Click-crosslinked hydrogels and methods of use thereof

Abstract

The disclosure provides click-crosslinked hydrogels comprising alginates and native extracellular matrices, such as collagen, and methods of use.

Description

[0001] CLICK-CROSSLINKED HYDROGELS AND METHODS OF USE THEREOF

[0002] RELATED APPLICATIONS

[0003]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 626,437, filed on January 29, 2024, the entire contents of which are hereby incorporated herein by reference.

[0004] BACKGROUND

[0005]

[0002] A hydrogel is a polymer gel comprising a network of crosslinked polymer chains.

[0006] The network structure of hydrogels allows them to absorb significant amounts of water. Some hydrogels are highly stretchable and elastic; others are viscoelastic. Uses of hydrogels include cell delivery vehicles, cancer vaccines, tissue engineering (e.g., as scaffolding), and protein / small molecule delivery vehicles. Hydrogels are also useful as structural materials. Alginates serve as attractive polymers for multiple hydrogel applications and can be used to make bulk hydrogels, cryogels and microgels. However, these polymers do not provide the needed ligands for adhesion, and must be modified with motifs such as RGD (fibronectin mimetic peptide) or GFOGER (collagen mimetic peptide) to facilitate cell engagement.

[0007] Native extracellular matrices such as collagens have been incorporated into bulk alginate gels in the form of interpenetrating networks (IPNs), where the two polymers are physically associated. However, the physically incorporated natural polymers can diffuse out over time, and may not be conducive for cryogel manufacture.

[0008]

[0003] There is a need for covalently crosslinked, hybrid hydrogels that are capable of being created using finely tunable and simple chemical reactions that are nontoxic to cells, that can occur at a rapid rate under biological conditions, that are more stretchable and / or stronger than currently existing hydrogels, and that are capable of being produced in a cost-effective way.

[0009] SUMMARY

[0010]

[0004] In some aspects, the present disclosure provides a hydrogel comprising a first polymer and a second polymer, wherein: the first polymer comprises alginate, and the second polymer comprises collagen; the first polymer and the second polymer are present in the hydrogel at a ratio of from about 3:7 w / w to about 4: 1 w / w; the hydrogel comprises pores having an average pore size of about 1 pm to about 200 pm; and wherein the first polymer is connected to the second polymer by linkers of formula

[0011] (A): wherein bond = is a single or a double bond;

[0012] R1is -Co-C6alkyl-NH-, -Co-C6alkyl -O-, or -Co-C3alkyl-C(0)-;

[0013] R2is a bond, aryl, or heteroaryl, wherein aryl and heteroaryl are optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci-Ce alkyl)amino, or di(Ci-Ce alkyl)amino;

[0014] R3is -Co-Ce alkyl-NH-, -Co-Ce alkyl-O-, or -Co-C3alkyl-C(0)-; and

[0015] R4is hydrogen, Ci-Ce alkyl, aryl, or heteroaryl, wherein aryl and heteroaryl are optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci-Ce alkyl)amino, or di(Ci-Ce alkyl)amino.

[0016]

[0005] In some embodiments, the first polymer is connected to the second polymer by linkers of formula (A’): first polymer second polymer (A’) wherein the variables are as defined in Formula (A).

[0017]

[0006] In some embodiments, the average pore size is from about 1 pm to about 50 pm, about 1 pm to about 25 pm, 5 pm to about 100 pm, about 5 pm to about 10 pm, about 15 pm to about 50 pm, about 25 pm to about 75 pm, about 40 pm to about 80 pm, about 25 pm to about 100 pm, about 50 pm to about 150 pm, or about 75 pm to about 200 pm.

[0018]

[0007] In some embodiments, the average pore size is about 25 pm to about 75 pm.

[0019]

[0008] In some embodiments, the first polymer and the second polymer are present in the hydrogel at a ratio of about 3:7 w / w, about 2:3 w / w, about 1 : 1 w / w, about 3:2 w / w, about 7:3 w / w or about 4: 1 w / w.

[0020]

[0009] In some embodiments, the total gel percentage ranges from about 0.8% to about 3%.

[0021]

[0010] In some embodiments, in the linkers of Formula (A) or (A’): bond = is a single bond;

[0022] R1is -Ci-C6alkyl-NH-, or -Co-C3alkyl-C(0)-;

[0023] R2is a bond or aryl optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci-Ce alkyl)amino, or di(Ci-Ce alkyl)amino;

[0024] R3is -Ci-C6alkyl-NH-, or -Co-C3alkyl-C(0)-; and

[0025] R4is hydrogen, Ci-Ce alkyl, or heteroaryl, wherein heteroaryl is optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci-Ce alkyl)amino, or di(Ci-Ce alkyl)amino.

[0026] [OH] In some embodiments, R1and R3are both -methyl-NH-; or R1and R3are both -C(O)-.

[0012] In some embodiments, the linkers of formula (A) are of formula (I): or of formula (II): or of formula (III): wherein the linkers of formula (I), (II), or (III) are optionally substituted.

[0027]

[0013] In some embodiments, the hydrogel is a cryogel.

[0028]

[0014] In some embodiments, the hydrogel further comprises a therapeutic or a diagnostic agent. In some embodiments, the therapeutic or a diagnostic agent comprises a cell, a biologic, or an adjuvant. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a T cell or a stem cell.

[0029]

[0015] In some embodiments, the biologic comprises a protein, a peptide, a nucleic acid, a lipid or a carbohydrate. In some embodiments, the biologic is an antibody or an antigenbinding fragment thereof, a growth factor or a fragment thereof, a hormone, a neurotransmitter, a neurotransmitter receptor, a growth factor receptor, a signaling molecule, an interferon, an interleukin, a chemokine, a cytokine, a colony stimulating factor, a chemotactic factor, an MMP-sensitive substrate, or an extracellular matrix component. In some embodiments, the growth factor or a fragment thereof is selected from the group consisting of Transforming Growth Factor-a (TGF-a), TGF-pi, TGF-P2, Fibroblast Growth Factor (FGF), Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), Epidermal Growth Factor (EGF), Platelet Derived Growth Factor (PDGF), Insulin-like Growth Factor (IGF), Hepatocyte Growth Factor (HGF) and a Bone Morphogenetic Protein (BMP).

[0030]

[0016] In some aspects, the present disclosure also provides a method of delivering a cell and / or a biologic to a subject in need thereof, the method comprising administering to the subject the hydrogel of the disclosure.

[0031]

[0017] In some embodiments, the cell comprises a T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the biologic comprises Interleukin-2 (IL-2), FMS-like tyrosine kinase 3 ligand (FLT3L), or Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF).

[0032]

[0018] In some aspects, the present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject the hydrogel of the disclosure.

[0019] In some embodiments, the hydrogel comprises a cell, wherein the cell is a T-cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the method further comprises administering an adjuvant to the subject. In some embodiments, the adjuvant comprises CpG oligodeoxynucleotides, Polyinosinic:polycytidylic acid (Poly (I:C)), Monophosphoryl-Lipid A (MPLA), Lipopolysaccharide (LPS) or Mannan. In some embodiments, the method further comprises an antigen-free vaccine component, e.g., FMS- like tyrosine kinase 3 ligand (FLT3L) and / or Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF). In some embodiments, the cancer is selected from the group consisting of a hematological malignancy, and a solid tumor cancer. In some embodiments, the cancer is a skin cancer e.g., melanoma, pancreatic cancer, breast cancer, glioblastoma, or leukemia.

[0033]

[0020] In some aspects, the present disclosure also provides a method of treating cancer in a subject, the method comprising administering to the subject a hydrogel comprising a first polymer and a second polymer, wherein: the first polymer comprises alginate, and the second polymer comprises collagen or gelatin; wherein the first polymer is connected to the second polymer by linkers of formula (A):

[0034] wherein bond = is a single or a double bond;

[0035] R1is -Co-C6alkyl-NH-, -Co-C6alkyl -O-, or -Co-C3alkyl-C(0)-;

[0036] R2is a bond, aryl, or heteroaryl, wherein aryl and heteroaryl are optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci- Ce alkyl)amino, or di(Ci-Ce alkyl)amino;

[0037] R3is -Co-Ce alkyl-NH-, -Co-Ce alkyl-O-, or -Co-C3alkyl-C(0)-; and R4is hydrogen, Ci-Ce alkyl, aryl, or heteroaryl, wherein aryl and heteroaryl are optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci-Ce alkyl)amino, or di(Ci-Ce alkyl jamino.

[0038]

[0021] In some embodiments, hydrogel further comprises a T cell. In some embodiments, the T cell is a CD8+ T cell.

[0039]

[0022] In some embodiments, the method further comprises administering a cytokine to the subject. In some embodiments, the cytokine is selected from the group consisting of FMS-like tyrosine kinase 3 ligand (FLT3L), Granulocyte Macrophage Colony- Stimulating Factor (GM-CSF) and Interleukin-2 (IL-2). In some embodiments, the cytokine is comprised in the hydrogel.

[0040]

[0023] In some embodiments, the method further comprises administering an adjuvant to the subject.

[0041]

[0024] In some embodiments, the cancer is selected from the group consisting of a hematological malignancy and a solid tumor cancer.

[0042]

[0025] In some embodiments, the hydrogel comprises pores having an average pore size of about 1 pm to about 200 pm. In some embodiments, the average pore size is from about 1 pm to about 50 pm, about 1 pm to about 25 pm, 5 pm to about 100 pm, about 5 pm to about 10 pm, about 15 pm to about 50 pm, about 25 pm to about 75 pm, about 40 pm to about 80 pm, about 25 pm to about 100 pm, about 50 pm to about 150 pm, or about 75 pm to about 200 pm. In some embodiments, the average pore size is about 25 pm to about 75 pm.

[0043]

[0026] In some embodiments, the first polymer and the second polymer are present in the hydrogel at a ratio of from about 3:7 w / w to about 4: 1 w / w. In some embodiments, the first polymer and the second polymer are present in the hydrogel at a ratio of about 3:7 w / w, about 2:3 w / w, about 1 : 1 w / w, about 3:2 w / w, about 7:3 w / w or about 4: 1 w / w. In some embodiments, the total gel percentage of the hydrogel ranges from about 0.8% to about 3%.

[0044]

[0027] In some embodiments, the first polymer is connected to the second polymer by linkers of formula (A’): first polymer second polymer (A’) wherein the variables are as defined in Formula (A).

[0045]

[0028] In some embodiments, bond = is a single bond;

[0046] R1is -Ci-C6alkyl-NH-, or -Co-C3alkyl-C(0)-;

[0047] R2is a bond or aryl optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci-Ce alkyl)amino, or di(Ci-Ce alkyl)amino;

[0048] R3is -Ci-C6alkyl-NH-, or -Co-C3alkyl-C(0)-; and

[0049] R4is hydrogen, Ci-Ce alkyl, or heteroaryl, wherein heteroaryl is optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci-Ce alkyl)amino, or di(Ci-Ce alkyl)amino.

[0050]

[0029] In some embodiments, R1and R3are both -methyl-NH-; or R1and R3are both -C(O)-.

[0051]

[0030] In some embodiments, the linkers of formula (A) are of formula (I):

[0052] or of formula (II): or of formula (III):

[0053]

[0031] In some embodiments, the hydrogel is a cryogel.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055]

[0032] Figure 1A is a schematic depicting the method of synthesis of alginate-collagen hybrid hydrogels. Figure IB is a representative bulk picture of a tunable, rod-shaped alginate-collagen hybrid hydrogel. Figure 1C is a representative SEM image of an alginatecollagen hybrid hydrogel showing its macroporous structure. Scale bar on the lower left represents 10 pm. Figure ID is a representative SHG image of a pristine alginate-collagen hybrid hydrogel showing pore size distribution. Scale bar on the lower left represents 50 pm. Figure IE is a violin plot showing quantification of pore size distribution in alginate- collagen hybrid hydrogel.

[0033] Figure 2A shows scatter plots of hydrogel shape recovery as a function of alginate to collagen weight ratio. Figure 2B shows scatter plots of interconnected porosity as a function of alginate to collagen weight ratio. Figure 2C shows scatter plots of hydrogel shape recovery as a function of total gel percentage at 60% Alginate-40% Collagen. Figure 2D shows scatter plots of interconnected porosity as a function of total gel percentage at 60% Alginate-40% Collagen.

[0056]

[0034] Figure 3A is schematic depicting the method adopted for tunable release of bioactive factors from alginate-collagen hybrid hydrogels. Figure 3B is a plot of the amount of GM- CSF released from alginate-collagen hybrid hydrogels over time, with or without laponite, in the presence or absence of “wicked” conditions. Figure 3C is a plot of the amount of IL-2 released from alginate-collagen hybrid hydrogels over time, with or without laponite, in the presence or absence of “wicked” conditions. Figure 3D is a plot of the amount of CpG released from alginate-collagen hybrid hydrogels over time, with or without PEI condensation, in the presence or absence of “wicked” conditions.

[0057]

[0035] Figure 4A shows violin plots comparing T cell migration speeds in alginate-only or alginate-collagen hybrid hydrogels. Figure 4B shows IVIS images of mice subcutaneously injected with T cells with or without alginate-collagen hybrid hydrogels.

[0058]

[0036] Figure 5A is a schematic depicting the method followed for therapeutic studies in mice. Figure 5B shows Kaplan-Meier curves comparing survival of mice treated with T cells delivered in alginate-collagen hybrid hydrogel, and with T cells administered intravenously or by direct peritumoral injection with preconditioning. Figure 5C shows Kaplan-Meier curves comparing survival of mice treated with T cells delivered in alginate- collagen hybrid hydrogel, and with T cells administered intravenously or by direct peritumoral injection without preconditioning. Figure 5D shows Kaplan-Meier curves comparing survival of mice treated with T cells delivered in alginate-collagen hybrid hydrogels and antigen-free vaccination, with T cells delivered in alginate-collagen hybrid cryogels alone, and with antigen-free vaccination alone.

[0059]

[0037] Figure 6A is a schematic depicting the method of synthesis of alginate-gelatin hybrid hydrogels. Figure 6B shows bar graphs comparing the relative extent of norbornene modification present on gelatin polymers with different extents of modification. Figure 6C shows bar graphs comparing the storage moduli of alginate-gelatin hybrid hydrogels fabricated using gelatin with different extents of norbornene modification. Figure 6D shows bar graphs comparing the crossover points (the time of transition of a gel from liquid-like to solid-like behavior) of alginate-gelatin hybrid hydrogels fabricated using gelatin with different extents of norbomene modification.

[0060]

[0038] Figure 7A shows scatter plots of percentage shape recovery for alginate-gelatin hybrid hydrogels with varying extents of norbornene modification on gelatin, alginatecollagen ratios, and the total hydrogel percentage. Figure 7B shows scatter plots of percentage interconnected porosity for alginate-gelatin hybrid hydrogels with varying extents of norbornene modification on gelatin, alginate-collagen ratios, and the total gel percentage.

[0061] DETAILED DESCRIPTION

[0062] Definitions

[0063]

[0039] Before the disclosed methods and materials are described, it is to be understood that the aspects described herein are not limited to specific embodiments, apparatuses, or configurations, and as such can, therefore, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0064]

[0040] Throughout this specification, unless the context requires otherwise, the words “comprise” and “include” and variations (e.g., “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element or step or group of components, features, elements or steps.

[0065]

[0041] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context dictates otherwise.

[0066]

[0042] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Quantitative values described herein using the modifier “about” can include ranges of ± 10% of the stated value.

[0067]

[0043] As used herein, the term “aryl” represents an aromatic ring system having a single ring (e.g., phenyl) which is optionally fused to other aromatic hydrocarbon rings or non-aromatic hydrocarbon rings. “Aryl” includes ring systems having multiple condensed rings and in which at least one is carbocyclic and aromatic, (e.g., 1,2,3,4-tetrahydronaphthyl, naphthyl). Examples of aryl groups include phenyl, 1 -naphthyl, 2-naphthyl, indanyl, indenyl, dihydronaphthyl, fluorenyl, tetralinyl, and 6,7,8,9-tetrahydro-5J7-benzo[a]cycloheptenyl. In certain examples, aryl groups include those having a first carbocyclic, aromatic ring fused to an aromatic or aliphatic heterocycle, for example, 2, 3 -dihydrobenzofuranyl. The aryl groups herein may be “optionally substituted”, e.g., be unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, Ci-Cealkyl, Ci- Cealkoxy, (Ci-CealkyQamino, and di(Ci-C6alkyl)amino.

[0068]

[0044] As used herein, the term “alkyl” means a saturated hydrocarbon having a designed number of carbon atoms, such as 1 to 6 carbons (i.e., inclusive of 1 and 6), 1 to 6 carbons, 1 to 3 carbons, or 1, 2, 3, 4, 5 or 6. Alkyl group may be straight or branched and depending on context, may be a monovalent radical or a divalent radical (i.e., an alkylene group). In the case of an alkyl or alkyl group having zero carbon atoms (i.e., “Coalkyl”), the group is simply a single covalent bond if it is a divalent radical or is a hydrogen atom if it is a monovalent radical. For example, the moiety “-(Co-Cealkyl)-O-” signifies connection of an oxygen through a single bond or an alkylene bridge having from 1 to 6 carbons and Co-Csalkyl represents a bond, methyl, ethyl, and propyl moi eties. Examples of “alkyl” include, for example, methyl, ethyl, propyl, isopropyl, butyl, iso-, sec- and tert-butyl, pentyl, and hexyl.

[0069]

[0045] The term “alkoxy” represents an alkyl group of indicated number of carbon atoms attached to the parent molecular moiety through an oxygen bridge. Examples of “alkoxy” include, for example, e.g., methoxy, ethoxy, propoxy, and isopropoxy.

[0070]

[0046] The terms “halogen” or "halo" indicate fluorine, chlorine, bromine, and iodine.

[0047] As used herein, the term “heteroaryl” refers to an aromatic mono- or bi-cyclic ring system of 3-14 atoms ring system containing at least one heteroatom selected from nitrogen, oxygen and sulfur in an aromatic ring. Most commonly, the heteroaryl groups will have 1, 2, 3, or 4 heteroatoms. The heteroaryl may be fused to one or more non-aromatic rings, for example, cycloalkyl or heterocycloalkyl rings. In one embodiment of the present compounds the heteroaryl group is bonded to the remainder of the structure through an atom in a heteroaryl group aromatic ring. In another embodiment, the heteroaryl group is bonded to the remainder of the structure through a non-aromatic ring atom. Examples of heteroaryl groups include, for example, pyridyl, pyrimidinyl, quinolinyl, benzothienyl, indolyl, indolinyl, pyridazinyl, pyrazinyl, isoindolyl, isoquinolyl, quinazolinyl, quinoxalinyl, phthalazinyl, imidazolyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, furanyl, thienyl, pyrrolyl, oxadiazolyl, thiadiazolyl, benzofl, 4]oxazinyl, triazolyl, tetrazolyl, isothiazolyl, naphthyridinyl, isochromanyl, chromanyl, tetrahydroisoquinolinyl, isoindolinyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isobenzothienyl, benzoxazolyl, pyridopyridinyl, benzotetrahydrofuranyl, benzotetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, pteridinyl, benzothiazolyl, imidazopyridinyl, imidazothiazolyl, dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl, dihydrobenzisothiazinyl, benzopyranyl, benzothiopyranyl, chromonyl, chromanonyl, pyridinyl-A-oxide, tetrahydroquinolinyl, dihydroquinolinyl, dihydroquinolinonyl, dihydroisoquinolinonyl, dihydrocoumarinyl, dihydroisocoumarinyl, isoindolinonyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl A-oxide, pyrimidinyl A -oxi de, pyridazinyl A-oxide, pyrazinyl A-oxide, quinolinyl A-oxide, indolyl A-oxide, indolinyl A- oxide, isoquinolyl A-oxide, quinazolinyl A-oxide, quinoxalinyl A-oxide, phthalazinyl A- oxide, imidazolyl A-oxide, isoxazolyl A-oxide, oxazolyl A-oxide, thiazolyl A-oxide, indolizinyl A-oxide, indazolyl A-oxide, benzothiazolyl A-oxide, benzimidazolyl A-oxide, pyrrolyl A-oxide, oxadiazolyl A-oxide, thiadiazolyl A-oxide, triazolyl A-oxide, tetrazolyl A- oxide, benzothiopyranyl S-oxide, benzothiopyranyl S,S-dioxide. Preferred heteroaryl groups include pyridyl, pyrimidyl, pyrazinyl, quinolinyl, indolyl, pyrrolyl, furanyl, thienyl, imidazolyl, pyrazolyl, indazolyl, thiazolyl and benzothiazolyl. The heteroaryl groups herein may be “optionally substituted”, z.e., unsubstituted or substituted with one or more substituents selected from the group consisting of halogen, hydroxy, Ci-Cealkyl, Ci- Cealkoxy, (Ci-C6alkyl)amino, and di(Ci-C6alkyl)amino.

[0048] As used herein the term “contacting” includes the physical contact of at least one substance to another substance, either directly or indirectly. An example of indirect contacting is injecting a hydrogel into a mammal to result in the hydrogel contacting a tissue.

[0071]

[0049] As used herein the term “sufficient amount” and “sufficient time” includes an amount and time needed to achieve the desired result or results, such as to dissolve a portion of the polymer.

[0072]

[0050] All percentages, ratios and proportions herein are by weight, unless otherwise specified. A weight percent (weight %, also as wt %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included (e.g., on the total amount of the active material).

[0073]

[0051] All temperatures are in degrees Celsius (°C) unless otherwise specified.

[0074] Hydrogels of the Disclosure

[0075]

[0052] The present disclosure provides hydrogels comprising a first polymer and a second polymer, wherein the first polymer comprises alginate and the second polymer comprises collagen or gelatin, wherein the first polymer is connected to the second polymer using click chemistry. The hydrogels of the present disclosure provide advantages, such as serving as reservoirs for localized T cell delivery and concomitant recruitment and activation of antigen presenting cells for host T cell priming, thereby enhancing adoptive T cell therapy for solid tumors.

[0076]

[0053] Alginates serve as attractive polymers for multiple applications and can be used to make bulk hydrogels, cryogels and microgels. However, alginates alone do not provide the needed ligands for adhesion, and must be modified with motifs such as RGD (fibronectin mimetic peptide) or GFOGER (collagen mimetic peptide) to facilitate cell engagement. Previously, components of native extracellular matrices, such as collagen, have been incorporated into bulk alginate gels in the form of interpenetrating networks (IPNs), where the alginate and collagen are physically associated. However, collagen can diffuse out of the IPNs over time, and thus, the IPNs may not be conducive for cryogel manufacture.

[0077]

[0054] Accordingly, the present disclosure provides hybrid hydrogels comprising a first polymer and a second polymer, wherein the first polymer comprises alginate and the second polymer comprises collagen or gelatin, and wherein the first polymer is connected to the second polymer using click chemistry. In some embodiments, the linkers connecting the first polymer and the second polymer are products of a reaction between a tetrazine moiety and a norbomene moiety. Because the in the hydrogels of the disclosure the first polymer is different from the second polymer, the hydrogels of the disclosure may also be referred to herein as “hybrid hydrogels”. In the hydrogels of the disclosure, alginate provides structural support to the hydrogel, while collagen or gelatin provides ligands for adhesion and interaction of cells.

[0078]

[0055] The first polymer comprised in the hydrogels of the disclosure is alginate. Alginate molecules are comprised of (l-4)-linked P-D-mannuronic acid (M units) and a L-guluronic acid (G units) monomers, which can vary in proportion and sequential distribution along the polymer chain. In some embodiments, alginate comprised in the hydrogels of the disclosure may be a reduced alginate or an oxidized alginate as described e.g., in WO 2017 / 075055, the entire contents of which are hereby incorporated herein by reference.

[0079]

[0056] The second polymer comprised in the hydrogels of the present disclosure may be collagen. Collagen is a protein found in the extracellular matrix and is ubiquitously expressed in connective tissues. Collagens help tissues to withstand stretching. There are at least 16 types of collagen, and the most abundant type is Type I collagen (also called collagen-I). Collagen (e.g., collagen-I) is present in most tissues, primarily bone, tendon, and skin. The collagen molecules pack together, forming thin, long fibrils. Collagen (e.g., collagen I) is isolated, e.g., from rat tail. The fundamental structure of collagen-I is a long (-300 nm) and thin (-1.5 nm diameter) protein made up of three coiled subunits: two al(I) chains and one a2(I). Each subunit contains 1050 amino acids and the subunits are wound around each other to form a right-handed triple helix structure. See, e.g., “Collagen: The Fibrous Proteins of the Matrix.” Molecular Cell Biology. Lodish et al., eds. New York: W.H. Freeman. Section 22.3(2000); and Venturoni et al. Biochemical and Biophysical Research Communications 303 (2003) 508-513. The al chain of collagen-I has a molecular weight of about 140 kDa. The a2 chain of collagen-I has a molecular weight of about 130 kDa. Collagen-I as a trimer has a molecular weight of about 400 kDa. Collagen-I as a dimer has a molecular weight of about 270 kDa. In some examples, the collagen in the hydrogels described herein include fibrillar collagen. Exemplary types of collagen include non-fibrillar and fibrillar collagen, collagen types I-III, V, XI, XXIV, and XXVII. See, e.g., Exposito, et al. Int. J. Mol. Sci. 11(2010):407-426.

[0080]

[0057] The second polymer comprised in the hydrogels of the present disclosure may also be gelatin. Gelatin is a protein derived from the hydrolysis of collagen. It is biocompatible, has low immunogenicity and cell-responsive properties, thereby making it an attractive candidate for use as a synthetic extracellular scaffold.

[0081]

[0058] The polymers comprised in the hydrogels of the disclosure (e.g., alginate, collagen, or gelatin) can be oxidized, reduced, or neither, or a mixture thereof. In some cases, oxidized polymers or partially oxidized polymers are biodegradable. For example, hydrogels comprising oxidized or partially oxidized alginate are biodegradable.

[0082]

[0059] As used herein, the term “tetrazine moiety” includes molecules that comprise 1, 2,4,5- tetrazine substituted with suitable spacer for linking to the first polymer or the second polymer, and optionally further substituted with one or more substituents at any available position.

[0083]

[0060] As used herein, the term “norbomene moiety” includes norbomadiene and norbomene groups further comprising suitable spacer for linking to the first polymer or the second polymer, and optionally further substituted with one or more substituents at any available position.

[0084]

[0061] In some embodiments, the first polymer is connected to the second polymer by linkers of formula (A): a. wherein i. bond is a single or a double bond; ii. R1is -Co-C6alkyl-NH, -Co-C6alkyl -O-, or -Co-C3alkyl-C(0)-; iii. R2is a bond, aryl, or heteroaryl, wherein aryl and heteroaryl are optionally substituted with halogen, hydroxy, Ci-Cealkyl, Ci-Cealkoxy, (Ci-C6alkyl)amino, or di(Ci-C6alkyl)amino; iv. R3is -Co-Cealkyl-NH, -Co-Cealkyl-O-, or -Co-C3alkyl-C(0)-; and v. R4is hydrogen, Ci-Cealkyl, aryl, or heteroaryl, wherein aryl and heteroaryl are optionally substituted with halogen, hydroxy, Ci-Cealkyl, Ci-Cealkoxy, (Ci- Cealkyl)amino, or di(Ci-C6alkyl)amino.

[0085]

[0062] In some embodiments, the linkers of formula (A) are of formula (I): or by formula (II): or by formula (III): wherein the linkers of formula (I), (II), or (III) are optionally substituted at any suitable position.

[0086]

[0063] In some embodiments, in the linkers of formula (A) bond is a single bond. In another embodiment, bond is a double bond.

[0087]

[0064] In some embodiments, in the linkers of formula (A) R1is: a. NH, -Ci-C6alkyl-NH-, -O-, -Ci-C6alkyl -O-, -C(O)-, or -Ci-C3alkyl-C(O)-; b. -Co-C6alkyl-NH; c. -Ci-C6alkyl-NH-; d. -C1-C3 alkyl-NH-; e. -methyl-NH- or -pentyl-NH-; f. -Co-C6alkyl-O-; g. -Ci-C6alkyl-O-; h. -C1-C3 alkyl-O-; i. -methyl-O- or -pentyl-O-; j. -C0-C3 alkyl-C(O)-; k. -C(O)-; l. -methyl -C(O)-; or m. the same as R3.

[0088]

[0065] In some embodiments, in the linkers of formula (A) R2is a bond.

[0089]

[0066] In some embodiments, in the linkers of formula (A) R2is a. aryl or heteroaryl, each optionally substituted; b. optionally substituted aryl; c. phenyl; d. optionally substituted heteroaryl; or e. pyridyl, pyrimidyl, or pyrazinyl.

[0090]

[0067] In some embodiments, in the linkers of formula (A) R3is: a. -NR2N-, -CI-C6alkyl-NR2N-, -O-, -Ci-C6alkyl -O-, -C(O)-, or -Ci-C3alkyl- C(O)-; b. -Co-C6alkyl-NR2N-; c. -C1-C6alkyl-NR2N-; d. -C1-C3 alkyl-NR2N-; e. -methyl-NH- or -pentyl-NH-; f. -Co-C6alkyl-O-; g. -Ci-C6alkyl-O-; h. -C1-C3 alkyl-O-; i. -methyl-O- or -pentyl-O-; j. -C0-C3 alkyl-C(O)-; k. -C(O)-; l. -methyl -C(O)-; or m. the same as R1.

[0091]

[0068] In some embodiments, R4is hydrogen.

[0092]

[0069] In some embodiments, R4is: a. Ci-Ce alkyl, aryl, or heteroaryl, wherein aryl and heteroaryl are optionally substituted; b. aryl or heteroaryl, wherein aryl and heteroaryl are optionally substituted; c. optionally substituted aryl; d. phenyl; e. optionally substituted heteroaryl; or f. pyridyl, pyrimidyl, or pyrazinyl.

[0093]

[0070] In some embodiments, R4is Ci-Ce alkyl, C1-C3 alkyl, or methyl.

[0094]

[0071] In some embodiments, the hydrogel of the disclosure comprises a plurality of linkers of formula (A); or formula (I), formula (II), or formula (III).

[0095]

[0072] In some embodiments, in the hydrogels of the disclosure, the first polymer which is alginate is connected to the second polymer, which is collagen or gelatin, by linkers of formula (A’): first polymer second polymer (A’) wherein R1, R2, R3and R4are as defined for Formula (A). Thus, in some embodiments, a hydrogel of the disclosure is generated by reacting together alginate which has been modified with a tetrazine moiety, and collagen or gelatin which has been modified with a norbornene moiety. When alginate modified with a tetrazine moiety, e.g., tetrazine, is reacted with a collagen or gelatin modified with a norbomene moiety, e.g., norbornene, this leads to generation of a robust hydrogel at cryogelation conditions.

[0096]

[0073] In view of the present disclosure, the methods and compositions described herein can be configured by the person of ordinary skill in the art to achieve a desired goal. In general, the disclosed compositions and methods provide several unmet advantages. For example, as the click alginate-collagen and alginate-gelatin hybrid hydrogels are biocompatible and provide a bio-orthogonal cross-linking reaction, there are many potential applications of these modified biopolymers, ranging from in vitro cell, protein, and drug encapsulation and release, to in vivo hydrogels with long-term stability. Though previous polymer systems may be capable of some of these applications, click polymers of the disclosure provide additional capabilities outside of canonical polymer hydrogels that were previously unattainable due to incompatible chemistries or contaminants.

[0097]

[0074] In some embodiments, polymers, e.g., alginate, collagen or gelatin polymers, are modified with tetrazine or norbornene moieties that can subsequently be covalently crosslinked to form click-crosslinked hybrid hydrogels, e.g., click alginate-gelatin or click alginate-collagen hydrogels. In some embodiments, alginate is modified with a tetrazine moiety, and collagen or gelatin is modified with a norbomene moiety.

[0098]

[0075] Click-crosslinked hydrogels are capable of encapsulating cells, proteins, and other biological molecules with minimal damage. For example, the viability of a population of cells following encapsulation into a hydrogel described herein in at least about 10%, e.g., at least about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95% or greater. Viability of cells can be determined by standard methods in the art.

[0099]

[0076] The cross-linking reaction has been previously shown by others to be highly specific, bio-orthogonal, and quick (see, e.g., Devaraj et al. Bioconjugate Chem. 19.12(2008):2297- 2299; Karver et al. Bioconjugate Chem. 22.11(2011):2263-2270; and Alge et al.

[0100] Biomacromol. 14.4(2013): 949-953), allowing for incorporation of cells with high postencapsulation viability. Methods of preparing a hybrid hydrogel described herein are also provided by the disclosure. For example, schematic illustrations of methods for preparing hybrid hydrogels are shown in Figures 1 A, 3 A, 6A and 8A.

[0101]

[0077] The mechanical properties of the hydrogels described herein, e.g, alginate-collagen hydrogels or alginate-gelatin hydrogels, can be tuned for a set polymer concentrations via the degree of substitution of norbomene or tetrazine groups on the polymer chains or the ratio of norbomene to tetrazine groups. This tuning allows for the creation of elastic hydrogels with Young’s moduli ranging from about 50 to about 50,000 Pa.

[0078] In some embodiments, weight the first polymer, i.e., alginate, in the hydrogels of the disclosure may constitute about 30% to about 80% of the total weight of the polymers present in the hydrogel, e.g., the total weight of the first polymer and the second polymer.

[0102] Conversely, weight of the second polymer, i.e., collagen or gelatin, may constitute about 20% to about 70% of the total weight of the polymers present in the hydrogel, e.g., the total weight of the first polymer and the second polymer. Thus, in some embodiments, hydrogels of the disclosure may comprise the first polymer, i.e., alginate, and the second polymer, i.e., collagen or gelatin, at the ratio ranging from about 3:7 w / w to about 4: 1 w / w, e.g., about 3:7 w / w, about 2:3 w / w, about 1 : 1 w / w, about 3 :2 w / w, about 7:3 w / w or about 4: 1 w / w. In some embodiments, hydrogels of the disclosure may comprise the first polymer, i.e., alginate, and the second polymer, i.e., collagen or gelatin, at the ratio ranging from about 3:7 w / w to about 7:3 w / w, about 2:3 w / w to about 4: 1 w / w, about 3:7 w / w to about 1 : 1 w / w, about 1 : 1 w / w to about 4: 1 w / w. In some embodiments, changing the ratio of alginate to collagen or gelatin in the hydrogels of the disclosure may affect shape deformability of the hydrogel.

[0103]

[0079] The hydrogels of the disclosure may comprise pores large enough for a cell to travel through. For example, the hydrogel of the disclosure may comprise pores having an average diameter of about 1 pm to 250 pm, e.g., from about 1 pm to about 50 pm, about 1 pm to about 25 pm, 5 pm to about 100 pm, about 5 pm to about 10 pm, about 15 pm to about 50 pm, about 25 pm to about 75 pm, about 40 pm to about 80 pm, about 25 pm to about 100 pm, about 50 pm to about 150 pm, or about 75 pm to about 200 pm. In some embodiments, the average pore size is about 25 pm to about 75 pm, e.g., about 50 pm.

[0104]

[0080] As used herein, the term “total gel percentage” of a hydrogel refers to the concentration of polymers, e.g., the first polymer and the second polymer, present in a hydrogel mixture prior to gelation, expressed as % w / w. The total gel percentage is a determinant of hydrogel stiffness. In some embodiments, a hydrogel of the disclosure is characterized by a total gel percentage of about 0.8% to about 3%, e.g., about 0.8% to about 1.5%, about 0.8% to about 2.5%, about 1% to about 1.5%, about 1.5% to about 2.5%, about 1.5% to about 2%, about 2% to about 3% or about 2.5% to about 3%. In some embodiments, changing the total gel percentage in the hydrogels of the disclosure may have an effect on the interconnected porosity of the hydrogels.

[0081] As used herein, the term “interconnected porosity” of a hydrogel refers to the network of interconnected pores, which is related to the spongy behavior of the hydrogel. Gels with high interconnected porosity are typically easier to deform. Further, interconnected porosity facilitates migration of cells through a hydrogel. Interconnected porosity of hydrogels is measured by first weighing intact hydrogels (“Mhydrated”), followed by wicking hydrogels with Kimwipes™ for 15 seconds to remove the excess buffer from hydrogel pores. The wicked hydrogels are then re-weighed (“Mwicked”) and interconnected porosity is calculated as the mass of water wicked away relative to the total hydrated mass, as follows:

[0105] (Mhydrated — Mwicked) / Mhydrated

[0106]

[0082] In some embodiments, a hydrogel of the disclosure is characterized by an interconnected porosity that lies within a range of about 0.2 to about 0.99, e.g., about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.85, about 0.9, about 0.92, about 0.94, about 0.96, about 0.98, or about 0.99.

[0107]

[0083] In some embodiments, a hydrogel of the disclosure is in the form of a rod-based structure. The rod-based structure allows easy injection of the hydrogel into a subject.

[0108]

[0084] The polymers in the hydrogels of the disclosure may be about 1-90% crosslinked, e.g., at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, 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% crosslinked, at least about 75% crosslinked, at least about 80% crosslinked, at least about 85% crosslinked or at least about 90% crosslinked. Ranges intermediate to the recited values are also intended to be part of this invention. For example, the polymers of the hydrogel may be about 1% to about 10%, about 7% to about 15%, about 12% to about 20%, about 15% to about 30%, about 20% to about 40%, about 30% to about 50%, about 45% to about 65% or about 50% to about 90% crosslinked.

[0109]

[0085] The term “% crosslinked”, used interchangeably with the term “crosslinking density”, refers to the number of moles of click moieties conjugated per moles of alginate monomers that have reacted with each other.

[0110]

[0086] In some cases, the hybrid hydrogels described herein have favorable mechanical properties. For example, upon compression or dehydration, the hydrogel maintains structural integrity, i.e., after compression or dehydration, the hydrogel regains its shape after it is rehydrated or the shear forces of compression are removed / relieved. The hydrogel also maintains structural integrity in that it is flexible (i.e., not brittle) and does not break under sheer pressure.

[0111]

[0087] Additionally, the hydrogels described herein are defect resistant, i.e., the durable gel is not prone to development of tears. But even if a defect arises, the gel maintains its toughness and does not fail.

[0112]

[0088] An advantage of the hybrid hydrogels described herein is that they are biocompatible to cells (e.g., show tunable degradation, and produce no inflammation in cells) over long periods of time, e.g., 3 days, 7 days, 14 days, 28 days 56 days, 112 days, or 224 days.

[0113]

[0089] The biocompatible hybrid hydrogels described herein offer significant advantages, particularly in medical applications. For example, drug delivery hydrogels or cell delivery hydrogels that are used for muscle generation or regeneration are subject to application of energy / stresses. Because the hydrogels described herein are more mechanically robust, more durable, and are characterized by a higher fracture resistance compared to prior hydrogels, they are more suitable for such applications involving muscle tissue. Other applications are also improved with the use of the tough hydrogels. For example, materials used in surgical procedures (e.g., wraps, meshes), cartilage replacementjoint replacement, orthopedic / orthochondral defect repair (e.g., bone or cartilage fillers), spinal procedures (e.g., nuclear propulsus spinal surgery), ophthamological uses (e.g., optically-clear, flexible, durable lenses, contact lens or implantable lens), as well as non-medical uses (e.g., fillers in cosmetic surgical procedures).

[0114]

[0090] In some embodiments, the hydrogels of the disclosure are injectable. In some embodiments, the hydrogels of the disclosure are shape deformable.

[0115]

[0091] As used herein, the term “shape deformable” is a mechanical property of the hydrogel related to the extent to which a hydrogel can self-deform back to its original shape, following its prior deformation on application of a temporary external force. In some embodiments, a hydrogel of the disclosure deforms during injection and regains its shape following injection. Shape deformability or shape recovery is estimated by first weighing an intact hydrogel (“Mhydrated”), followed by wi eking the hydrogel with Kimwipes™ for 15 seconds to remove excess buffer, rehydrating the hydrogel in DPBS and then re-weighing the rehydrated hydrogel (“Mrehydrated”). Shape recovery of the hydrogel is quantified as:

[0116] Mrehydrated / Mhydrated

[0117]

[0092] In some embodiments, a hydrogel of the disclosure is characterized by shape recovery that lies within a range of about 0.1 to about 1.0, e.g. about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.85, about 0.9, about 0.92, about 0.94, about 0.96, about 0.98, about 0.99, or about 1.0.

[0118] Cryogels

[0119]

[0093] In some embodiments, hydrogels of the present disclosure may be cryogels. Cryogels are a class of materials with a highly porous interconnected structure that are produced using a cryotropic gelation (or cryogelation) technique. Cryogelation is a technique in which the polymerization-crosslinking reactions are conducted in quasi-frozen reaction solution.

[0120] During freezing of the polymer (e.g., alginate, collagen, or gelatin) solution, the polymers are expelled from the ice concentrate within the channels between the ice crystals, so that the reactions only take place in these unfrozen liquid channels. After polymerization and, after melting of ice, a porous material is produced whose microstructure is a negative replica of the ice formed. Ice crystals act as porogens. Pore size is tuned by altering the temperature of the cryogelation process. For example, the cryogelation process is typically carried out by quickly freezing the solution at -20°C. Lowering the temperature to, e.g., -80 °C, would result in more ice crystals and lead to smaller pores.

[0121]

[0094] The advantage of the “cryogels”, as compared to conventional macroporous hydrogels obtained by phase separation, is their high mechanical stability. They are very tough, and can withstand high levels of deformations, such as elongation and torsion; they can also be squeezed under mechanical force to drain out their solvent content. The improved mechanical properties of alginate-collagen and alginate-gelatin hybrid cryogels originate from the high crosslinking density of the unfrozen liquid channels of the reaction system. Thus, after polymerization, the gel channels with high polymer content are perfect materials for building the pore walls. Methods of Preparing a Hydrogel

[0122]

[0095] The present disclosure also provides a method for preparing a hydrogel comprising: a) providing a first polymer, i.e., alginate, comprising a tetrazine moiety and a second polymer, i.e., collage or gelatin, comprising at least one norbomene moiety. An example of a first polymer comprising a tetrazine moiety is shown below, with the number of tetrazine moieties denoted by “f ’, where t can be any integer between 1 and 100,000:

[0123] An example of a second polymer comprising a norbomene moiety is shown below, with the number of norbomene moieties denoted by “n”, wherein n can be any integer between 1 and 100,000:

[0124] b) contacting the second polymer with the first polymer to form a cross-linked polymer having crosslinks of formula (A). In one embodiment, the crosslinks of formula (A) are of the form of formula (I), or formula (II), or formula (III):

[0125] wherein the linkers of formula (I), (II), or (III) are optionally substituted at any suitable position.

[0126]

[0096] In some embodiments, each molecule of the first polymer comprises about 1-50,000 tetrazine moieties, e.g., about 1-10,000, about 1-5000, about 1-1000, about 5000-50,000, about 5000-10,000, about 1000-10,000, about 1000-5000, about 500-5000, about 500-1000, or about 1-500 tetrazine moieties. In some embodiments, each molecule of the second polymer comprises about 1-50,000 norbornene moieties, e.g., about 1-10,000, about 1-5000, about 1-1000, about 5000-50,000, about 5000-10,000, about 1000-10,000, about 1000-5000, about 500-5000, about 500-1000, or about 1-500 norbomene moieties. In some embodiments, step b) of the method comprises contacting a second polymer with a first polymer at a ratio of about 1 : 104 to about 10: 1 (second polymer : first polymer). For example, the ratio of the second polymer to the first polymer is about 1 : 10, or about 1 :9, or about 1 :8, or about 1 :7, or about 1 :6, or about 1 :5, or about 1 :4, or about 1 :3, or about 1 :2, or about 1 : 1, or about 2: 1, or about 3: 1, or about 4: 1, or about 5: 1, or about 6: 1, or about 7: 1, or about 8: 1, or about 9: 1, or about 10: 1.

[0127]

[0097] In some cases, the tetrazine moiety is coupled to the polymer by reacting the polymer with benzyl amine tetrazine and a coupling agent. In other examples, the tetrazine moiety is coupled to the alginate by reacting the polymer with benzyl alcohol tetrazine or benzoic acid tetrazine and a coupling agent. Exemplary coupling agents include, but are not limited to, 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), carbonyl diimidazole, N,N’- dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), and N,N,N',N - tetramethyl-O-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU).

[0128]

[0098] In some cases, the norbornene moiety is coupled to the polymer by reacting the polymer with norbomene methanamine and a coupling agent. In other examples, the norbomene moiety is coupled to the polymer by incubating the polymer with norbornene methanol or norbomene carboxylic acid and a coupling agent. Exemplary coupling agents include, but are not limited to, EDC, carbonyl diimidazole, DCC, DIC, and HBTU. In some cases, N-Hydroxysuccinimide (NHS) is also included in the coupling reaction.

[0129]

[0099] The norbornene modified polymers (e.g., norbornene modified alginate) can also be modified with thiol-containing molecules or proteins via thiol-ene photochemistry either before or after crosslinking into a hydrogel. For example, the thiol-ene chemistry occurs before crosslinking. In such cases, the thiol reacts with all of the norbornene functional groups on the polymer(s). In other cases, there remain unreacted norbomene groups on the polymer(s) after reaction with the thiol. The reactivity can be controlled by varying the amount of thiol containing compound and the amount of polymer containing norbomene groups (e.g., incubating a smaller number of moles of thiol with a larger number of moles of polymer containing norbornene. Alternatively, reactivity can be controlled by varying the degree of substitution of the polymer(s) with norbomene groups and, e.g., incubating a smaller number of moles of thiol with a larger number of moles of total norbornene groups. In such a way, unreacted norbornene groups on the polymer after the thiol-ene chemical reaction are available to react with tetrazine in the crosslinking reaction. In some examples, the thiol does not react with the crosslinked norbornene-tetrazine product after gelation.

[0130]

[0100] A variety of polymers suitable for the click conjugation of the disclosure allow for a drug delivery platform that can be configured for specific drug delivery goals through the material specific characteristics given herein. Exemplary applications include use as a dermal filler, in drug delivery, as a wound dressing, for postsurgical adhesion prevention, as a method of treating cancer, and for repair and / or regenerative medical applications such as cell therapy (e.g., immunoisolated cell therapy), gene therapy, tissue engineering, immunotherapy.

[0131] Hydrogels of the Disclosure Comprising a Therapeutic or Diagnostic Agent

[0132]

[0101] Hydrogels, e.g., cryogels of the present disclosure may further comprise a therapeutic or a diagnostic agent. For example, hydrogels of the disclosure may be used to entrap or encapsulate a therapeutic or diagnostic agent. As used herein, the term “therapeutic or diagnostic agent” that may be comprised in the hydrogels of the invention includes any agent that may be used to treat, prevent or diagnose a disorder in a subject in need thereof. A therapeutic or diagnostic agent may be a cell, e.g., a mammalian cell, such as a human mesenchymal stem cell (hMSC), or a biologic. The biologic may be a peptide, a protein, a DNA molecule, an RNA molecule, a PNA molecule, an antibody or a vaccine. Exemplary therapeutic agents include, but are not limited to, those found in Harrison ’s Principles of Internal Medicine, 13thEdition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY;

[0133] Physicians’ Desk Reference, 50thEdition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8thEdition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, USP XII NF XVII, 1990; current edition of Goodman and Oilman’s The Pharmacological Basis of Therapeutics,' and current edition of The Merck Index, the entire contents of all of which are incorporated herein by reference.

[0134]

[0102] A therapeutic or diagnostic agent that is entrapped or encapsulated in the polymer structure of the hydrogels of the disclosure may or may not be chemically linked to it. A therapeutic or a diagnostic agent may be incorporated into the hydrogel through its addition to the gel mixture before the preparation of the hydrogel, e.g., cryogelation. A therapeutic or a diagnostic agent entrapped or encapsulated in the hydrogel of the disclosure may be released from the hydrogel by diffusion or gel degradation over time. For example, a therapeutic or diagnostic agent having a relatively low molecular weight (less than 10 kDa molecular mass), e.g., CpG oligonucleotides, may be released from a hydrogel by diffusion. A therapeutic or diagnostic agent having a relatively high molecular weight (e.g., greater than about 10 kDa, e.g., 10-50 kDa), e.g., proteins, large DNAs, e.g., plasmid DNA, or cells may be released from a hydrogel of the disclosure via degradation of the hydrogel. In some embodiments, the rate of release of a therapeutic or diagnostic agent from a hydrogel of the disclosure may be tuned by processes such as their adsorption or condensation prior to cryogelation, as described in the Examples. Laponite or related materials may be used for adsorption, whereas condensation can occur in the presence of molecules such as polyethyleneimine (PEI).

[0135]

[0103] In some embodiments, hydrogels of the disclosure comprising a therapeutic or a diagnostic agent, e.g., a polypeptide or an oligonucleotide, can provide sustained release of the therapeutic or a diagnostic agent. In some embodiments, a hydrogel of the disclosure may comprise a polypeptide, e.g., a cytokine, such as a granulocyte macrophage colonystimulating factor (GM-CSF) or interleukin-2 (IL-2) and may provide a sustained release of the polypeptide, such as GM-CSF or IL-2 from the hydrogel. In some embodiments, a polypeptide, such as GM-CSF or IL-2, has been adsorbed onto a matrix, such as laponite, and has been added to the a mixture of the first polymer and the second polymer prior to gelation. In some embodiments, a hydrogel of the disclosure comprising a polypeptide, e.g., GM-CSF or IL-2 adsorbed onto a matrix, e.g., laponite, can provide sustained release of the polypeptide for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days.

[0136]

[0104] In some embodiments, a hydrogel of the disclosure comprises an oligonucleotide, e.g., a synthetic oligonucleotide containing unmethylated CpG motifs (also referred to herein as “CpG”), which may act as an adjuvant. In some embodiments, the CpG has been condensed with a cationic polymer, such as polyethylenemine (PEI) and has been added to a mixture of the first polymer and the second polymer prior to gelation. In some embodiments, a hydrogel of the disclosure comprising an oligonucleotide, e.g., CpG, condensed with a cationic polymer, e.g., PEI, can provide sustained release of the oligonucleotide, e.g., CpG, for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days.

[0137]

[0105] In some embodiments, a therapeutic or diagnostic agent encapsulated by the hydrogel of the invention may comprise a compound selected from the group consisting of an oligosaccharide; a polysaccharide; a peptide; a protein; a peptide analog; a peptide derivative; a peptidomimetic; an antibody (polyclonal or monoclonal); an antigen binding fragment of an antibody; a nucleic acid, e.g., an oligonucleotide, an antisense oligonucleotide, siRNAs, shRNAs, a ribozyme, an aptamer, microRNAs, a pre-microRNAs, iRNAs, plasmid DNA (e.g. a condensed plasmid DNA), modified RNA, a nucleic acid analog or derivative; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof. The nucleic acid may comprise one or more unnatural nucleotides. The peptide or the protein may comprise one or more unnatural amino acids.

[0106] As used herein, the term “peptide” is used in its broadest sense to refer to compounds containing amino acids, amino acid equivalents or other non-amino groups, while still retaining the desired functional activity of a peptide. Peptide equivalents can differ from conventional peptides by the replacement of one or more amino acids with related organic acids (such as PABA), amino acids or the like or the substitution or modification of side chains or functional groups. The peptides can be linear or cyclic. A peptide can be modified to include one or more of D-amino acids, beta-amino acids, chemically modified amino acids, naturally occurring non-proteogenic amino acids, rare amino acids, and chemically synthesized compounds that have properties known in the art to be characteristic of an amino acid.

[0138]

[0107] As used herein, the term “nucleic acid” or “oligonucleotide” means at least two nucleotides, including analogs or derivatives thereof, that are covalently linked together. Exemplary oligonucleotides include, but are not limited to, single-stranded and doublestranded siRNAs and other RNA interference reagents (RNAi agents or iRNA agents), shRNA (short hairpin RNAs), antisense oligonucleotides, aptamers, ribozymes, and microRNAs (miRNAs). The nucleic acids can be single stranded or double stranded. The nucleic acid can be DNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of uracil, adenine, thymine, cytosine and guanine. An RNA molecule may be selected from the group consisting of an mRNA, an RNAi, an siRNA, an shRNA, a microRNA, an isRNA, a IncRNA and an antisense RNA. In some embodiments, an oligonucleotide may be a synthetic oligonucleotide containing unmethylated CpG motifs (CpG).

[0139]

[0108] The nucleic acid may also include one or more unnatural nucleotides. For example, the nucleic acid can comprise one or more nucleic acid modifications known in the art. For example, the nucleic acids can comprise one or more backbone modifications, e.g., phosphoramide (Beaucage et al., Tetrahedron 49(10): 1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970)), phosphorothioate, phosphorodithioate, O- methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), or peptide nucleic acid linkages (see Egholm, J. Am. Chem. Soc. 114: 1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31 : 1008 (1992); and Nielsen, Nature, 365:566 (1993), the entire contents of all of which are herein incorporated by reference. The nucleic acids can also include modifications to nucleobase and / or sugar moieties of nucleotides. Exemplary sugar modifications at the sugar moiety include replacement of 2’ -OH with halogens (e.g., fluoro), O-mehtyl, O-methoxyethyl, NH2, SH and S-m ethyl.

[0140]

[0109] In some embodiments, the term “therapeutic or diagnostic agent” comprises biological material, for example, an extracellular matrix material such as fibronectin, vitronection and laminin; a cytokines; a growth factor; a differentiation factor, a nucleic acid; a protein; a peptide; an antibody or a fragment thereof or an antigen binding portion thereof, or a cell.

[0141] [HO] Suitable growth factors and cytokines that may be incorporated into the hydrogels of the invention include, but are not limited, to stem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage stimulating factor (GM-CSF), stromal cell-derived factor- 1, steel factor, VEGF, TGFp, platelet derived growth factor (PDGF), angiopoeitins (Ang), epidermal growth factor (EGF), bFGF, HNF, NGF, bone morphogenic protein (BMP), fibroblast growth factor (FGF), hepatocye growth factor, insulin-like growth factor (IGF-1), interleukin (IL)-3, IL-la, IL-ip, IL-6, IL-7, IL-8, IL-11, and IL-13, colonystimulating factors, thrombopoietin, erythropoietin, fit3 -ligand, and tumor necrosis factor a (TNFa). Other examples are described in Dijke et al., “Growth Factors for Wound Healing”, Bio / Technology, 7:793-798 (1989); Mulder GD, Haberer PA, Jeter KF, eds. Clinicians' Pocket Guide to Chronic Wound Repair. 4th ed. Springhouse, PA: Springhouse Corporation; 1998:85; Ziegler T.R., Pierce, G.F., and Herndon, D.N., 1997, International Symposium on Growth Factors and Wound Healing: Basic Science & Potential Clinical Applications (Boston, 1995, Serono Symposia USA), Publisher: Springer Verlag.

[0142] [Ill] Trophic factors are factors whose continued presence improves the viability or longevity of a cell. Trophic factors include, without limitation, platelet-derived growth factor (PDGP), neutrophil-activating protein, monocyte chemoattractant protein, macrophage- inflammatory protein, platelet factor, platelet basic protein, and melanoma growth stimulating activity; epidermal growth factor, transforming growth factor (alpha), fibroblast growth factor, platelet-derived endothelial cell growth factor, insulin-like growth factor, glial derived growth neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, bone growth / cartilage-inducing factor (alpha and beta), bone morphogenetic proteins, interleukins (e.g., interleukin inhibitors or interleukin receptors, including interleukin 1 through interleukin 10), interferons (e.g., interferon alpha, beta and gamma), hematopoietic factors, including erythropoietin, granulocyte colony stimulating factor, macrophage colony stimulating factor and granulocyte-macrophage colony stimulating factor; tumor necrosis factors, and transforming growth factors (beta), including beta-1, beta-2, beta-3, inhibin, and activin.

[0143]

[0112] The therapeutic or diagnostic agent, e.g., a protein or a peptide, can be recombinantly produced, or purified from a protein composition. The active agent, if a TGF-P such as a BMP, or other dimeric protein, can be homodimeric, or can be heterodimeric with other BMPs (e.g., a heterodimer composed of one monomer each of BMP -2 and BMP-6) or with other members of the TGF-P superfamily, such as activins, inhibins and TGF-pi (e.g., a heterodimer composed of one monomer each of a BMP and a related member of the TGF-P superfamily). Examples of such heterodimeric proteins are described for example in Published PCT Patent Application WO 93 / 09229, the content of which is incorporated herein by reference.

[0144]

[0113] In some embodiments, the therapeutic or diagnostic agent can be one or more of Hedgehog, Frazzled, Chordin, Noggin, Cerberus and Follistatin proteins. These families of proteins are generally described in Sasai etal., (1994) Cell 791779-790 (Chordin); PCT Patent Publication W094 / 05800 (Noggin); and Fukui et al., Devel. Biol. 159: 1-31 (1993) (Follistatin). Hedgehog proteins are described in WO96 / 16668; WO96 / 17924; and WO95 / 18856. The Frazzled family of proteins is a recently discovered family of proteins with high homology to the extracellular binding domain of the receptor protein family known as Frizzled. The Frizzled family of genes and proteins is described in Wang et al., Biol. Chem. 271 :44684476 (1996). The therapeutic or diagnostic agent can also include other soluble receptors, such as the truncated soluble receptors disclosed in PCT patent publication WO95 / 07982. From the teaching of WO95 / 07982, one skilled in the art will recognize that truncated soluble receptors can be prepared for numerous other receptor proteins. The above publications are hereby incorporated by reference herein.

[0145]

[0114] In some embodiments, a hydrogel of the disclosure comprises a cell. Cells that may be comprises in hydrogels of the disclosure include, but are not limited to, stem cells (e.g., embryonic stem cells, mesenchymal stem cells, and bone-marrow derived stem cells), chrondrocytes progenitor cells, pancreatic progenitor cells, myoblasts, fibroblasts, keratinocytes, neuronal cells, glial cells, astrocytes, pre-adipocytes, adipocytes, vascular endothelial cells, hair follicular stem cells, endothelial progenitor cells, mesenchymal cells, neural stem cells and smooth muscle progenitor cells. In some embodiments, a cell comprised in the hydrogel of the disclosure is an immune cell, such as a T-cell, e.g., a CD4+ T-cell or a CD8+ T-cell. In one embodiments, the cell comprised in the hydrogel of the disclosure is a CD8+ T-cell.

[0146]

[0115] In some embodiments, the cell may be a genetically modified cell. A cell may be genetically modified to express and secrete a desired compound, e.g, a bioactive agent, a growth factor, differentiation factor, cytokines, and the like. Methods of genetically modifying cells for expressing and secreting compounds of interest are known in the art and easily adaptable by one of skill in the art.

[0147]

[0116] Differentiated cells that have been reprogrammed into stem cells can also be used. For example, human skin cells reprogrammed into embryonic stem cells by the transduction of Oct3 / 4, Sox2, c-Myc and Klf4 (Junying Yu, et. aL, Science, 2007, 318: 1917-1920 and Takahashi K. et. al., Cell, 2007, 131 : 1-12).

[0148]

[0117] Cells useful for incorporation into the hydrogels of the present disclosure may come from any source, e.g., a mammal. For example, the cell can be from a human, a rat or a mouse. Human cells include, but are not limited to, human cardiac myocytes-adult (HCMa), human dermal fibroblasts-fetal (HDF-f), human epidermal keratinocytes (HEK), human mesenchymal stem cells-bone marrow, human umbilical mesenchymal stem cells, human hair follicular inner root sheath cells, human umbilical vein endothelial cells (HUVEC), and human umbilical vein smooth muscle cells (HUVSMC), human endothelial progenitor cells, human myoblasts, human capillary endothelial cells, human neural stem cells and human immune cells, e.g., human T-cells, such as CD8+ T cells. Exemplary rat and mouse cells include, but not limited to, RN-h (rat neurons-hippocampal), RN-c (rat neurons-cortical), RA (rat astrocytes), rat dorsal root ganglion cells, rat neuroprogenitor cells, mouse embryonic stem cells (mESC) mouse neural precursor cells, mouse pancreatic progenitor cells, mouse mesenchymal cells and mouse endodermal cells.

[0118] In some embodiments, a hydrogel of the disclosure comprises a cell, e.g., an immune cell, such as a T-cell. In some embodiments, an immune cell, e.g., a T-cell may be loaded into a pre-formed hydrogel of the disclosure by briefly wicking the hydrogel and rehydrating it in a concentrated solution of T-cells. In some embodiments, T-cells may migrate from the hydrogels of the disclosure at speed which is faster than speed of T-cell migration from a comparator hydrogel, e.g., an alginate-only hydrogel. In some embodiments, when a hydrogel of the disclosure comprising a T-cell is injected into a subject, e.g., a mouse or a human, the T-cell persists in the subject for a longer period of time than a T-cell that has been directly injected into a subject.

[0149]

[0119] In some embodiments, tissue culture cell lines can be used in the hydrogels described herein. Examples of cell lines include, but are not limited to, Cl 66 cells (embryonic day 12 mouse yolk), C6 glioma Cell line, ULI (cardiac muscle cell line), AML12 (nontransforming hepatocytes), HeLa cells (cervical cancer cell line) and Chinese Hamster Ovary cells (CHO cells). An ordinary skill artisan in the art can locate, isolate and expand such cells. In addition, the basic principles of cell culture and methods of locating, isolation and expansion and preparing cells for tissue engineering are described in “Culture of Cells for Tissue Engineering” Editor(s): Gordana Vunjak-Novakovic, R. Ian Freshney, 2006 John Wiley & Sons, Inc., and Heath C. A., Trends in Biotechnology, 2000, 18:17-19, content of both of which is herein incorporated by reference in its entirety.

[0150]

[0120] In one embodiment, the biologic may be a peptide, e.g., a peptide having a molecular weight of 250 kDa or less. In a further embodiment, the peptide is an angiogenesis factor, e.g., FGF, VEGF, VEGFR, IGF, NRP-1, Angl, Ang2, PDGF, PDGFR, TGF-P, endoglin, a TGF-P receptor, MCP-1, integrin, an integrin ligand (e.g., an RGD peptide), VE-cadherin, CD31, ephrin, plasminogen activator, plasminogen activator inhibitor-1, eNOS, COX-2, AC133, ID1 or ID3. In a specific embodiment, the peptide encapsulated by the hydrogels of the present invention is VEGF.

[0151]

[0121] The therapeutic or diagnostic agent which may be comprised in the hydrogel of the invention may be a STING adjuvant, a CRISPR-Cas 9 reagent and an adjuvant-loaded subcellular vesicle derived from disrupted cancer cells.

[0152]

[0122] In one embodiment, the therapeutic or diagnostic agent may also be a vaccine. Adjuvant

[0153]

[0123] The term “adjuvant” encompasses substances that accelerate, prolong, or enhance the immune response to an antigen. In some embodiments an adjuvant serves as a lymphoid system activator that enhances the immune response in a relatively non-specific manner, e g., without having any specific antigenic effect itself. For example, in some embodiments an adjuvant stimulates one or more components of the innate immune system. In certain embodiments, an adjuvant enhances antigen-specific immune responses when used in combination with a specific antigen or antigens, e.g., as a component of a vaccine. Adjuvants include, but are not limited to, aluminum salts (alum) such as aluminum hydroxide or aluminum phosphate, complete Freund's adjuvant, incomplete Freund's adjuvant, surface active substances such as lysolecithin, pluronic polyols, Amphigen, Avridine, bacterial lipopolysaccharides, 3-O-deacylated monophosphoryl lipid A, synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof (see, e.g., U.S. Pat. No. 6, 113,918), L121 / squalene, muramyl dipeptide, polyanions, peptides, saponins, oil or hydrocarbon and water emulsions, particles such as ISCOMS (immunostimulating complexes), etc. In some embodiments an adjuvant stimulates dendritic cell maturation. In some embodiments an adjuvant stimulates expression of one or more costimulator(s), such as B7 or a B7 family member, by antigen presenting cells (APCs), e.g., dendritic cells. In some embodiments an adjuvant comprises a CD40 agonist. In some embodiments, a CD40 agonist comprises an anti-CD40 antibody. In some embodiments, a CD40 agonist comprises a CD40 ligand, such as CD40L. In some embodiments an adjuvant comprises a ligand for a Toll-like receptor (TLR). In some embodiments, an agent is a ligand for one or more of TLRs 1-13, e.g., at least for TLR3, TLR4, and / or TLR9. In some embodiments, an adjuvant comprises a pathogen-derived molecular pattern (PAMP) or mimic thereof. In some embodiments, an adjuvant comprises an immunostimulatory nucleic acid, e.g., a double-stranded nucleic acid, e.g., double-stranded RNA or an analog thereof. For example, in some embodiments, an adjuvant comprises polyriboinosinic:polyribocytidylic acid (polylC). In some embodiments an adjuvant comprises a nucleic acid comprising unmethylated nucleotides, e.g., a single-stranded CpG oligonucleotide. In some embodiments, an adjuvant comprises a cationic polymer, e.g., a poly(amino acid) such as poly-L-lysine, poly-L-arginine, or poly-L-ornithine. In some embodiments an adjuvant comprises a nucleic acid (e.g., dsRNA, polylC) and a cationic polymer. For example, in some embodiments, an adjuvant comprises polylC and poly-L-lysine. In some embodiments, an adjuvant comprises a complex comprising polylC, poly-L-lysine, and carboxymethylcellulose (referred to as polylCLC). In some embodiments, an adjuvant comprises a CD40 agonist and a TLR ligand. For example, in some embodiments an adjuvant comprises (i) an anti-CD40 antibody and (ii) an immunostimulatory nucleic acid and / or a cationic polymer. In some embodiments, an adjuvant comprises an anti-CD40 antibody, an immunostimulatory nucleic acid, and a cationic polymer. In some embodiments, an adjuvant comprises (i) an anti-CD40 antibody and (ii) poly(IC) or poly(ICLC). In certain embodiments, an adjuvant is pharmaceutically acceptable for administration to a human subject. In certain embodiments an adjuvant is pharmaceutically acceptable for administration to a non-human subject, e.g., for veterinary purposes.

[0154] Antigen-Free Vaccine

[0155]

[0124] An “antigen-free vaccine”, as used herein, refers to a vaccine composition that does not comprise an antigen before administration of the vaccine composition to a subject. Without wishing to be bound by any theory, it is hypothesized that the antigen-free vaccine attracts, traps, captures, or otherwise acquires a cancer antigen to or near the vaccine composition after administering to the subject, and subsequently exposes the cancer antigen to an immune cell, thereby generating cancer-specific and / or subject-specific immune response and / or preventing or treating cancer.

[0156]

[0125] Active specific immunotherapy involves the priming of the immune system in order to generate a T-cell response against tumor-associated antigens. One example of the active specific approach is adoptive T-cell therapy, which involves the ex vivo cultivation of T cells with demonstrated activity against a specific target cancer antigen. Cells are obtained from the subject, purified, and cultured. Such ex vivo cultivation increases the frequency of these T cells to achieve therapeutic levels. The cells are then infused back into the patient via injectable alginate-based hybrid hydrogels.

[0157]

[0126] In some aspects, the methods disclosed herein relate to the administration of antigen- free vaccines comprised in the hydrogels of the disclosure in the treatment of cancer in a subject. In certain embodiments, the anti gen -free vaccines are FMS-like tyrosine kinase 3 ligand (FLT3L) or Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF). As demonstrated in the Examples provided herein, anti-tumor protection was significantly enhanced when the hydrogels of the disclosure acted as in situ antigen-free vaccine sites in addition to T cell delivery depots.

[0158] Methods of the Disclosure

[0159]

[0127] In some embodiments, the present disclosure provides methods of treating cancer in a subject in need thereof that comprise administering to the subject a hydrogel of the disclosure comprising a T-cell. In some embodiments, the hydrogels of the present disclosure comprise a CD8+ T-cell. In some embodiments, a hydrogel of the disclosure comprising a T-cell, e.g., a CD8+ T-cell is administered directly into a tumor.

[0160]

[0128] In some embodiments, a hydrogel of the disclosure comprising a T-cell is administered to a subject for treating cancer in combination with an antigen-free vaccine. In some embodiments, the antigen-free vaccine comprises FLT3L and / or GM-CSF. In some embodiments, a hydrogel of the disclosure comprises a T-cell, e.g., a CD8+ T-cell and an antigen-free vaccine component, e.g., FLT3L and / or GM-CSF. In other embodiments, a hydrogel of the disclosure comprising a T-cell, e.g., a CD8+ T-cell, is administered separately from the antigen-free vaccine, e.g., FLT3L and / or GM-CSF.

[0161]

[0129] In some embodiments, the cancer is selected from the group consisting of a hematological tumor or a solid tumor. In some embodiments, the cancer is a skin cancer e.g., melanoma, pancreatic cancer, breast cancer, glioblastoma, or leukemia.

[0162]

[0130] As used herein, the term “treating” cancer in a subject in need thereof includes achieving, partially, substantially or completely, one or more of the following: ameliorating, improving or achieving a reduction in the severity of at least one symptom or indicator associated with cancer, e.g., a reduction in the size of a tumor in the subject; and arresting the progression or worsening of cancer in the subject.

[0163] Administration of Hydrogels of the Disclosure

[0164]

[0131] Syringes and needles are typically used to introducing the hydrogels of the disclosure into the body. The term "syringe" technically refers to the reservoir (that holds the liquid) and the plunger (which pushes the liquid out of the reservoir). The "needle" is the part that enters the body, e.g., into a vein, under the skin, or into muscle or other tissue. The word "syringe" is also sometimes used to refer to the entire reservoir / plunger / needle combination. They come in a variety of sizes, e.g., a common reservoir size is lee (1 cubic centimeter (cc) = 1 milliliter), with a 25 gauge needle size or smaller.

[0165]

[0132] The needle gauge refers to the size of the bore or hole in the needle. The higher the gauge, the thinner the needle (and the smaller the hole). A 28 gauge needle (abbreviated 28G) is therefore thinner than a 25 gauge needle, which is in turn thinner than an 18 gauge needle. Insulin needles are typically 1 / 2 inch in length and tuberculin needles are typically 5 / 8 of an inch in length. As inscribed on packaging, needle length appears after the gauge number: "28G 1 / 2" refers to a 28 gauge needle that is 1 / 2 inch long.

[0166]

[0133] Larger gauge (frequently 23 G or 21G), longer needles are often used for intramuscular injections. Muscle syringes are typically 1 cc in volumes, but larger volumes are sometimes, e.g., 2 to 5 ccs syringes, depending on the application. Larger volumes and larger bores are appropriate for delivery of cryogels for larger scale muscle repair or regeneration, e.g., after extensive or traumatic laceration of tissue such as injuries incurred in battle or car / plane accidents. Intravenous injectors or needles are used for fine or delicate tissue therapy, e.g., cosmetic dermal filler administration. Such applications typically use shorter needles no larger than 25G.

[0167]

[0134] As used herein, the term "subject" means any human or animal. In certain aspects, the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g, Rhesus macaques. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiment, the subject is a mammal (e.g., a primate or human). In one embodiment, the subject is a human. In some embodiments, the mammal is a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow, and is not limited to these examples. Mammals other than humans can be advantageously used, for example, as subjects that represent animal models of, for example, a hematological malignancy. In addition, the methods described herein can be used to treat domesticated animals and / or pets. A subject can be male or female.

[0135] In certain embodiments, a subject can be one who has been previously diagnosed with or otherwise identified as suffering from or having a condition, disease, or stem cell disorder. A "subject in need" of treatment for a particular condition (e.g., a cancer) can be a subject having that condition, diagnosed as having that condition, or at increased risk of developing that condition relative to a given reference population. In some embodiments, the methods of treatment described herein comprise selecting a subject diagnosed with, suspected of having, or at risk of developing a hematological malignancy or being immunocompromised. In some embodiments, the methods described herein comprise selecting a subject diagnosed with, suspected of having, or at risk of developing a non-malignant disease, for example a non- malignant disease described herein.

[0168] EXAMPLES

[0169] Example 1: Synthesis of alginate-collagen hybrid hydrogels

[0170]

[0136] The goal of this experiment was to synthesize alginate-collagen hybrid hydrogels. In this experiment, alginate and rat tail collagen type 1 were first modified with tetrazine and norbomene respectively, via inverse electron demand Diels-Alder click reactions, and then reacted under cryogelation conditions (-12°C to -18°C) to form macroporous alginate- collagen hybrid hydrogels, in accordance with the scheme shown in Figure 1A.

[0171] Synthesis of tetrazine-modified alginate (Alg-T)

[0172]

[0137] Click alginate biopolymers were modified with 3-(p-benzylamino)-l,2,4,5-tetrazine by first allowing high molecular weight alginate, MW = 265 kDa (Protanol LF 20 / 40; FMC Technologies) to dissolve in stirred buffer containing 0.1 M MES, 0.3 M NaCl, pH 6.5 at 0.5% w / v. Next, N-hydroxysuccinimide (NHS; Sigma-Aldrich) and l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC; Sigma-Aldrich) were added in 5X molar excess of the carboxylic acid groups of alginate. Tetrazine was then added at 1 mmol per gram of alginate to make Alg-T. The coupling reaction was stirred at room temperature for 24 hours, after which the reaction was quenched with hydroxylamine (Sigma- Aldrich) and dialyzed in 12-14 kDa MWCO dialysis tubing (Spectrum Labs) for 4 days against a decreasing salt gradient from 150 mM to 0 mM NaCl in diH2O. The purified Alg-T polymers were treated with activated charcoal, sterile filtered (0.22 pm), and freeze-dried. This resulted in purified Alg-T polymers with a 5% degree of substitution of the available carboxylic acid groups of alginate.

[0173] Synthesis of norbornene-modified collagen (Col-Nb)

[0174]

[0138] Rat Tail Collagen Type I (Coming) was modified with 5-Norbornene-2-acetic acid succinimidyl ester (Nb-NHS; Sigma Aldrich) using a 1 : 10 ratio of Nb-NHS: collagen (w / w). First, rat tail collagen was neutralized with NaOH to pH 7.2-7.5, buffered with 10X DPBS, and diluted to an initial concentration of 2 mg / ml. Next, Nb-NHS was dissolved to 2 mg / ml in DMSO and diluted 10-fold in IX PBS. An equal volume of Nb-NHS solution was then added to the neutralized collagen under continual stirring, resulting in a final collagen concentration of 1 mg / ml. The reaction proceeded for 5 hours at 4 °C to delay collagen gelation, and was quenched with 0.1 N acetic acid to re-acidify the collagen solution. Norbornene-modified collagen (Col-Nb) was then dialyzed against 0.025 N acetic acid for 4 days, filtered through a 0.45-pm filter, and lyophilized.

[0175] Synthesis of alginate-collagen hybrid hydrogels

[0176]

[0139] To prepare click alginate-collagen hybrid hydrogels, lyophilized Col-Nb was dissolved to a concentration of 9 mg / ml in 0.025 N acetic acid at 4 °C for at least 48 hours. Alg-T was freshly dissolved to 2 % wt / vol and cooled at 4 °C. Dissolved Col-Nb was neutralized with cold 1 N NaOH, buffered with cold 10X DPBS and balanced with cold milliQ water, after which Alg-T was added. The hydrogel mixture was immediately pipetted into a 2 mm diameter Tygon tubing (VWR) at 50 pl hydrogel mixture per 1 cm tubing, and placed in a -15 °C freezer overnight for cryo-polymerization. After cryogelation, hydrogels were thawed at room temperature and ejected by gently flushing the tygon tubing with 400 pl DPBS, to yield 0.75% wt / vol alginate-collagen hybrid hydrogel, comprised of 60% alginate and 40% collagen. Hydrogels with different ratios of alginate to collagen or total polymer content were fabricated by varying the relative final concentrations of the alginate or collagen (while keeping the total polymer amount constant), or by increasing the total amount of polymer, respectively.

[0177] Characterization of alginate-collagen hybrid hydrogels

[0140] The synthesis described above yielded injectable, shape deformable scaffolds as shown in Figure IB. Moreover, the length of the hydrogel could be adjusted for specific applications. The macroporous structure of the alginate-collagen hybrid hydrogels was confirmed by Scanning Electron Microscope (SEM) imaging, as shown in Figure 1C. The pore size distribution of pristine hydrogels was determined by second-harmonic imaging microscopy. Figure ID shows a representative second-harmonic generation (SHG) image. Quantification of pore sizes revealed a distribution ranging from 1 pm to 250 pm, with a mean pore size of 42 pm. The distribution of pore sizes in the hydrogel is shown as a violinplot in Figure IE.

[0178]

[0141] Thus, alginate-collagen hybrid hydrogels that formed injectable, shape deformable macroporous scaffolds were synthesized by the methods described in this example. The collagen provides adhesion ligands while the alginate provides structural support to the gel. Furthermore, the gels were rod-shaped, which enhanced scalability, as the length of the gels could be easily tuned.

[0179] Example 2: Mechanical properties of alginate-collagen hybrid hydrogels

[0180]

[0142] The goal of this experiment was to evaluate the mechanical properties of alginate- collagen hybrid hydrogels as a function of their composition. To this end, shape recovery of hydrogels following shear stress, as well as their interconnected porosity were quantified while systematically varying two parameters: the alginate-collagen ratio, and the total hydrogel percentage at 60% alginate-40% collagen. Interconnected porosity of hydrogels was measured by first weighing intact hydrogels (“Mhydrated”), followed by wicking hydrogels with Kimwipes™ for 15 seconds to remove the excess buffer from hydrogel pores. The wicked hydrogels were then re-weighed (“Mwicked”) and interconnected porosity was quantified as:

[0181] (Mhydrated — Mwicked) / Mhydrated

[0182]

[0143] Shape recovery was estimated by first weighing intact hydrogels (“Mhydrated”), followed by wicking the hydrogels with Kimwipes™ for 15 seconds to remove excess buffer, rehydrating the hydrogels in DPBS and then re-weighing the rehydrated hydrogels (“Mrehydrated”). Shape recovery of the hydrogel was quantified as:

[0183] Mrehydrated / Mhydrated

[0144] While the shape recovery ratio did not vary significantly as the alginate was increased from 30% to 50%, it was observed to increase by almost 2.5-fold at 60% alginate relative to 50%. These results are shown in Figure 2A. However, no significant changes were observed in the interconnected porosity of the hydrogels over this alginate composition range, as shown in Figure 2B. Next, the total hydrogel percentage was varied from 0.65% to 0.90%, for a hydrogel composed of 60% alginate and 40% collagen. While the shape recovery ratio showed a slight increase with increasing the hydrogel percentage from 0.65% to 0.90%, the interconnected porosity showed a significant decrease from 0.65% to 0.90% hydrogel percentage. These results are shown in Figure 2C (shape recovery) and Figure 2D (interconnected porosity).

[0184]

[0145] Thus, these experiments showed that changing the ratio of alginate to collagen significantly altered hydrogel shape deformability, while total gel percentage influenced the interconnected porosity of the gels.

[0185] Example 3: Tunable release of bioactive factors from alginate-collagen hybrid hydrogels

[0186]

[0146] The goal of this experiment was to determine if incorporated bioactive factors such as cytokines and adjuvants could be tunably released from the alginate-collagen hybrid hydrogels. To this end, cytokines and adjuvants were adsorbed onto laponite or condensed with polyethyleneimine (PEI), respectively, and added to the hydrogel solution before cryogelation, as shown in the schematic in Figure 3A. For “wicked” conditions, hydrogels were first briefly wicked and rehydrated in buffer.

[0187]

[0147] Temporal profiles of the amount of bioactive factor released from the hydrogel revealed that adsorption onto 50 pg laponite could tune the release of GM-CSF and IL-2, when compared to conditions with no adsorption. As shown in Figure 3B, sustained release of the cytokine GM-CSF from the hydrogel was observed over the course of at least 12 days, when it was adsorbed onto 50 pg laponite. Similarly, the cytokine IL-2 also displayed sustained release over a course of 7 days, as shown in Figure 3C. Measurements of the amount of the adjuvant CpG released over a time course of 15 days revealed that the release rate could be tuned when CpG was condensed with PEI, as compared to CpG release in the absence of PEI, as shown in Figure 3D.

[0148] These results demonstrate that the release profiles of bioactive factors including cytokines and adjuvants can be tuned by adsorption onto laponite (IL-2, GM-CSF) or by polyethyleneimine condensation (CpG).

[0188] Example 4: In vivo persistence of T cells loaded in alginate-collagen hybrid hydrogels

[0189]

[0149] The goal of this experiment was to evaluate the in vitro migration speed and the in vivo persistence of T cells encapsulated in alginate-collagen hybrid hydrogels. To this end, T cells were loaded into pre-formed hydrogels by briefly wicking the gels and rehydrating them in a concentrated solution of T-cells.

[0190]

[0150] T cells loaded into the alginate-collagen hybrid hydrogels exhibited enhanced migration relative to those in alginate only gels in vitro. These results are shown in Figure 4A. Next, T cell loaded hydrogels were injected subcutaneously into mice, and the bioluminescence of luciferase-expressing T cells was measured by an In Vivo Imaging System (IVIS) at Day 0 and Day 5 post injection. IVIS images, as shown in Figure 4B, revealed the enhanced persistence of T cells loaded in hydrogels at the injection site as compared to T cells that were directly injected.

[0191]

[0151] Thus, the loading of T cells in alginate-collagen hybrid hydrogels enhances local T cell persistence in vivo, and their migration speed in vitro.

[0192] Example 5: Therapeutic studies of tumor control by T cells loaded in alginate-collagen hybrid hydrogels

[0193]

[0152] The goal of this experiment was to evaluate the tumor control mediated by T cells loaded into alginate-collagen hybrid hydrogels. For this, 1 x 105B16-F10 melanoma cells were subcutaneously injected into female C57 / BL6 mice on day 0. Next, on day 5, when the tumors were palpable, a total of 4 x 106pmel-1 CD8+ T cells were delivered either through direct injection, or through peritumoral injection of T cells loaded into alginate-collagen hybrid hydrogels. For studies where preconditioning was performed, mice were subjected to sub-lethal irradiation on day 4. Mice were then monitored for tumor growth and survival. A schematic of the method followed for the therapeutic studies is shown in Figure 5A.

[0194]

[0153] Kaplan-Meier curves were plotted to compare the survival of mice under different experimental conditions. These graphs are shown in Figure 5B (with preconditioning), and Figure 5C (without preconditioning). The results revealed that peritumoral injection of T cells loaded into hydrogels provided superior therapeutic benefit compared to standard T cell delivery approaches against established B16-F10 tumors, irrespective of whether or not mice were preconditioned by sub-lethal irradiation before treatment. Next, hydrogel -mediated T cell delivery was combined with antigen-free vaccination. The vaccine component utilized either FLT3L or GM-CSF for the recruitment of antigen presenting cells. Figure 5D shows Kaplan-Meier curves comparing the survival of mice treated with integrated hydrogel mediated T cell delivery combined with antigen-free vaccination, with survival of mice treated with either hydrogel mediated T cell delivery or antigen-free vaccination alone. These data show that the protection was radically enhanced when the hydrogels acted as in situ antigen-free vaccine sites in addition to T cell delivery depots, irrespective of the factor used to recruit antigen presenting cells to the tumors. Notably, 10 of 16 and 13 of 16 mice treated with integrated T cell delivery plus antigen-free FLT3L or GMCSF vaccines, respectively, completely rejected their primary tumors.

[0195]

[0154] Thus, the therapeutic studies presented in this example revealed that T cells loaded into alginate-collagen hybrid hydrogels provided significantly better tumor control than T cells delivered through direct peritumoral injection or intravenous infusion. Furthermore, the anti-tumoral response was further enhanced when localized T cell delivery was integrated with in situ antigen-free vaccination.

[0196] Example 6: Synthesis of alginate-gelatin hybrid hydrogels

[0197]

[0155] Alginate-gelatin hybrid hydrogels were fabricated by modifying alginate and fish- derived gelatin with tetrazine and norbornene, respectively, after which they were reacted overnight at -20°C to form hydrogels, as shown in the schematic in Figure 6A. Similar to the alginate-collagen gels, gelatin provides adhesion ligands for cell engagement, while the alginate provides structural integrity to the hydrogels.

[0198]

[0156] The relative amounts of norbomene present on gelatin polymers with different extents of modification were quantified by NMR experiments. These results are shown in Figure 6B. Next, the storage moduli and crossover points of alginate-gelatin hybrid hydrogels fabricated using gelatin with different extents of modification were measured. As shown in Figure 6C, the storage modulus for gelatin with high norbornene modification was almost 4- fold higher than for gelatin with low norbomene modification. Measurements of the crossover point, z.e., the time of transition of a gel from liquid-like to solid-like behavior revealed a longer transition time for gelatin with low levels of modification, as shown in Figure 6D.

[0199]

[0157] Thus, reacting gelatin-norbomene with alginate-tetrazine under cryogelation conditions resulted in robust macroporous, injectable hydrogels with shape deformability. The physical properties of these gels could be varied by tuning the extent of gelatin modification.

[0200] Example 7: Mechanical profiles of alginate-gelatin hybrid hydrogels

[0201]

[0158] The goal of this experiment was to evaluate the mechanical properties of the alginate- gelatin hydrogel as a function of its composition. To this end, shape recovery of hydrogels following shear stress, as well as their interconnected porosity were quantified as a function of three parameters: the extent of norbomene modification on gelatin, the alginate-collagen ratio, and the total hydrogel percentage.

[0202]

[0159] The percentage shape recovery showed no significant difference between hydrogels composed of 2% total polymer comprising 45% alginate and 55% gelatin, with those composed of 1.2% total polymer comprising 80% alginate and 20% gelatin. Furthermore, a hydrogel composed of 2% total polymer comprising 45% alginate and 55% gelatin with low levels of modification showed almost complete shape recovery following shear stress. These results are shown in Figure 7A. Different trends were observed when the percentage interconnected porosity was measured. Here, for hydrogels composed of 2% total polymer comprising 45 % alginate and 55% gelatin, gels comprised of gelatin with high levels of modification displayed almost 4-fold higher interconnected porosity than those comprised of gelatins with low levels of modification. These results are shown in Figure 7B.

[0203]

[0160] Thus, these experiments showed that the mechanical and physical properties of alginate-gelatin hybrid hydrogels could be varied by tuning the extent of gelatin modification and the ratio of alginate to gelatin.

Claims

CLAIMS1. A method of treating cancer in a subject, the method comprising administering to the subject a hydrogel comprising a first polymer and a second polymer, wherein: the first polymer comprises alginate, and the second polymer comprises collagen or gelatin; wherein the first polymer is connected to the second polymer by linkers of formulawherein bond is a single or a double bond;R1is -Co-C6alkyl-NH-, -Co-C6alkyl -O-, or -Co-C3alkyl-C(0)-;R2is a bond, aryl, or heteroaryl, wherein aryl and heteroaryl are optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci-Ce alkyljamino, or di(Ci-Ce alkyljamino;R3is -Co-Ce alkyl-NH-, -Co-Ce alkyl-O-, or -Co-C3alkyl-C(0)-; andR4is hydrogen, Ci-Ce alkyl, aryl, or heteroaryl, wherein aryl and heteroaryl are optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci-Ce alkyljamino, or di(Ci-Ce alkyljamino.

2. The method of claim 1, wherein the hydrogel further comprises a T cell.

3. The method of claim of 2, wherein the T cell is a CD8+ T cell.

4. The method of any one of claims 1-3, further comprising administering a cytokine to the subject.

5. The method of claim 4, wherein the cytokine is selected from the group consisting of FMS-like tyrosine kinase 3 ligand (FLT3L), Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) and Interleukin-2 (IL-2).

6. The method of claim 4 or 5, wherein the cytokine is comprised in the hydrogel.

7. The method of any one of claims 1-6, further comprising administering an adjuvant to the subject.

8. The method of any one of claims 1-7, wherein the cancer is selected from the group consisting of a hematological malignancy and a solid tumor cancer.

9. The method of any one of claims 1-8, wherein the hydrogel comprises pores having an average pore size of about 1 pm to about 200 pm.

10. The method of claim 9, wherein the average pore size is from about 1 pm to about 50 pm, about 1 pm to about 25 pm, 5 pm to about 100 pm, about 5 pm to about 10 pm, about 15 pm to about 50 pm, about 25 pm to about 75 pm, about 40 pm to about 80 pm, about 25 pm to about 100 pm, about 50 pm to about 150 pm, or about 75 pm to about 200 pm.

11. The method of claim 10, wherein the average pore size is about 25 pm to about 75 pm.

12. The method of any one of claims 1-11, wherein the first polymer and the second polymer are present in the hydrogel at a ratio of from about 3:7 w / w to about 4: 1 w / w.

13. The method of any one of claims 1-12, wherein the first polymer and the second polymer are present in the hydrogel at a ratio of about 3:7 w / w, about 2:3 w / w, about 1 : 1 w / w, about 3:2 w / w, about 7:3 w / w or about 4: 1 w / w.

14. The method of any one of claims 1-13, wherein the total gel percentage of the hydrogel ranges from about 0.8% to about 3%.

15. The method of any one of claims 1-14, wherein the first polymer is connected to the second polymer by linkers of formula (A’): first polymer second polymer(A’) wherein the variables are as defined in Formula (A).

16. The method of any one of claims 1-15, wherein: bond = is a single bond;R1is -Ci-C6alkyl-NH-, or -Co-C3alkyl-C(0)-;R2is a bond or aryl optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci-Ce alkyl)amino, or di(Ci-Ce alkyl)amino;R3is -Ci-C6alkyl-NH-, or -Co-C3alkyl-C(0)-; andR4is hydrogen, Ci-Ce alkyl, or heteroaryl, wherein heteroaryl is optionally substituted with halogen, hydroxy, Ci-Ce alkyl, Ci-Ce alkoxy, (Ci-Ce alkyl)amino, or di(Ci-Ce alkyl)amino.

17. The method of to claim 16, wherein R1and R3are both -methyl -NH-; or R1and R3are both -C(O)-.

18. The method of any one of claims 1-17, wherein the linkers of formula (A) are of formula (I):or of formula (II):or of formula (III):

19. The method of any one of claims 1-18, wherein the hydrogel is a cryogel.

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