HA-VS was dissolved in a pH-adjusted Avastin solution, the pH of which was approximately 7, by adding 1 / 10 volume of 0.4 M NaHPO buffer, unless otherwise specified. DX-SH was dissolved directly in the Avastin solution.

[0204] 4.2 Preparation of micronized hydrogel

[0205] The dissolved HA-VS and DX-SH were dissolved in the Avastin solution as described in Example 3.1 and mixed thoroughly. Approximately 400 μL was transferred to 20 mL of oil phase and stirred at maximum speed using a conventional vortex mixer at ambient temperature for 1 hour to form a micronized hydrogel (microgel). The oil phase was a mixture of SPAN-80 / TWEEN-80 / n-heptane in a volume ratio of 2:1:97. After briefly spinning down the microgel, the supernatant oil phase was discarded. The microgel was washed successively with excess absolute ethanol and DI water six times each. The microgel was collected after each washing step using centrifugation at less than 5000 rpm. Avastin was then added to the microgel, which was then stored at 4°C until use.

[0206] Alternatively, the particles can be prepared by a microfluidic device.

[0207] 4.3 Preparation of micronized hydrogel in macroscopic hydrogel

[0208] HA-VS was dissolved in 0.1 M phosphate buffer (pH 7.4), and DX-SH was dissolved in DI water. The two components were thoroughly mixed and temporarily stored on ice. This mixture was used as the macrogel precursor. The microgel prepared according to Example 3.2 was transferred to a centrifuge tube. Excess Avastin solution was removed by pipetting, and the microgel was weighed. The liquid macrogel precursor was added to the microgel in a 1:1 weight ratio and mixed. For in vivo studies, the mixture was injected into rabbit vitreous chambers. For in vitro release and degradation / swelling studies, the mixture was pipetted onto a hydrophobic surface as approximately 30-50 μl hemispherical droplets and then incubated overnight in a humidified chamber at ambient temperature.

[0209] Example 5 Measurement of Hydrogel Swelling and Degradation

[0210] The hydrogel was placed in a 2 ml centrifuge tube and incubated at 37°C with 1 ml of PBS containing 0.02 w / v% NaN3 as the swelling buffer. At predetermined time points, the hydrogel was removed from the swelling buffer, carefully blotted dry with tissue paper, and weighed. The swelling ratio (Q w ) is the weight of the hydrogel before swelling (W0) versus time t (W t ) was defined as the wet weight at 100°C.

[0211] Example 6 Measurement of the hydrolysis rate of hydrolyzable polymers

[0212] The ester hydrolysis rates of these hydrolyzable hydrogel precursors were determined in DO as previously described. 1 H NMR was used to measure (Lau CML, Jahanmir G, Chau Y. “Local environment-dependent kinetics of ester hydrolysis revealed by direct 1 "H NMR measurement of degrading hydrogels." Acta Biomater. October 2019. Briefly, the sample polymer was dissolved in 0.2 M phosphate buffer (pD 7.7) prepared using DO (99.8 atomic % DO, J&K) as the solvent and incubated at 37 °C. 1 H NMR spectra were recorded periodically using a Varian mercury 300 MHz high-resolution NMR spectrometer VNMRJ 2.2 D (Agilent, USA). The ester hydrolysis rate was calculated from the change in the ester adjacent methyl integral.

[0213] The pKa values ​​were predicted using the online platform Chemicalize developed by ChemAxon (https: / / chemicalize.com / ).

[0214] Example 7 Controlling Hydrogel Degradation by Tuning Ester Linker Chemistry

[0215] The structures and calculated hydrolysis half-lives of various dextran-conjugated ester conjugates are summarized and compared (Table 2). 1 The t of esters is determined based on the characteristic chemical shifts of the methyl groups adjacent to the ester in the H NMR spectrum. 0.5 was measured. [Table 2]

[0216] DX-O-DTT has simple ester chemistry, with the ester directly attached to the dextran pyranose. DX-O(Me)-DTT differs from DX-O-DTT in that it has one more carbon atom next to the carbonyl group and a methyl group. The increased methyl group, along with its hydrophobic and electron-donating effects, increased the degradation time from 8 hours to 2 weeks (Figure 5). To further increase the hydrolysis half-life, the more hydrophobic thiol donor PDT was used instead of DTT. The increased hydrophobicity contributes to the ester t 0.5 The time to maturity was further extended from approximately 5.6 days to approximately 7.4 days (Table 2).

[0217] All of the above hydrogel formulations degraded within a few weeks, which was too short for most controlled release applications. -This is attributed to the limitation that only the carbonyl side of the ester linker is modified, which affects only the carbonyl sensitivity to hydroxyl groups. However, the tendency for the alkyloxy side to leave remains unchanged. Considering that the most acidic pKa of the dextran hydroxy group is 11.8 at 37°C (see Larsen C. "Macromolecular prodrugs. XIII. Determination of the ionization constant of dextran by potentiometric titration and from kinetic analysis of the hydrolysis of dextran indomethacin ester conjugates". Int J Pharm. 1989 52(1):55-61), any modification, such as the addition of an electron-withdrawing neighboring group that increases the pKa of the hydroxyl, would extend the half-life, and vice versa. Following this principle, two leaving groups, 1-(hydroxymethylthio)-4-mercapto-2,3-butanediol and (3-mercaptopropylthio)methanol, with pKa values ​​of 15.8 and 15.6, respectively, were designed.

[0218] The experimentally measured half-life of DX-DTT-VMA-DTT was approximately 54 d, and that of DX-DTT-VMA-PDT was 53 d. The results are in good agreement with the general understanding of the reaction mechanism, in that a poorer leaving group (R-OH) extended the ester half-life. In addition, when the alkyloxy side was stabilized with DTT, hydrophobic modulators on the carbonyl side showed a synergistic effect, as the half-life of DX-PDT-VMA-DTT was approximately 113 d (Table 2).

[0219] Example 8: Preventing covalent binding of proteins to polymer networks

[0220] 8.1 Protein Integrity Analysis by Native SDS-PAGE

[0221] The IgG-loaded (F—IgG and bevacizumab) hydrogels in Table 3 were obtained as described in Example 3.1 with various VS polymer / SH polymer ratios.

[0222] The hydrogel was placed in a 2 ml tube, and 1 ml of PBS was added to the gel. The tube was then incubated at 37°C until the gel was completely degraded. Non-reducing SDS-PAGE of the degradation products was performed to evaluate the MW of the proteins after degradation. F-IgG and F-IgG in PBS were dissolved in 30% DX-VS in 5% DM, while Avastin and DX-VS in 5% DM with Avastin at 30% were used as controls. SDS-PAGE experiments were performed using precast 4-15% gradient gels (BeyoGel Plus PAGE, Beyotime, China) with the Mini-PROTEAN system (Bio-Rad Laboratories, USA) according to the manufacturer's guidelines. Proteins were stained with Coomassie Blue (BeyoBlue, China) for prestained protein ladders (BeyoColor 6.5-270 kDa, Beyotime, China) (or FITC-IgG, imaged in UV mode for F-IgG). To quantify the percentage of free protein in the gel, the band intensity of released IgG was compared to that of unencapsulated IgG protein using ImageJ 1.52 following an online tutorial ( https: / / di.uq.edu.au / community-and-alumni / sparq-ed / sparq-ed-services / using-imagej-quantify-blots ).

[0223] 8.2 Experimental results

[0224] To assess the protein size after release from fully degraded hydrogels, non-reducing SDS-PAGE was performed (Figures 6-7). Hydrolytic hydrogels were prepared by mixing DX 40 kJ-VS and DX 40 kJ / (Me)-DTT, both with 5% DM, at different concentrations (Table 3 and Figure 6). The loading and incubation of F-IgG were the same as described above. After all hydrogels were completely degraded, a crude mixture of F-IgG and the degradation products was subjected to non-reducing SDS-PAGE analysis without purification. Native F-IgG and F-IgG with native DX 40 kJ were included as controls. The PAGE gels were imaged under bright field (Figure 6A) and UV (Figure 6B), respectively. Most protein was captured in the wells when the VS polymer / SH polymer mass ratio (hereafter referred to as the VS / SH ratio) was higher than 0.67 from chemical conjugation to the hydrogel precursor. Reducing the VS / SH ratio to 0.67 was effective in preventing undesired VS-amine coupling and preserving the loaded proteins in their native conformation because thiols have a higher reactivity selectivity for vinyl sulfones than for amines. Because the commercially available F-IgG is polyclonal and contains BSA as a stabilizer (not mentioned in the product description, but clarified by technical support), multiple bands were observed on the SDS-PAGE gel.

[0225] The monoclonal antibody bevacizumab released from hydrogels consisting of DX 40 kJ-VS and DX 40 kJ-O(Me)-DTT with 5% DM was analyzed at different concentrations using the same method. The results were similar: for protein to not bind to the polymer, the VS / SH ratio should be less than 1 (Table 4 and Figure 7). Compared to lane 7, the amounts of free protein in lanes 2 to 6 were 99.1%, 97%, 98.9%, 90.7%, and 12.3%, respectively. [Table 3] [Table 4]

[0226] Example 9 Measurement of protein release from hydrogel depots

[0227] The hydrogels were placed in 2 ml or 4 ml tubes, and 1 ml of PBS containing 0.02 w / v% NaN3 was used as the release buffer. NaN3 was added to prevent bacterial growth in the release buffer during long-term incubation. Unless otherwise specified, the pH of PBS was 7.4. In some cases, the pH was adjusted to 4.5. At each time point, the release buffer was removed and replaced with fresh buffer. The drug concentration in the release buffer was measured using Bradford's Assay (Bio-Rad Laboratories, Inc., California, USA) according to the manufacturer's instructions. The concentration of F-IgG was measured spectrophotometrically at 490 / 520 nm excitation / emission using a 96-well plate. Fluorescence intensity-IgG concentration standard curves were established using PBS at pH 4.5 and pH 7.4, respectively.

[0228] Example 10. Controlling the initial release by manipulating the hydrogel mesh size

[0229] The average mesh size of the hydrogel (ξ avg ) and its polydispersity are theoretically considered to be important parameters governing the diffusion behavior of solute molecules in polymer networks. We investigated the cumulative release of the model protein bevacizumab from non-hydrolytic hydrogels with varying polymer concentrations, and compared the initial release and ξ avg The relationship between ξ avg was regulated by changing the polymer concentration only in the relaxed state (Table 5). The molecular weight and DM were the same in the different groups. By increasing the polymer concentration from 9% to 30% w / v, the initial release rate on the first day was controlled from 90% to only 10% (Figure 8). [Table 5]

[0230] Example 11 Sustained protein release by cross-linking degradation

[0231] The protein release behavior consisted of two phases. In the initial phase, the protein was released from the hydrogel, and the release rate was related to the polymer concentration. All hydrogel formulations showed a second phase in which the protein was not released from the gel or was released at a very slow rate.

[0232] Hydrolytic gels using DX 40 kO (Me)-DTT to crosslink DX-VS were synthesized as described in Example 3. The formulations of polymer concentration and VS / SH ratio are shown in Table 6. F-IgG was used as a model protein for all hydrogels. [Table 6]

[0233] The ester in DX-O(Me)-DTT, when crosslinked with DX-VS, has a hydrolysis half-life of approximately 5.6 d in solution (Table 6) and 2.9 d in hydrogel form (at 37°C at pH 7.4) (see au CML, Jahanmir G, Chau Y. Chau Y. "Local environment-dependent kinetics of ester hydrolysis revealed by direct 1H NMR measurement of degrading hydrogels," Acta Biomater, October 2019).

[0234] Because the polymer matrix of hydrogels is highly hydrated, the hydrolytic cleavage rate is expected to be ubiquitous throughout the hydrogel. Therefore, hydrolytic hydrogels degrade primarily in the bulk rather than at the surface. Random cleavage of esters in crosslinks results in a decrease in the effective number of crosslinks. According to Flory's model (see Peppas NA, Lustig SR, Solute Diffusion in Hydrophilic Network Structures. In: Peppas NA, ed. Hydrogels in Medicine and Pharmacy. Vol. 1. Fundamentals. Boca Raton, FL: CRC Press; 1986:57–83), a decrease in the number of crosslinks decreases the elastic energy of the gel, and the swelling ratio increases accordingly. Therefore, we used the change in swelling ratio over time as an indicator of bulk erosion (Figure 9A).

[0235] In all formulations, a portion of the protein was not released or was released at an almost undetectable rate for the nondegradable gels (Figure 8), whereas all of the protein was released in the hydrogels containing hydrolytic crosslinks (Figure 9B). When the DM and molecular weight of the precursor polymers were kept constant, increasing the total polymer concentration from 15% to 30% w / v reduced the initial release and resulted in a smoother degradation-driven release profile (Figure 9B).

[0236] When the total polymer concentration was maintained at 30% w / v, the DM was increased from 5% to 8%, the molecular weight of DX-VS increased from 40 kDa to 150 kDa, and the gel lifetime was extended from 12 to more than 30 days ( Figure 9A ).

[0237] The release behavior of loaded IgG was similar between the degradable and non-hydrolyzable hydrogels in the early stage, and then the release curves diverged. IgG molecules were gradually released from the hydrolyzable hydrogel until the mesh structure completely collapsed, whereas the IgG release rate was very low for the non-hydrolyzable hydrogel (Figure 9B).

[0238] We further investigated the relationship between hydrogel degradation and protein release by changing the pH of the release buffer. Lowering the pH from 7.4 to 4.5 suppressed OH-catalyzed hydrolytic cleavage, which was reflected in the change in swelling rate. Except for the initial release phase, the protein release rate significantly decreased at pH 4.5 but significantly accelerated at pH 7.4.

[0239] Alternating pH between 7.4 and 4.5 resulted in a fast / slow release pattern of IgG depending on the pH change.

[0240] These data suggested that IgG release was degradation-dependent.

[0241] Example 12: Controlling protein release rate by blending different hydrolyzable scaffolds

[0242] Two hydrolytic hydrogel formulations, a rapidly degrading component A (linker S 2, t 0.5 =5.6 d) and slowly degradable component B (linker L 2, t 0.5 =54 d) was prepared as described in Example 3. The formulation is shown in Table 7. [Table 7]

[0243] All other formulation parameters remained the same. The polymer concentration was controlled at 30% w / v, and the VS / SH ratio was 0.5. Components A and B were mixed in two ratios: 25 A / 75 B and 50 A / 50 B, resulting in two hybrid hydrogels. Figure 10 shows the cumulative release of F-IgG (A) and the corresponding hydrogel swelling (B) at 37 °C and pH 7.4. Data are presented as mean ± standard deviation (n = 3).

[0244] Example 13 In vivo pharmacokinetics of protein-encapsulated hydrogels

[0245] Hydrogels 1, 2, and 3 were prepared as described in Example 3. Hydrogel 1, consisting of HA-VS and DX-DTT-VMA-DTT, had a polymer mass ratio of 1:2 and a total polymer concentration of 23%. Hydrogel 2, consisting of a micronized hydrogel in a macroscopic hydrogel, was prepared. The micronized hydrogel consisting of HA-VS and DX-DTT-VMA-DTT had a polymer mass ratio of 1:2 and a total polymer concentration of 23%, while the macroscopic hydrogel consisting of HA-VS and DX-DTT-VMA-DTT had a polymer mass ratio of 1:2 and a total polymer concentration of 18%. The hydrogel consisting of HA-VS and DX-DTT-VMA-DTT was micronized at a polymer mass ratio of 1:2 and a total polymer concentration of 23%. The formulations of Hydrogels 1, 2, and 3 are shown in Table 8.

[0246] Hydrogel formulations 1 and 2 were able to release bevacizumab in vitro for at least 3 months. The in vitro release kinetics of formulation 3 was not measured because particles may have been removed by pipetting during release measurements. However, because it is the same as the microgel of formulation 2 (Figure 11B), it is expected to continue to release protein similar to formulations 1 and 2. [Table 8]

[0247] 13.1 Intravitreal injection in rabbits

[0248] Female New Zealand White rabbits weighing 3-4 kg were used. Before all procedures, the rabbits were anesthetized with an intramuscular injection of a ketamine / medetomidine mixture. The hydrogel precursor was prepared as previously described and cooled in an ice bath. After thorough mixing, the mixture was loaded into an insulin syringe with a 29-gauge, 12 mm-long needle. Prior to injection, the corneas of the anesthetized rabbits were topically anesthetized with alcaine and then disinfected with Tobrex. Approximately 40 μL of bevacizumab containing approximately 1 mg was intravitreally injected into the pars plana eye, 3 mm posterior to the superior temporal limbus. To prevent post-injection infection, tobramycin ointment was applied to the ocular surface. Bolus injections of PBS or bevacizumab (Avastin) were also performed.

[0249] The retina, fundus, and intravitreal hydrogel were visually inspected periodically using a fundus imaging system (Volk iNview, Volk Optical, USA) attached to an iPhone 6S with the operating system iOS 9 (Apple, USA). Before the examination, rabbits were anesthetized and the pupils were dilated with Mydriacyl™. The superior, inferior, temporal, and nasal regions near the optic disc were recorded.

[0250] IOP was measured using a tonometer (TonoVet, icare, Finland) according to the manufacturer's instructions. The mean IOP was calculated from six measurements for each eye at each time point.

[0251] 13.2 Protein Measurement in Rabbit Eyes

[0252] Approximately 150 μL of aqueous humor was collected from the anterior chamber at each time point using an insulin syringe with a 31-gauge needle. The collected samples were diluted with an equal volume of PBS containing 2% w / v bovine serum albumin (BSA) and stored in a -80°C freezer until analysis. Bevacizumab in the aqueous humor was quantified by sandwich enzyme-linked immunosorbent assay (ELISA) according to Yu et al. (see Y. Yu, X. Lin, Q. Wang, M. He, and Y. Chau, "Long-term therapeutic effect in nonhuman primate eyes from a single injection of anti-VEGF controlled release hydrogel," Bioeng.Transl.Med., 2019). Briefly, lyophilized VEGFA-165 was dissolved in water at 100 μg / mL as a stock solution and diluted with PBS to a coating concentration of 0.3 μg / mL. PBS containing 0.05% v / v TWEEN 20 was used as the wash buffer. The blocking buffer used was 1% w / v BSA in PBS. The bevacizumab standard, aqueous samples, and IgG-HRP were similarly diluted with 1% BSA.

[0253] A high-affinity 96-well plate was coated overnight at 4°C with 90 μL of Avastin / PBS 0.25 μg / mL. After blocking with 350 μL 1% BSA for 2 hours, the bevacizumab standard and 100 μL of aqueous body fluid samples were incubated for an additional 2 hours, followed by 1 hour of incubation with 100 μL of IgG-HRP at 1 μg / mL. After each step, each well was washed three times with 300 μL of wash buffer. All incubation steps, except for coating, were performed at ambient temperature. Then, 100 μL of TMB was added to each well and incubated in the dark for 15–30 minutes, depending on color intensity. After a sufficient blue color developed, 50 μL of 2M HCl was added to each well to stop the reaction and develop a yellow color. Standards were measured in triplicate, and aqueous samples were measured in duplicate. The absorbance at 450 nm was measured using a Varioskan LUX plate reader (ThermoFisher), and a bevacizumab concentration standard curve was fitted to the absorbance using the 5-parameter logistic (5PL) algorithm using SkanIt 6.0 (ThermoFisher).

[0254] 13.3 Experimental Results

[0255] After bolus administration, intraocular concentrations decreased with first-order elimination kinetics. The calculated half-life was 4 days. In contrast, for all three hydrogel formulations, the elimination rate significantly decreased after approximately 40 days. At day 57, aqueous concentrations of bevacizumab were no longer detectable in the bolus injection group but remained detectable in all hydrogel groups, indicating that the hydrogels were able to release the protein into the eye over several months (Figure 11A). Simulations of intraocular bevacizumab concentrations after bolus administration were performed based on first-order elimination kinetics and the calculated half-life.

[0256] Example 14 In vivo biocompatibility of protein-encapsulated hydrogels in rabbit eyes

[0257] Injection of the three gels (formulations 1, 2, and 3 in Table 8) into rabbit eyes demonstrates that the gels are compatible with the animals both short-term and long-term. No gross changes in retinal structure or media clarity were observed with any of the three formulations (Figure 12).

[0258] Example 15 Degradation and protein release of hydrogels formed by HA-MI and DX-SH

[0259] 15.1 Swelling Test: 27 kDa HA-MI obtained in Example 3, 3% DM, and 18% DM were dissolved in phosphate buffer (PB) at 120 mg / ml, where 3% DM HA-MI was dissolved in 0.02 M PBS and 18% DM HA-MI was dissolved in 0.1 M PBS. 40 kDa and 5% DM DEX-SH were dissolved in PB at 240 mg / ml. After complete dissolution, both solutions were cooled in a refrigerator for 15 minutes before being mixed in a 1:1 volume ratio (MI resin / SH resin mass ratio or MI / SH ratio = 0.5). The formed gel was weighed to its initial mass and then transferred to 2 ml of PBS containing 0.03% sodium azide. The swelling test was performed in an incubator at 37°C. At each time point, the gel was blotted dry and weighed, and the buffer was replaced with fresh PBS containing 0.03% sodium azide.

[0260] The results are shown in Figure 17, which indicates that the polymer is degradable and the gel lifetime of the hydrogel is over 300 hours.

[0261] 15.2 Release Tests

[0262] HA-MI (27 kDa, 18% DM, 8 mg) was dissolved in 67 μL of PBS. DEX-SH (40 kDa, 6% DM, 16.13 mg) was dissolved in 67 μL of Avastin. After complete dissolution, both solutions were cooled in an icebox for 15 minutes. 20 μL of each HA-MI and DEX-SH solution was mixed (MI / SH ratio = 0.5) on a piece of parafilm at room temperature, and the gel was incubated at 37°C for 30 minutes to form a gel. The gel was then weighed and transferred to a centrifuge tube. PBS containing 0.03% sodium azide was used as the release buffer. At each time point, the buffer was removed and replaced with fresh buffer. Protein concentration was measured by Bradford assay according to the manufacturer's instructions (Biorad).

[0263] The results are shown in Figure 18, which showed that protein was cumulatively released from the hydrogel over a period of more than 200 hours.

[0264] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. While the present invention has been described with reference to the above specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. Furthermore, it is to be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It is to be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the present invention. It is therefore contemplated that the present invention also embraces such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A composition comprising at least one first hydrogel-forming polymer and at least one second hydrogel-forming polymer, the first hydrogel-forming polymer is capable of reacting with the second hydrogel-forming polymer to form a hydrogel; The hydrogel is degradable, allowing for sustained release of the targeted agent; wherein the first hydrogel-forming polymer comprises a first hydrogel-forming polymer derivative; the first hydrogel-forming polymer derivative comprises a first modification, the first hydrogel-forming polymer derivative being electrophilic; the second hydrogel-forming polymer comprises a second hydrogel-forming polymer derivative; the second hydrogel-forming polymer derivative comprises a second modification, the second hydrogel-forming polymer derivative being nucleophilic; the weight ratio of said first hydrogel-forming polymer to said second hydrogel-forming polymer is less than 1; the first hydrogel-forming polymer and / or the second hydrogel-forming polymer are selected from the group consisting of polysaccharides, derivatives of polysaccharides, and any combination thereof; The composition.

2. 2. The composition of claim 1, wherein the first modification is selected from the group consisting of vinyl, acryloyl, alkene, thiol ester, isocyanate, isothiocyanate, alkyl halide, sulfonyl halide, epoxide, imido ester, fluorophenyl ester, carbonate, carbodiimide, disulfide, aziridine, and any combination thereof.

3. The composition according to any one of claims 1 to 2, wherein the first modification is selected from vinyl sulfone, maleimide, acrylate, methacrylic acid, epoxide and any combination thereof.

4. 4. The composition of claim 1, wherein the second modification is selected from the group consisting of thiols, amines, azides, hydrazides, dienes, hydrazines, hydroxylamines, and any combination thereof.

5. 5. The composition of any one of claims 1 to 4, wherein the first hydrogel-forming polymer and / or the second hydrogel-forming polymer is selected from the group consisting of hyaluronic acid, chitosan, chondroitin sulfate, alginate, carboxymethylcellulose, dextran, derivatives thereof, and any combination thereof.

6. 6. The composition of any one of claims 1 to 5, wherein the first hydrogel-forming polymer and / or the second hydrogel-forming polymer is selected from the group consisting of dextran, hyaluronic acid, derivatives thereof, and any combination thereof.

7. The composition according to any one of claims 1 to 6, wherein the hydrogel is hydrolyzable without the involvement of a degrading enzyme.

8. The composition of any of claims 1 to 7, wherein at least one of the first hydrogel-forming polymer and / or the second hydrogel-forming polymer comprises a degradable linker.

9. The composition of claim 8 , wherein the degradable linker comprises a hydrolyzable functional group.

10. 10. The composition of claim 9, wherein the hydrolyzable functional groups are selected from ester groups, anhydride groups, and amide groups.

11. The composition of claim 10, wherein the ester group is selected from an oxyester group and a thiol ester group.

12. 12. The composition of any one of claims 1 to 11, wherein the first hydrogel-forming polymer derivative has a first average degree of modification (first DM) of less than about 40% and the second hydrogel-forming polymer derivative has a second average degree of modification (second DM) of less than about 40%.

13. 13. The composition of any one of claims 1 to 12, wherein the ratio of the first DM to the second DM is from about 3:1 to about 1:

3.

14. the first hydrogel-forming polymer derivative is a dextran derivative modified with one or more vinyl sulfone groups, a hyaluronic acid derivative modified with one or more vinyl sulfone groups, or a combination thereof; The composition of any one of claims 1 to 13, wherein the second hydrogel-forming polymer derivative is a dextran derivative modified with one or more thiol groups, a hyaluronic acid derivative modified with one or more thiol groups, or a combination thereof.

15. 15. The composition of any one of claims 1 to 14, wherein the first hydrogel-forming polymer and the second hydrogel-forming polymer have a weight average molecular weight of from about 1 kDa to about 500 kDa.

16. The composition according to any one of claims 1 to 15, wherein the composition is a powder.

17. 16. The composition of any one of claims 1 to 15, wherein the composition is a liquid composition and the concentration of the first hydrogel-forming polymer and / or the second hydrogel-forming polymer in the liquid composition is from about 1% w / v to about 50% w / v.

18. 18. A hydrogel for sustained release of a targeted agent formed from the composition of any one of claims 1 to 17.

19. 20. The hydrogel of claim 18, further comprising a targeting agent.

20. The hydrogel of any one of claims 18 to 19, wherein the targeting agent comprises a polymer.

21. The hydrogel of any one of claims 18 to 20, wherein the targeting agent comprises a macromolecule having a molecular weight of at least about 80 kDa.

22. 22. The hydrogel of any one of claims 18 to 21, wherein at least about 20% of the targeting agent is free targeting agent not bound to the hydrogel.

23. The hydrogel of any one of claims 18 to 22, wherein the targeting agent comprises a protein or polypeptide.

24. 24. The hydrogel of any one of claims 18-23, wherein less than about 50% of the targeting agent is cumulatively released from the hydrogel within a first 24 hours, and the remaining portion of the targeting agent is cumulatively released from the hydrogel within about 1 to about 36 months.

25. 25. The hydrogel of any one of claims 18 to 24, comprising macroscopic and micronized hydrogels.

26. A method for preparing a hydrogel according to any one of claims 18 to 25, comprising: Providing a composition according to any one of claims 1 to 17; mixing the composition with a buffer to form a polymer solution; and Subjecting the polymer solution to conditions that permit the formation of a hydrogel.

27. 27. The method of claim 26, wherein subjecting the polymer solution to conditions that allow for the formation of a hydrogel comprises injecting the polymer solution into a subject in need thereof.

28. 27. The method of claim 26, wherein subjecting the polymer solution to conditions that permit the formation of a hydrogel comprises incubating the composition at about 1°C to about 45°C.

29. The method of any one of claims 26 to 28, wherein the polymer solution further comprises a targeting agent.

30. A method for preparing a composition according to any one of claims 1 to 16, comprising: grafting the precursor polymers with degradable linkers to obtain a first hydrogel-forming polymer and / or a second hydrogel-forming polymer; and The first hydrogel-forming polymer and / or the second hydrogel-forming polymer may be mixed and allowed to react under conditions that allow the first hydrogel-forming polymer and / or the second hydrogel-forming polymer to form a hydrogel.

31. 18. A method for sustained release of a targeted agent comprising mixing the composition of any one of claims 1 to 17 with a targeted agent to obtain a mixture and subjecting said mixture to conditions permitting the formation of a hydrogel capable of sustained release of said targeted agent.

32. 26. A method for sustained release of a targeted agent comprising encapsulating the targeted agent in a hydrogel according to any one of claims 18 to 25.

33. A kit comprising: a) a composition according to any one of claims 1 to 17; and b) a targeting agent to be released by the hydrogel formed using the composition of a).

34. Use of a composition according to any one of claims 1 to 17 for the manufacture of a hydrogel.

Images