Disulfide hydrogels

Abstract

Provided are various formulations, which rapidly form into disulfide-linked hydrogels (e.g., PEG hydrogels) in 30 seconds or less. These rapidly forming hydrogels were also able to conform and adhere to mucosal tissues via PEG-mucin entanglements and hydrogen bonding. The crosslinked hydrogels are formed from disulfide exchange between a multivalent thiol component and a multivalent disulfide component. Also provided are methods of making and methods of using the crosslinked hydrogels.

Description

DISULFIDE HYDROGELSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 671.202, filed on July 13. 2024, the disclosure of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under contract no.EB030834 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 13, 2025 is named “070919_00157_ST26.xml”, and is 2.665 bytes in size.BACKGROUND OF THE DISCLOSURE

[0004] Mucosal tissues such as the eye, stomach, nose, lung, and vagina are often sites of inflammatory’ and infectious diseases making them a desirable site for therapeutic delivery. Moreover, the mucosa may also be useful as a non-invasive route for drug delivery to local and distal sites as these tissues possess a large surface area that is highly vascularized and immunologically active.

[0005] However, mucosal tissues possess natural defense mechanisms to facilitate the rapid clearance of potentially infectious or irritating materials from these sites which can lead to reduced drug bioavailability' when locally' administered. This has motivated use of formulations with enhanced viscosity' (e.g., ointments, creams, gels) to act as a local drug depot for sustained delivery. Thus, biomaterial systems have been sought to provide these benefits for delivery of existing drugs and others under clinical development.

[0006] Delivery of therapeutic-loaded gels to the cervicovaginal, ocular, nasal, and rectal routes have often necessitated design of in situ forming hydrogels which can be administered in a liquid form and rapidly transform upon administration to act as a local drug depot. This can be achieved with thermosensitive polymers such as Pluronic and / use of click chemistry' including dibenzocyclooctyne (DBCO)-azide and norbomene-tetrazine for rapidhydrogel assembly. A drawback of the latter approach using click chemistry' is these gels are not reversible and contain crosslinks which will remain intact such that these gels will not be naturally degraded once they have delivered a therapeutic payload. Sulfhydryl crosslinked hydrogel biomaterials can also be constructed using maleimide or vinyl sulfone as reactive groups. While highly stable, sulfhydry l crosslinked gels ty pically form on the order of minutes which is less than ideal for in situ gelation. As an alternative, sulfide-dipyridyl disulfide reactions can also occur rapidly but, in general, they are underexplored for hydrogel formulations used in drug delivery. These crosslinks can also be reversed under reducing conditions and are likely to degrade over time once administered due to biological tissues. Moreover, this cross-linking mirrors that found with mucus gels and prior work has shown hydrogel attachment to mucosal tissues may be facilitated via reactions with available cysteinesBRIEF SUMMARY OF THE DISCLOSURE

[0007] In situ gelling polymeric biomaterials have proven useful as drug delivery vehicles to enable sustained release at sites of disease or injury. However, if delivered to mucosal tissues, such as the eyes, nose, gastrointestinal, and cervicovaginal tract, these gels must also possess the ability7to adhere to an epithelium coated in mucus. Tow ards this end, we report a new rapid in situ gelling polyethylene glycol-based hydrogel. Unlike other chemistries that enable rapid gel formation which form via irreversible covalent bonds, we use a bio-reducible linker allowing the gels to be naturally degraded over several days once administered. Described herein are various formulations, which rapidly form into disulfide- linked hydrogels (e.g., PEG hydrogels) in 30 seconds or less. These rapidly forming hydrogels were also able to conform and adhere to mucosal tissues via entanglement and hydrogen bonding. Controlled release of protein-based cargoes from the gels w as achieved over several hours whereas 40 nm nanoparticle-based cargos w ere retained over 24 hours. These rapid in situ forming gels were well-tolerated by mammalian cells and were retained in the nasal cavity of the mice for up to 1 week. These studies support further testing and development of rapid in situ forming gels for drug delivery to improve therapeutic retention and efficacy at mucosal sites.

[0008] The present disclosure provides crosslinked hydrogels. The crosslinked hydrogels are formed from disulfide exchange between a multivalent thiol component and a multivalent disulfide component. Also provided are methods of making and methods of using the crosslinked hydrogels. The crosslinked hydrogels may be referred to as hydrogels.

[0009] In an aspect, the present disclosure provides crosslinked hydrogels. The crosslinked hydrogels may comprise, consist essentially of, or consist of an aqueous medium, as well as a crosslinked network of aliphatic groups attached via disulfides. The hydrogels may be formed from disulfide exchange with a multivalent thiol component and a multivalent disulfide component in an aqueous medium. As used throughout, the term “multivalent” refers to a compound or molecule having one or a plurality of specific functional groups. For example, a multivalent thiol component has one or a plurality of thiol groups.

[0010] In an aspect, the present disclosure provides methods of making a crosslinked hydrogel of the present disclosure. Also included are methods of encapsulating cargo in the hydrogels.

[0011] In an example, a method of making a crosslinked hydrogel comprises mixing a first mixture comprising the multivalent thiol in an aqueous medium with a second mixture comprising the multivalent disulfide in an aqueous medium such that disulfide exchanges occur between the multivalent thiol and the multivalent disulfide thus forming a crosslinked hydrogel. The two mixtures can be mixed on a substrate or in a vessel. Alternatively, the two mixtures could be mixed via a syringe (e.g.. each mixture is in a separate syringe and mixed as or while the mixtures are deposited are a substrate, see, e.g., FIG. 1).

[0012] In an aspect, the present disclosure provides methods of using hydrogels of the present disclosure. The hydrogels may be used to deliver cargo or as three-dimensional cell scaffolds.

[0013] For example, the hydrogels may be used to deliver cargo to an individual. For example, the hydrogel may be formed on a surface of the individual. Alternatively, the hydrogel may be delivered subcutaneously. For example, the cargo can be a therapeutic agent. Examples of therapeutic agents include, but are not limited to, small molecules and biologies. Examples of cargo also include drugs, small molecules, peptides, proteins, enzymes, antibodies, DNA, RNA, siRNA, nanoparticles (e.g., nanoparticles loaded with small molecules, such as, for example, progesterone / Fluticasone loaded PLGA nanoparticles), drug nanocrystals (e.g., progesterone / Fluticasone nanocrystals), liposomes (e.g., liposomes loaded with small molecules), viral vectors, bacteriophages, mammalian and bacterial cell therapies, and the like, and combinations thereof. The surface may be a mucosal tissue, such as, for example mucosal tissue of an eye, gastrointestinal tract, nose, lung, vagina, uterus, buccal, or urinary bladder. Other areas of therapeutic delivery include, but are not limited to, subcutaneous, intradermal, intramuscular, cancer related tissues, and intraperitoneal.BRIEF DESCRIPTION OF THE FIGURES

[0014] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0015] FIG. 1. Schematic illustrating the preparation of rapid in situ forming PEG hydrogels.

[0016] FIG. 2A. Physical characterization of rapid in situ forming PEG gels. Storage modulus (G’j for each gel at frequency of 1 Hz. (n=3), ****p <0.0001 for one-way ANOVA with Tukey’s multiple comparison test.

[0017] FIG. 2B. Physical characterization of rapid in situ forming PEG gels.Estimated pore size size for particle tracking microrheology using densely PEGylated 100 nm nanoparticles as probes. * p <0.05, ***p <0.001, **** ? <0.0001 for Kruskal-Wallis test.

[0018] FIG. 2C. Physical characterization of rapid in situ forming PEG gels.Equilibrium swelling ratio after immersion in PBS. (n=3), *p <0.05 for Kruskal-Wallis test.

[0019] FIG. 2D. Physical characterization of rapid in situ forming PEG gels.Degradation profile of PEG gels after immersion in PBS for 30 hours.

[0020] FIG. 2E. Physical characterization of rapid in situ forming PEG gels.Dissolution of PEG gels after 10 minutes in PBS or under reducing conditions in DTT.

[0021] FIG. 2F. Physical characterization of rapid in situ forming PEG gels. SEM images of 20kD PEG gels at 200x magnification.

[0022] FIG. 2G. Physical characterization of rapid in situ forming PEG gels. SEM images of 20kD PEG gels at 450x magnification.

[0023] FIG. 3A. Self-healing of rapid in situ forming PEG gels. Photographs of self- healing behavior for PEG gels.

[0024] FIG. 3B. Self-healing of rapid in situ forming PEG gels. Amplitude sweep demonstrating linear viscoelastic region below 100% strain.

[0025] FIG. 3C. Self-healing of rapid in situ forming PEG gels. Cyclic strains of 0.5% (low, unshaded area) and 200% (high, shaded area) at a frequency of 10 rad / sec.

[0026] FIG. 3D. Self-healing of rapid in situ forming PEG gels. Cyclic strains of 0.5% (low, unshaded area) and 500% (high, shaded area) at a frequency of 10 rad / sec.

[0027] FIG. 3E. Self-healing of rapid in situ forming PEG gels. Cyclic strains of 0.5% (low, unshaded area) and 1000% (high, shaded area) at a frequency of 10 rad / sec.

[0028] FIG. 4A. Mucoadhesive properties of rapid in situ forming PEG hydrogels. Schematic illustrating potential mechanisms enabling PEG gel adhesion to mucosal tissues.

[0029] FIG. 4B. Mucoadhesive properties of rapid in situ forming PEG hydrogels. Image of PEG gels following application to porcine intestinal tissue.

[0030] FIG. 4C. Mucoadhesive properties of rapid in situ forming PEG hydrogels. Mucoadhesive strength as measured by pull-apart test for PEG gels & 4% w / v chitosan immediately (n=3), * p <0.05 for on-way ANOVA with Tukey’s multiple comparison test. Data shows mean ± SD.

[0031] FIG. 4D. Mucoadhesive properties of rapid in situ forming PEG hydrogels. 24 hours (n=3), non-significant (ns) as per Kruskal-Wallis test after application to porcine intestinal tissue. Data shows mean ± SD.

[0032] FIG. 5. Release kinetics of model therapeutic cargo from rapid forming PEG hydrogels. Cumulative mass release profiles of bovine serum albumin (BSA), immunoglobulin (IgG), and 40 nm nanoparticles (NP) from PEG hydrogels with either 10 kDa or 20 kDA PEG-SH and PEG-OPSS over 24 hours (n=3). Data shows mean ± SD.

[0033] FIG. 6A. Biocompatibility7of rapid forming PEG gels. Viability of HEK-293 cells following treatment with 4-arm PEG solutions (n=5). * p <0.05, **p <0.01, *** ?<0.001, and **** ? <0.0001 for one-way ANOVA with Dunnett’s multiple comparison test. Data shows mean ± SD.

[0034] FIG.6B. Biocompatibility of rapid forming PEG gels. Viability7of HEK-293 cells following treatment with PEG hydrogels (n=4). * p <0.05, ** <0.01, ***p <0.001, and **** / ? <0.0001 for one-way ANOVA with Dunnett’s multiple comparison test. Data shows mean ± SD.

[0035] FIG. 6C. Biocompatibility of rapid forming PEG gels. Live / Dead fluorescent staining following treatment with PEG hydrogels.

[0036] FIG. 7A. In vivo nasal retention of rapid forming PEG gels. IVIS images of mice at different time points following administration of either PBS or rapid forming PEG hydrogels loaded with near infrared labeled, 100 nm polystyrene nanoparticles.

[0037] FIG. 7B. In vivo nasal retention of rapid forming PEG gels. Quantification of total radiation efficiency from IVIS images, used to assess in vivo retention of PEG hydrogels over time. Data shows mean ± SD.

[0038] FIG. 7C. In vivo nasal retention of rapid forming PEG gels. Body weight of mice monitored throughout the study to evaluate overall health and tolerability7of PEG hydrogels. Data shows mean ± SD.

[0039] FIG. 8A. Bulk rheological properties of rapid in situ forming PEG gels. Storage modulus (G’) as a function of frequency at 10% strain (n=3).

[0040] FIG. 8B. Bulk rheological properties of rapid in situ forming PEG gels. Loss modulus (G”) as a function of frequency at 10% strain (n=3).

[0041] FIG. 8C. Bulk rheological properties of rapid in situ forming PEG gels.Storage modulus (G’) as a function of frequency at 10% strain (n=3).

[0042] FIG. 8D. Bulk rheological properties of rapid in situ forming PEG gels. Loss modulus (G”) as a function of frequency at 10% strain (n=3).

[0043] FIG. 9A. Bulk rheological properties of control gels used for mucoadhesion. Storage modulus (G’) and Loss modulus (G”) as a function of frequency at 10% strain of 2% w / v 4-arm PEG-DBCO lOkD crosslinked with 2%w / v 4-arm PEG- Azide lOkD (n=3).

[0044] FIG. 9B. Bulk rheological properties of control gels used for mucoadhesion. Storage modulus (G’) and Loss modulus (G”) as a function of frequency at 10% strain of 4% w / v chitosan (n=3).

[0045] FIG. 9C. Bulk rheological properties of control gels used for mucoadhesion. Flow sweep (n=l) showing no instant crosslinking between mucin chains and PEG-Thiol or PEG-OPS S indicating mucoadhesion at 0 hr. is driven by polymer-mucin chain entanglement and hydrogen bonding.

[0046] FIG. 10A. Micro rheological properties of lOkD PEG gels at 0 hr. and 24 hr. after mixing PEG-SH and PEG-OPSS solutions. Estimated pore size based on analysis of mean square displacement (MSD) at r=ls.

[0047] FIG. 10B. Micro rheological properties of lOkD PEG gels at 0 hr. and 24 hr. after mixing PEG-SH and PEG-OPSS solutions. (B) Complex microviscosity ( / *) at a frequency co = 1 Hz calculated from measured MSD. * p <0.05, **p <0.01, ***p <0.001, **** <0.0001 for Kruskal -Wallis test.

[0048] FIG. 11. Mucoadhesive properties of 0.5% w / v 4-arm PEG-SH lOkD and 1% w / v 4-arm PEG-OPSS lOkD gels at 0 hr. and 24hr. after application to porcine intestinal tissue (n=3). * p <0.05 for Welch’s t test.

[0049] FIG. 12A. Release kinetics of model therapeutic cargo from different formulations of rapid forming PEG hydrogels. Cumulative release profile of (A) BSA from lOkD PEG gels (n=3) over 24 hours.

[0050] FIG. 12B. Release kinetics of model therapeutic cargo from different formulations of rapid forming PEG hydrogels. Cumulative release profile of IgG from lOkD PEG gels (n=3) over 24 hours.

[0051] FIG. 12C. Release kinetics of model therapeutic cargo from different formulations of rapid forming PEG hydrogels. Cumulative release profile of 20 nm nanoparticles (NP) from 20kD PEG gels (n=3) over 24 hours.

[0052] FIG. 13A. Biocompatibility of rapid forming 5kD PEG gels. Viability' of HEK-293T cells following treatment with 4-arm PEG solutions (n=4) * p <0.05 ***p <0.001 for Kruskal-Wallis test.

[0053] FIG. 13B. Biocompatibility of rapid forming 5kD PEG gels. Viability of HEK-293T cells following treatment with PEG hydrogels (n=4) ****p <0.0001 for one-way ANOVA with Tukey’s multiple comparison test.

[0054] FIG. 14A. FTIR Spectrum of rapid forming PEG gels at lOkD.

[0055] FIG. 14B. FTIR Spectrum of rapid forming PEG gels at 20kD.DETAILED DESCRIPTION OF THE DISCLOSURE

[0056] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, process step, and electronic changes may be made without departing from the scope of the disclosure.

[0057] As used herein, unless otherwise indicated, ‘"about”, “substantially”, or “the like”, when used in connection with a measurable variable (such as. for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including, but not limited to, those w ithin experimental error (which can be determined by, e.g., a given data set, an art accepted standard, etc. and / or with, e.g., a given confidence interval (e.g. 90%. 95%. or more confidence interval from the mean), such as, for example, variations of + / - 10% or less, +1-5% or less, + / -1% or less, and + / -0. 1% or less of and from the specified value), insofar such variations in a variable and / or variations in the alternatives are appropriate to perform in the instant disclosure. As used herein, the term “about” may mean that the amount or value in question is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off. measurement error, or the like, or other factors known to those of skill in the art such that equivalent results or effects are obtained. In general, an amount, size, composition,parameter, or other quantity or characteristic, or alternative is “about"’ or “the like,"’ whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0058] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “0. 1% to 5%” should be interpreted to include not only the explicitly recited values of 0.1% to 5%, but also, unless otherwise stated, include individual values (e g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0059] The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0060] As used herein, unless otherwise stated or indicated, “s” refers to second(s). “min” refers to minute(s), and “h” refers to hour(s).

[0061] The phrase “therapeutically effective amount” is used herein to mean an amount sufficient to reduce by at least about 15 percent, preferably by at least 50 percent, more preferably by at least 90 percent, and most preferably prevents oxidative stress in the individual. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in the individual.

[0062] As used herein, unless otherwise stated, the term “group” refers to a chemical entity that is monovalent (i.e. , has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalently bonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like).Illustrative examples of groups include:

[0063] As used herein, unless otherwise indicated, the term “aliphatic” or “aliphatic groups” refers to branched or unbranched hydrocarbon groups that, optionally, contain one or more degree(s) of unsaturation. Degrees of unsaturation can arise from, but are not limited to, cyclic aliphatic groups. For example, the aliphatic groups / moieties are a Ci6 to C40 aliphatic group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., Ci6, 617, Cis, C19, C20, 621, C22, 623, 624, C25, C26, C27, C28, 629, C30, 631, C32, C33, C34, C35, C36, C37, C38, C39, and C40). Aliphatic groups include, but are not limited to, alkyd groups, alkene groups, and alkyne groups. The aliphatic group can be unsubstituted or substituted with one or more substituent(s). Examples of substituents include, but are not limited to, various substituents such as, for example, halogens (-F, -Cl, -Br. and -I), azide group, aliphatic groups (e g., alkyl groups, alkene groups, alkyne groups, and the like), aryl groups, hydroxyl groups, alkoxide groups, carboxylate groups, carboxylic acid groups, ether groups, ester groups, amide groups, thioether groups, thioester groups, and the like, and combinations thereof.

[0064] As used herein, unless otherwise indicated, the term “alkyl” or “alkyl group” refers to branched or unbranched, linear saturated hydrocarbon groups and / or cyclic hydrocarbon groups. Examples of alkyl groups include, but are not limited to, methy l groups, ethyl groups, propyl groups, butyl groups, isopropyl groups, tert-butyl groups, cyclopropyl groups, cyclopentyl groups, cyclohexyl groups, and the like. Alkyl groups are saturated groups, unless it is a cyclic group. For example, an alkyl group is a Ci to C40 alkyd group, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., 61, 62, 63, 64, 65, 66, 67, 6s, 69, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624. 625, 626, 627. 628, 629, 630. 631, 632, 633, 634, 635, 636, 637, 638. 639, and 640). The alkyl group may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to, halogens (-F, -61, -Br, and -I), aliphatic groups(e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group), aryl groups, halogenated aryl groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and combinations thereof.

[0065] As used herein, the term "cycloalkyl" or “cycloalkyl group’' refers to a cyclic hydrocarbon group, e.g.. cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl groups. Cycloalkyl groups can be saturated or partially unsaturated ring systems optionally substituted with, for example, one to three substituents. Each substituent is independently chosen from alkyl, -NH2, oxo (=0), phenyl, haloalkyl (e.g., -CF3), halo (e.g., -F, -Cl, -Br, -I), alkoxy, and -OH groups. Additionally, alkyl substituents may be substituted with various other functional groups. Additional non-limiting examples include aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g., trifluoromethyl group), aryl groups, halogenated aryl groups, alkoxide groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and combinations thereof.

[0066] As used herein, unless otherwise indicated, the term “aryl” or "aryl group” refers to C5 to C30 aromatic or partially aromatic carbocyclic groups, including all integer numbers of carbons and ranges of numbers of carbons therebetween (e.g., C5, Ce, C7, Cs, C9, C10, C11, C12. C13. C14, C15, C16. C17, C18, C19, C20, C21. C22, C23, C24. C25, C26, C27. C28, C29, and C30). An aryl group may also be referred to as an aromatic group. The aryl groups may comprise polyaryl groups such as, for example, fused rings, bi ary l groups, or a combination thereof. The aryl group may be unsubstituted or substituted with one or more substituents. Examples of substituents include, but are not limited to. halogens (-F, -Cl, -Br, and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), and groups, alkoxides, carboxylates, carboxylic acids, ether groups, and the like, and combinations thereof. Examples of ary l groups include, but are not limited to, phenyl groups, biary I groups (e.g., bipheny l groups and the like), fused ring groups (e.g., naphthy l groups and the like), hydroxybenzyl groups, tolyl groups, xylyl groups, and the like.

[0067] As used herein, the term “heteroaryl” or “heteroaryl group” refers to a monocyclic or bicyclic ring system comprising one or two aromatic rings and containing at least one nitrogen or oxygen atom in an aromatic ring. Unless otherwise indicated, a heteroaryl group can be unsubstituted or substituted with one or more, and in particular one or two, substituents. Non-limiting examples of substituents include halogens (-F, -Cl, -Br,and -I), aliphatic groups (e.g., alkyl groups, alkenyl groups, alkynyl groups, and the like), halogenated aliphatic groups (e.g.. trifluoromethyl group), aryl groups, halogenated aryl groups, alkoxide groups, amine groups, nitro groups, carboxylate groups, carboxylic acids, ether groups, alcohol groups, alkyne groups (e.g., acetylenyl groups and the like), and the like, and combinations thereof. Examples of heteroaryl groups include, benzofuranyl, thienyl, fury l, pyridyl, oxazolyl, quinolyl, thiophenyl, isoquinolyl, indolyl, triazinyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl groups, and substituents analogs of any of the foregoing heteroaryl groups.

[0068] In situ gelling polymeric biomaterials have proven useful as drug delivery' vehicles to enable sustained release at sites of disease or injury'. However, if delivered to mucosal tissues, such as the eyes, nose, gastrointestinal, and cervicovaginal tract, these gels must also possess the ability to adhere to an epithelium coated in mucus. Towards this end, we report a new rapid in situ gelling polyethylene glycol-based hydrogel. Unlike other chemistries that enable rapid gel formation which form via irreversible covalent bonds, we use a bio-reducible linker allowing the gels to be naturally degraded over several days once administered. Described herein are various formulations, which rapidly form into disulfide- linked hydrogels (e.g., PEG hydrogels) in 30 seconds or less. These rapidly forming hydrogels were also able to conform and adhere to mucosal tissues via entanglement and hydrogen bonding. Controlled release of protein-based cargoes from the gels was achieved over several hours whereas 40 nm nanoparticle-based cargos were retained over 24 hours. These rapid in situ forming gels were well-tolerated by mammalian cells and were retained in the nasal cavity of the mice for up to 1 week. These studies support further testing and development of rapid in situ forming gels for drug delivery to improve therapeutic retention and efficacy at mucosal sites.

[0069] The present disclosure provides crosslinked hydrogels. The crosslinked hydrogels are formed from disulfide exchange between a multivalent thiol component and a multivalent disulfide component. Also provided are methods of making and methods of using the crosslinked hydrogels. The crosslinked hydrogels may be referred to as hydrogels.

[0070] In an aspect, the present disclosure provides crosslinked hydrogels. The crosslinked hydrogels may comprise, consist essentially of, or consist of an aqueous medium, as well as a crosslinked network of aliphatic groups attached via disulfides. The hydrogels may be formed from disulfide exchange with a multivalent thiol component and a multivalent disulfide component in an aqueous medium. As used throughout, the term "‘multivalent’'refers to a compound or molecule having one or a plurality of specific functional groups. For example, a multivalent thiol component has one or a plurality of thiol groups.

[0071] The crosslinked hydrogel comprises water and one or more crosslinked networks linked via disulfide bonds. The hydrogel can comprise various amounts of water. In various examples, a hydrogel comprises 80 to 99%, such as 85 to 99.9% by weight (based on the total weight of the composition) water. In an example, the hydrogel comprises about 91 to 99.9% by weight water. In various embodiments, the hydrogel comprises 99 to 99.5% weight water.

[0072] Various multivalent thiol components may be used. For example, the multivalent thiol component may comprise two or more thiol groups (e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, or 16 thiols). The multivalent thiol component may comprise one or more polyethylene glycol groups, each polyethylene glycol group comprising one or more thiol groups. For example, a multivalent thiol component may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the following groups:where n is greater than or equal to 0 (e.g., 1 to 10,000) and if there is more than one of the previous groups, each n may be the same or different. In various examples, a multivalent thiol component may have the following structure:In various examples, a multivalent thiol component may have a molecular weight of greater than 0. 1 kDa (e.g., 0.1 to 500 kDa). In various examples, the multivalent thiol component may be mixed w th a disulfide component. Various disulfide components may be used. Examples of disulfide components include, but are not limited to. dipyridyldisulfide (DPS),5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 2.2-dibenzothiazolyl disulfide, or the like.Without intending to be bound by any particular theory, it is considered that the multivalent thiol component may participate in a disulfide exchange with the disulfide component (e.g., the multivalent thiol component may participate in a disulfide exchange with DTNS such that a multivalent disulfide is formed).

[0073] Various multivalent disulfide components may be used. For example, the multivalent disulfide component may comprise two or more disulfide groups (e.g., 2. 3, 4, 5.6, 7, 9, 10, 1 1, 12, 13, 14, 15, or 16 disulfides). The multivalent disulfide components may comprise one or more polyethylene glycol groups, each polyethylene glycol group may comprise one or more disulfide groups. For example, a multivalent disulfide component may comprise one or more of the following groups:wherein n is greater than or equal to 0 (e.g., 0 to 10,000) and R is an aliphatic group, cyclic aliphatic group, aromatic group, aryl group, heteroaromatic group or heteroaryl group, R may be further substituted. In various examples, the R group is a pyridyl group. Thus, for example, a multivalent disulfide may comprise one or more of the following groups:In various examples, a multivalent disulfide may have the following structure:When R is a pyridyl group, a multivalent disulfide may have the following structure:In various examples, a multivalent disulfide component may have a molecular weight of greater than 0. 1 kDa (e.g., 0. 1 to 500 kDa). In various examples, a multivalent disulfide component may further comprise a cell-adhesion peptide (e.g., an RGD peptide, such as, for example cyclo(RGDfC) (SEQ ID NO: 1), which can form a disulfide bond with the multivalent disulfide, where lower case letters refer a D-amino acid). In various examples, the multivalent disulfide may be mixed with a disulfide (e.g., a non-multivalent disulfide). Various disulfides may be used. Examples of disulfides include, but are not limited to, dipyridyldisulfide (DPS), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 2,2-dibenzothiazolyl disulfide, or the like. Without intending to be bound by any particular theory, it is considered that the multivalent disulfide may participate in a disulfide exchange with the disulfide (e.g., the multivalent disulfide may participate in a disulfide exchange with DTNB).

[0074] In various examples, a crosslinked hydrogel may comprise one or more of the following crosslinks:

[0075] The crosslinked hydrogel can be formed from various amounts of the multivalent thiol and the multivalent disulfide. The crosslinked hydrogel can be formed from mixing a first mixture comprising the multivalent thiol and a second mixture comprising the multivalent disulfide. The concentration of the multivalent thiol in the first mixture may be 0. 1 to 99.9% w / v relative to the total volume of the first mixture, including all 0.1% w / v values therebetween (e.g., 0.1 to 5%, 0.1 to 10%, 0.1 to 20%, 0.1 to 25%, 0.1 to 30%, 0.1 to 40%, 0. 1 to 50%, 0. 1 to 60%, 0. 1 to 70%, 0.1 to 80%. 0. 1 to 90%, 0. 1 to 95%, or 0. 1 to 99%). The concentration of the multivalent disulfide in the first mixture may be 0. 1 to 99.9% w / v relative to the total volume of the second mixture, including all 0.1% w / v values therebetween (e.g., 0.1 to 5%, 0.1 to 10%, 0.1 to 20%, 0.1 to 25%, 0.1 to 30%, 0.1 to 40%, 0.1 to 50%, 0.1 to 60%, 0.1 to 70%, 0.1 to 80%, 0.1 to 90%, 0.1 to 95%, or 0.1 to 99%). Both the first and the second mixture comprise water. The water may be buffered to a pH of 5.0 to 8.0 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0). Various buffers may be used to buffer the water. For example, the buffered water may be phosphate buffered saline (PBS). Other suitable buffers include, but are not limited to, HEPES. MOPS. TRIS, MES hydrate, citrate buffer, or the like. In various examples, the multivalent thiol may comprise one or more disulfide bonds formed from a disulfide exchange with a disulfide (e.g., a non- multi valent disulfide). Various disulfides may be used. Examples of disulfides include, but are not limited to, dipyridyldisulfide (DPS), 5.5'-dithiobis(2-nitrobenzoic acid) (DTNB), 2.2- dibenzothiazolyl disulfide, or the like.

[0076] The hydrogel may encapsulate various cargo. For example, the cargo can be a therapeutic agent. Examples of therapeutic agents include, but are not limited to, small molecules and biologies. Examples of cargo also include drugs, small molecules, peptides, proteins, enzymes, antibodies, DNA, RNA, siRNA, nanoparticles (e.g.. nanoparticles loaded with small molecules, such as, for example, progesterone / Fluticasone loaded PLGA nanoparticles), drug nanocrystals (e.g., progesterone / Fluticasone nanocrystals), liposomes (e.g., liposomes loaded with small molecules), viral vectors, bacteriophages, mammalian and bacterial cell therapies, and the like, and combinations thereof.

[0077] Hydrogels of the present disclosure may have various desirable properties. For example, the hydrogels are degradable under reducing conditions (e.g., when in contact with a reductant such as, for example, DTT, glutathione, tris(2-carboxyethyl)phosphine (TCEP), or N-acetylcysteine). Depending on the reductant or environment, the hydrogel can degrade from over the course of minutes (e.g., 10 minutes or less) to a matter of hours to a matter of days (e g., less than 3 days). Additionally, the hydrogels may have a desirable storage modulus and loss modulus, of greater than IPa and 0. IPa respectively. Additionally, the hydrogels may adhere well to various substrates, such as, for example mucosal tissue, and cancer tissue. For example, the hydrogel may adhere to mucosal tissue with a mucoadhesive strength of greater than 100 Pa. Additionally, the hydrogels may be useful as cell growth scaffolds or to deliver cargo. For example, cargo (e.g., therapeutic agents) may be delivered over the course of hours or days (e.g., the cargo may be delivered over the course of 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or greater than 24 hours), allowing for the slow release of cargo. Without intending to be bound by any particular theory, it is considered that size of the cargo may affect the release rate and ultimately determine if the cargo is released due to diffusion or hydrogel degradation. In various examples, gels can also be crosslinked using enzyme responsive peptides to tune drug release or gel degradation.

[0078] In an aspect, the present disclosure provides methods of making a crosslinked hydrogel of the present disclosure. Also included are methods of encapsulating cargo in the hydrogels.

[0079] In an example, a method of making a crosslinked hydrogel comprises mixing a first mixture comprising the multivalent thiol in an aqueous medium with a second mixture comprising the multivalent disulfide in an aqueous medium such that disulfide exchanges occur between the multivalent thiol and the multivalent disulfide thus forming a crosslinked hydrogel. The two mixtures can be mixed on a substrate or in a vessel. Alternatively, the two mixtures could be mixed via a syringe (e.g., each mixture is in a separate syringe and mixed as or while the mixtures are deposited are a substrate, see. e.g., FIG. 1).

[0080] The first mixture and the second mixture can be mixed at various ratios to one another. For example, the ratios may be 0. 1-20 : 0. 1-20 (first mixture : second mixture, v / v). The concentration of the multivalent thiol in the first mixture may be 0.1 to 99.9% w / v relative to the total volume of the first mixture, including all 0.1% w / v values therebetween (e.g., 0.1 to 5%, 0.1 to 10%, 0.1 to 20%. 0.1 to 25%, 0.1 to 30%, 0.1 to 40%, 0.1 to 50%. 0.1 to 60%, 0.1 to 70%, 0.1 to 80%, 0.1 to 90%, 0.1 to 95%, or 0.1 to 99%). The concentration ofthe multivalent disulfide in the second mixture may be 0. 1 to 99.9% w / v relative to the total volume of the second mixture, including all 0.1% w / v values therebetween (e.g., 0.1 to 5%, 0.1 to 10%, 0.1 to 20%, 0.1 to 25%, 0.1 to 30%, 0.1 to 40%, 0.1 to 50%, 0.1 to 60%, 0.1 to 70%, 0.1 to 80%, 0.1 to 90%, 0.1 to 95%, or 0.1 to 99%).

[0081] Various aqueous mediums may be used. Both the first and the second mixture comprise water. The water may be buffered to a pH of 5.0 to 8.0 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4,5.5, 5.6, 5.7, 5.8. 5.9, 6.0, 6.1, 6.2. 6.3, 6.4, 6.5, 6.6. 6.7, 6.8, 6.9, 7.0. 7.1, 7.2, 7.3, 7.4. 7.5,7.6, 7.7, 7.8, 7.9, or 8.0). Various buffers may be used to buffer the water. For example, the buffered water may be phosphate buffered saline (PBS). Other suitable buffers include, but are not limited to, HEPES, MOPS. TRIS, MES hydrate, citrate buffer, or the like.

[0082] The first mixture and / or the second mixture may comprise one or more cargo. For example, the cargo can be a therapeutic agent. Examples of therapeutic agents include, but are not limited to, small molecules and biologies. Examples of cargo also include drugs, small molecules, peptides, proteins, enzy mes, antibodies, DNA, RNA, siRNA, nanoparticles (e.g., nanoparticles loaded with small molecules, such as, for example, progesterone / Fluticasone loaded PLGA nanoparticles), drug nanocrystals (e.g.. progesterone / Fluticasone nanocrystals), liposomes (e.g., liposomes loaded with small molecules), viral vectors, bacteriophages, mammalian and bacterial cell therapies, and the like, and combinations thereof. The cargo in the first mixture may be the same as the cargo in the second mixture. Alternatively, the cargo may the same in both mixtures. Alternatively, only the first mixture may comprise cargo. Alternatively, only the second mixture may comprise cargo. Upon mixing the two mixtures, the cargo will be distributed throughout the hydrogel.

[0083] In various examples, the hydrogel may be formed due at specific pH values or after a period of time. Without intending to be bound by any particular theory', it is considered that disulfide exchange optimally occurs at a pH of 8 or less. In various examples, the pH of the mixture formed from the first mixture and second mixture has a pH of 5.0 to 8.0 (e.g., 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5. 7.6, 7.7, 7.8, 7.9. or 8.0). In various examples, a hydrogel of the present disclosure may form in less than a minute. In various examples, a hydrogel of the present disclosure may form in less than 45 seconds. In various examples, a hydrogel of the present disclosure may form in 30 seconds or less. In various examples, a hydrogel of the present disclosure may form in 25 seconds or less. In various examples, a hydrogel of the present disclosure may form in 20 seconds or less. In various examples, a hydrogel of the presentdisclosure may form in 15 seconds or less. In various examples, a hydrogel of the present disclosure may form in 10 seconds or less. In various examples, a hydrogel of the present disclosure may form in 5 seconds or less. In various examples, the mixing of the first and second mixture may be done at a temperature of 4 to 45 °C, including every value and range therebetween (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37. 38. 39, 40, 41, 42, 43, 44, or 45 °C). In various examples, the hydrogel may form in a matter of seconds, minutes, hours, or days.

[0084] In an aspect, the present disclosure provides methods of using hydrogels of the present disclosure. The hydrogels may be used to deliver cargo or as three-dimensional cell scaffolds.

[0085] For example, the hydrogels may be used to deliver cargo to an individual. For example, the hydrogel may be formed on a surface of the individual. Alternatively, the hydrogel may be delivered subcutaneously. For example, the cargo can be a therapeutic agent. Examples of therapeutic agents include, but are not limited to, small molecules and biologies. Examples of cargo also include drugs, small molecules, peptides, proteins, enzymes, antibodies. DNA, RNA. siRNA, nanoparticles (e.g., nanoparticles loaded with small molecules, such as, for example, progesterone / Fluticasone loaded PLGA nanoparticles), drug nanocrystals (e.g., progesterone / Fluticasone nanocrystals), liposomes (e.g., liposomes loaded with small molecules), viral vectors, bacteriophages, mammalian and bacterial cell therapies, and the like, and combinations thereof. The surface may be a mucosal tissue, such as, for example mucosal tissue of an eye, gastrointestinal tract, nose, lung, vagina, uterus, buccal, or urinary bladder. Other areas of therapeutic delivery include, but are not limited to, subcutaneous, intradermal, intramuscular, cancer related tissues, and intraperitoneal.

[0086] In various examples, the hydrogel may be used as a three-dimensional scaffold for cell growth. Cells may be deposited in the hydrogel with growth medium to allow for the three-dimensional growth. In various other examples, the hydrogel could be used for diagnostic purposes (e.g., capturing pathogens and / or biomarkers for signal detection). Other examples include, progesterone / progesterone nanocrystal / PLGA nanoparticle loaded with progesterone-encapsulated gels for prevention of preterm birth, fluticasone / fluticasone nanocrystal / PLGA nanoparticle loaded with fluticasone - encapsulated gels for allergies and rhinosinusitis, and AAV9 encapsulated gels for nose-to-brain gene delivery.

[0087] The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in anembodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.

[0088] The following Statements are non-limiting examples of the present disclosure. They are not intended to be limiting in any way.Statement 1 . A crosslinked hydrogel formed from a mixture of multivalent thiol components and multivalent disulfide components in an aqueous medium, wherein the multivalent thiol components and the multivalent disulfide components create a crosslinked network via disulfide exchange between thiols of the multivalent thiol component and disulfides of the multivalent disulfide component .Statement 2. A crosslinked hydrogel according to Statement 1, wherein the multivalent thiol component comprises two or more thiol groups.Statement 3. A crosslinked hydrogel according to Statement 1 or Statement 2, wherein the multivalent thiol component comprises two to ten thiol groups (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10).Statement 4. A crosslinked hydrogel according to any one of the preceding Statements, wherein the multivalent thiol component comprises one or more polyethylene glycol groups. Statement 5. A crosslinked hydrogel according to any one of the preceding Statements, wherein the multivalent thiol component comprises one or more of the following groups:wherein n is greater than or equal to 0.Statement 6. A crosslinked hydrogel according to any one of the preceding Statements, wherein the multivalent thiol component has the following structure or is:wherein each n is greater than zero and each n is the same or different.Statement 7. A crosslinked hydrogel according to any one of the preceding Statements, wherein the multivalent thiol component has a concentration of 0. 1 to 99.9% w / v, including all 0.1% w / v values therebetween (e.g., 0.1 to 5%, 0.1 to 10%, 0.1 to 20%, 0.1 to 25%, 0.1 to 30%, 0.1 to 40%, 0.1 to 50%, 0.1 to 60%, 0.1 to 70%. 0.1 to 80%, 0.1 to 90%, 0.1 to 95%, or 0.1 to 99%).Statement 8. A crosslinked hydrogel according to any one of the preceding Statements, wherein the multivalent disulfide component comprises two or more disulfide groups.Statement 9. A crosslinked hydrogel according to any one of the preceding Statements, wherein the multivalent disulfide component comprises two to ten disulfide groups (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10).Statement 10. A crosslinked hydrogel according to any one of the preceding Statements, wherein the multivalent disulfide component comprises one or more polyethylene glycol groups.Statement 11. A crosslinked hydrogel according to any one of the preceding Statements. wherein the multivalent disulfide component comprises one or more of the following groups:wherein n is greater than or equal to 0 and R is an aliphatic group, cyclic aliphatic group, aromatic group, aryl group, heteroaromatic group, or heteroaryl group.Statement 12. A crosslinked hydrogel according to Statement 11. wherein R isStatement 13. A crosslinked hydrogel according to Statement 11. wherein the multivalent disulfide component is:wherein each n is greater than or equal to 0 and each n is the same or different.Statement 14. A crosslinked hydrogel according to Statement 13. wherein the multivalent disulfide component has the following structure or is:wherein each n is greater than or equal to 0 and each n is the same or different.Statement 15. A crosslinked hydrogel according to any one of the preceding Statements, wherein the multivalent disulfide component has a concentration of 0.1 to 99.9% w / v, including all 0.1% w / v values therebetween (e.g., 0.1 to 5%, 0.1 to 10%, 0.1 to 20%, 0.1 to 25%. 0. 1 to 30%, 0. 1 to 40%, 0. 1 to 50%, 0.1 to 60%. 0. 1 to 70%, 0. 1 to 80%, 0. 1 to 90%, 0.1 to 95%, or 0.1 to 99%).Statement 16. A crosslinked hydrogel according to any one of the preceding Statements, wherein the aqueous medium is water. Statement 17. A crosslinked hydrogel according to any one of Statements 1-16, wherein the aqueous medium is buffered water.Statement 18. A crosslinked hydrogel according to any one of the preceding Statements, wherein the aqueous medium has a pH of 5 to 8.Statement 19. A crosslinked hydrogel according to Statement 17 or Statement 18, wherein the buffered water is buffered by phosphate buffered saline (PBS), HEPES, MOPS, TRIS, MES hydrate, citrate buffer, or the like.Statement 20. A crosslinked hydrogel according to any one of the preceding claims, wherein the hydrogel further comprises one or more cargo.Statement 21. A crosslinked hydrogel according to Statement 20, wherein the one or more cargo are drugs, small molecules, peptides, proteins, enzymes, antibodies, DNA, RNA, siRNA, nanoparticles (e.g., nanoparticles loaded with small molecules, such as, for example, progesterone / Fluticasone loaded PLGA nanoparticles), drug nanocrystals (e.g., progesterone / Fluticasone nanocrystals), liposomes (e.g., liposomes loaded with small molecules), viral vectors, bacteriophages, mammalian and bacterial cell therapies, or the like, or any combination thereof.Statement 22. A method for making a crosslinked hydrogel according to any one of the preceding Statements, wherein a first mixture comprising the multivalent thiol components in an aqueous medium is mixed with a second mixture comprising the multivalent disulfide components in an aqueous medium such that disulfide exchange occurs between the multivalent thiol components and the multivalent disulfide components thus forming a crosslinked hydrogel.Statement 23. A method according to Statement 22, wherein the first mixture and second mixture are mixed in a ratio of 0. 1 to 20 : 0. 1 to 20 (first mixture : second mixture, v:v).Statement 24. A method according to Statement 22 or Statement 23, wherein the multivalent thiol component of the first mixture has a concentration of 0. 1 to 99.9% w / v, including all 0.1% w / v values therebetween (e.g., 0.1 to 5%, 0.1 to 10%, 0.1 to 20%, 0.1 to 25%, 0.1 to 30%, 0.1 to 40%, 0.1 to 50%, 0.1 to 60%, 0.1 to 70%, 0.1 to 80%, 0.1 to 90%, 0.1 to 95%, or 0. 1 to 99%).Statement 25. A method according to any one of Statements 22-24, wherein the multivalent thiol component comprises one or more disulfide bonds.Statement 26. A method according to any one of Statements 22-24, wherein the multivalent disulfide component of the second mixture has a concentration of 0. 1 to 99.9% w / v, including all 0.1% w / v values therebetween (e.g., 0.1 to 5%, 0.1 to 10%, 0.1 to 20%, 0.1 to 25%, 0.1 to 30%, 0.1 to 40%, 0.1 to 50%, 0.1 to 60%, 0.1 to 70%, 0.1 to 80%, 0.1 to 90%, 0.1 to 95%, or 0.1 to 99%).Statement 27. A method according to any one of Statements 22-26, wherein the aqueous medium of the first mixture is the same or different as the aqueous medium of the second mixture.Statement 28. A method according to Statement 27, wherein the aqueous medium is buffered water.Statement 29. A method according to Statement 28, wherein the buffered water is buffered with phosphate buffered saline (PBS), HEPES, MOPS, TRIS, MES hydrate, or the like.Statement 30. A method according to any one of Statements 22-29, wherein the first mixture and / or the second mixture further comprises one or more cargo.Statement 31. A method according to Statement 30, wherein the one or more cargo are drugs, small molecules, peptides, proteins, enzy mes, antibodies, DNA, RNA, siRNA, nanoparticles (e.g., nanoparticles loaded with small molecules, such as, for example, progesterone / Fluticasone loaded PLGA nanoparticles), drug nanocrystals (e.g.. progesterone / Fluticasone nanocrystals), liposomes (e.g., liposomes loaded with small molecules), viral vectors, bacteriophages, mammalian and bacterial cell therapies, or the like, or any combination thereof.Statement 32. A method according to any one of Statements 22-31, further comprises heating a mixture comprising the first mixture and the second mixture to a temperature of 4 to 45 °C, including every value and range therebetween (e.g., 4, 5. 6, 7, 8. 9, 10, 11, 12. 13. 14. 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 °C).Statement 33. A method of delivering a therapeutic to an individual comprising contacting a surface of an individual with a crosslinked hydrogel according to any one of Statements 1- 21, wherein the crosslinked hydrogel comprises one or more cargo.Statement 34. A method according to claim 30, wherein the crosslinked hydrogel is formed on the surface of the individual.Statement 35. A method according to Statement 33 or Statement 34, wherein the surface is a mucosal tissue.Statement 36. A method according to Statement 35, wherein the mucosal tissue is a mucosal tissue of an eye, gastrointestinal tract, nose, lung, vagina, uterus, buccal, or urinary bladder of the individual.

[0089] The following example is presented to illustrate the present disclosure. It is not intended to be limiting in any way.EXAMPLE

[0090] This example provides a description of hydrogels of the present disclosure.

[0091] As presented in this instant disclosure, hydrogel biomaterials were designed that form rapidly through thiol-pyridyl disulfide exchange. This material may comprise polyethylene glycol (PEG), a widely used polymer, and forms nearly instantaneously upon mixing under physiological conditions. Because the prepared hydrogels comprise thiols and pyridyl sulfides, it was hypothesized that adhesion of a PEG hydrogel to mucosal tissues may be favored through direct reaction with thiol groups on mucin biopolymers. To determine if potentially useful for drug deliver}' applications, the release rate of different types of cargo encapsulated within the PEG gels were assessed and biocompalibility was evaluated.

[0092] Materials. 4-arm PEG-SH (PEG-4SH) lOkD and 20kD was purchased from Laysan Bio. 4-arm PEG-OPSS (PEG-4OPSS) lOkD and 20kD was purchased from Creative PEG works. Dithiothreitol (DTT) and L-Glutathione reduced was obtained from Sigma Aldrich. Tetramethylrhodamine labeled BSA (TRITC-BSA) and Cyanine 5 conjugated IgG (Cy5-IgG) were purchased from Protein Mods. Resazurin reagent and Calcein AM were purchased from Biotium. Propidium Idodide (Invitrogen) FluoSpheres and cell culture reagents such as DMEM and fetal bovine serum were obtained from Thermo Fisher Scientific.

[0093] Hydrogel Synthesis. 4-arm thiol terminated PEG (PEG-4SH) dissolved in phosphate buffered saline (PBS) (>0.5% w / v) is mixed with equal volumes of 4-arm orthopyridyl disulfide terminated polyethylene glycol (PEG-4OPSS) dissolved in PBS (>0.5% w / v) for crosslinking and rapid gelation. Tube inversion method was used to measure gelation time at body temperature of 37 °C. Briefly, polymer solutions of 100 pL each are mixed in Eppendorf tubes placed on heat block at 37 °C. Tube was taken out and inverted to see gelation of the polymers every 5 sec. Time was noted when the solution stopped running on the walls of the tubes. It was repeated for all formulations of different polymer weight percentages and performed in triplicates.

[0094] Equilibrium swelling. 100 pL of cylindrical gels were made by mixing the polymer solutions in equal volumes in tip cutoff ImL syringes. Gels were then pushed out of the syringes into 5 mL of PBS and incubated at 37 °C for 24 hours to reach equilibrium swelling. After 24 hours, gels were taken out of PBS and weighed to measure mass of swollen network. Gels were then freeze dried to measure mass of dried network. Equilibrium swelling ratio was calculated using below equation.Mass of swollen network Swelling ratio=— - - . — - - —Mass of dried network

[0095] Degradation. Degradation of PEG hydrogels were measured in PBS, PBS+ dithiothreitol (DTT) and PBS+ glutathione. To measure degradation in PBS, 100 pL cylindrical gels were prepared and placed in 5mL PBS at 37 °C as described previously. Gels were weighed before immersing in PBS and at different time points after immersion in PBS. Percentage mass remaining was calculated using below equation. To measure degradation in reducing agents, 200 pL of 100 mM DTT or 100 mM glutathione in PBS was added to 100 pL gels prepared in Eppendorf tubes and incubated at 37 °C. Tube was inverted every minute for DTT and every ~10 hours for glutathione to check gel degradation. Time was noted when the entire gel transitioned to liquid state.Mass of hydrogel at time t Percentage mass remaining = - — — - - - : -Mass of hydrogel at time t=0

[0096] Scanning Electron Microscopy. Gels were prepared as described in the equilibrium swelling section. Briefly, gels composed of 2% w / v PEG-4SH and 2% w / v PEG- 4OPSS using 20 kDa polymers were incubated in PBS at 37 °C for 24 hours to achieve equilibrium swelling. Following incubation, the gels were freeze-dried, coated with carbon, and imaged using a Hitachi SU-70 Schottky field emission gun scanning electron microscope.

[0097] Fourier Transform Infrared Spectroscopy. To confirm crosslinking between the polymers, Fourier-transform infrared (FTIR) spectroscopy was performed using a Bruker VERTEX 70 FT-IR spectrometer equipped with a Platinum- ATR accessory and a diamond crystal plate. Spectra were collected for individual 10 kDa and 20 kDa polymers of PEG-4SH and PEG-4OPSS, as well as for freeze-dried hydrogels composed of 2% w / v PEG- 4SH and 2% w / v PEG-4OPSS synthesized using 10 kDa and 20 kDa polymers.

[0098] Bulk rheology. To confirm successful formation of PEG hydrogels and to understand polymer weight percentages on mechanical properties, bulk rheological measurements were performed using ARES G2 rheometer (TA instruments). PEG gel precursor solutions were loaded on to 25 mm diameter parallel plate at a gap of 1000 pm at 37 °C. Solution was allowed to gel and equilibrate to 37 °C for 5 min. Humidity chamber was used prevent solvent evaporation and consequent hydrogel drying. To determine the linear viscoelastic region of the fully formed gel, a strain sw eep measurement w as performed at 0.1- 10% strain at a frequency of 1 rad / s. To determine the elastic modulus, G'(OJ). and viscous modulus, G"(CD), a frequency sweep measurement is conducted within the linear viscoelasticregion of the gel, at 10% strain amplitude and angular frequencies from 0. 1 to 100 radians / s. To elucidate interaction between PEG solution with mucins, flow sweep was performed from a shear rate of 0.01 to 100 s'1using 40 nm cone-plate geometry.

[0099] Self-healing. 100 pL square gels were formed in 3D printed square molds. Two gels were pushed out of the molds and were brought together for 10 min before holding vertically to observe self-healing. One of the gels was kept transparent while rhodamine B was added to the other gel to aid in visualization. Response of the hydrogel to the application and removal of shear were examined in shear induced failure and recovery experiments using oscillatory time sweeps at a frequency of 10 rad / sec. Alternating high and low strain cycles were applied every 2 minutes, with high strains of 200%, 500%, and 1000% for the failure phase and a low strain of 1% for the recovery phase. The rheometer parameters were consistent with those described previously.

[0100] Particle tracking microrheology. Particle tracking microrheology was performed as previously described. Briefly, solutions are prepared with 1 pL of -0.002% w / v suspension of 100 nm fluorescent muco-inert nanoparticles and added into a 25 pL solution of PEG-4SH and PEG-4OPSS prior to gelation in a custom microscopy chamber, sealed with a cover slip, and equilibrated for 30 min at room temperature before imaging. Ten-second movies at 30 ms temporal resolution were acquired with a high-speed CMOS camera equipped on an inverted confocal microscope with a 63x / 1.4 NA oil objective. Movies were analyzed using custom written tracking software in MATLAB to extract 2D x, y-coordinates of MIP centroids over time. From these trajectories, time-averaged mean squared displacement (MSD;2A / -2(T)) ) as a function of lag time, r, is calculated as \A / -2(r)> = ( [x(t+r)-x(t)]2+ { [y(t+r)-y(t)]2j where x(t) and y(t) are the spatial coordinates of particles as a function of time t. Using the generalized Stokes- Einstein relation, measured MSD values are used to compute viscoelastic properties of the hydrogels (e.g., G'(co), G"(co), microviscosity). Hydrogel network pore size, was estimated based on MSD using the equation, Ar2(r) )1 / 2+ a. All measurements described were performed in n=3 PEG gel preparations per formulation.

[0101] Drug release. 0.2% w / v TRITC labeled bovine serum albumin (TRITC-BSA) or Cy5 labeled IgG (Cy5-lgG) was added to 2% w / v 4-arm PEG-SH solution and mixed with equal volumes of 3% w / v of 4-arm PEG-OPSS in tip cut-off syringes to make 100 pL cylindrical hydrogels. Resulting gels comprised of 0.1% w / v TRITC-BSA / Cy5-IgG, 1% w / v 4-arm PEG-SH and 1.5% w / v 4-arm PEG-OPSS were immersed in 1 mL of PBS andincubated at 37°C. For nanoparticle loading, 2% v / v FluoSpheres were encapsulated. Supernatants were collected at each time point and replaced with fresh buffer. To examine protein release, fluorescence of the collected supernatants was measured by UV / Vis spectroscopy using Tecan spark multimode microplate reader. Fluorescence Ex / Em wavelengths of 545 / 575 was used for TRITC-BSA quantification and Ex / Em wavelengths of 650 / 670 was used for Cy5-IgG quantification.

[0102] Mucoadhesion. Mucoadhesive strength of the gels was measured using a pullapart adhesion test. Square sections of 1 mm fresh porcine intestine were cut and 100 pL of PEG gel precursor solutions were applied on luminal side of the tissue. It was allowed to set for 5 min and apical side of the intestinal tissue was superglued to the clamps of a dynamic mechanical analyzer (TA Instruments. DMA Q800). Samples were initially isothermally compressed at a force of IN for 5 minutes to ensure the superglue dries and pulled at a rate of 0.5 N / min until failure. The adhesion strength of each sample was recorded and replicated three times. To visualize mucoadhesion, a solution of 1% w / v PEG-4SH, 2% w / v PEG- 4OPSS, and 0.01% w / v rhodamine B (200 pL total) was added onto a small section of intestinal tissue obtained from Animal Biotech Industries and allowed to set for 2 min before holding vertically to visualize and take photographs.

[0103] Biocompatibility. Biocompatibility of PEG polymers and PEG gels was evaluated on HEK 293T cells. Briefly. HEK 293T cells were cultured in DMEM supplemented with 10%FBS and 1% penicillin-streptomycin. Cells were seeded onto 96 well plate at a density of 20,000 cells per well. After allowing cell adherence overnight, cells were treated with 20 pL solution of polymers dissolved in PBS pH 7.4 in the concentration range of 1% - 4% w / v or 20 pL of PEG-4SH and PEG-4OPSS gels and incubated at 37 °C, 5% CO2 for 24 hours. Following the treatment, cell culture supernatant containing polymers or gels was removed and replaced with resazurin containing media. Cells with the resazurin reagent were incubated at 37 °C, 5% CO2 for 3 hours. 100 pL of cell culture supernatant containing resazurin was transferred to a 96-well black plate and fluorescence was measured at Ex / Em wavelengths of 570 / 585 nm. Cell viability was measured relative to the cells grown in media without any treatment. For live / dead staining, l x 105cells were seeded in an 8-well chambered cell culture slide. After allowing cell adherence overnight, 50 pL of 2% PEG-4SH and 2% PEG-4OPSS gels were added to each well and incubated at 37 °C, 5% CO2 for 24 hours. Following the treatment, live cell staining was performed using IpM calcein AM and 500 nM propidium iodide and cells were imaged using Zeiss confocal microscope.

[0104] In vivo gel retention. All experimental procedures conducted in mice were performed in accordance with the standards established by the US Animal Welfare Acts, set forth in NIH guidelines as well as the Policy and Procedures Manual of University of Maryland Institutional Animal Care and Use Committee (IACUC). These procedures are approved under IACUC protocol # R-MAY-22-25 at the University' of Maryland. Female BALB / c mice (6-8 weeks old, Charles River Laboratories) were used for the study. To prepare the hydrogel formulation, a 2% w / v PEG-4SH solution was mixed with near- infrared-labeled 100 nm polystyrene nanoparticles (excitation: 715 nm, emission: 755 nm). The PEG-4SH / nanoparticle solution and a 2% w / v PEG-4OPSS solution were loaded into a Twin-Syringe Delivery System (M-System, MedMix) for simultaneous mixing and delivery. Final concentration of nanoparticles in the gel was 1.5pg / pL which is approximately 2xlO10parti cl es / 10 pL. Mice were fully anesthetized using isoflurane and positioned on their stomachs during administration. To minimize the risk of nasal blockage, 10 pL of the combined liquid solution w as carefully administered dropwise into a single nostril of each mouse (n=3). To evaluate whether the hydrogel enhances cargo retention, a control group of mice (n=3) received an intranasal dose of nanoparticles suspended in PBS under identical conditions. Fluorescence signals from the hydrogel-encapsulated nanoparticles were measured at 0, 2, 5, and 48 hours, as w ell as at 7- and 15-days post-administration using IVIS Spectrum Imaging System. Background signal was recorded from untreated control mice (n=l). Throughout the study period, body weight was monitored to assess the overall health of the animals. At the end of the study, mice were euthanized, and nasal and lung tissues were harvested for ex vivo fluorescence imaging to evaluate nanoparticle retention and distribution.

[0105] Results and Discussion

[0106] Formulation of rapid in-situ forming PEG hydrogels. In this study, we developed a new method for rapid formation of polyethylene glycol (PEG) based hydrogels at physiological conditions of 37°C and pH of 7.4. These gels are also formed via bio- reducible disulfide linkers by combining thiol terminated 4-arm PEG (PEG-SH) and OPSS terminated 4-arm PEG (PEG-OPSS). An overview of crosslinking mechanism for 4-arm PEG-SH and 4-arm PEG-OPSS is depicted in FIG. 1. After being dissolved in phosphate buffered solution (PBS), we observed gel formation w ithin 30 seconds through rapid crosslinking via di-sulfide bond formation. Table 1 summarizes the time to gel as well as degradation time under reducing conditions for 6 lead formulations. Although a wide range ofweight percentages were tested and found to rapidly form gels as noted in Table 2, lead formulations were selected which retained stability in PBS for > 24 hours.

[0107] Table 1. Gelation and degradation times of rapid forming PEG gels.

[0108] Table 2. Gelation and degradation times of rapid forming PEG gels.

[0109] The disulfide reducing agent glutathione (GSH) is present throughout tissues in the body at a pM to mM concentration range. Thus, the degradation rate of PEG gels once administered in vivo is likely to be affected by the presence of GSH. Further, external disulfide reducing agents such as dithiothreitol (DTT) can be used for removal of the gel from the site of application as needed. The effect of these compounds was tested on PEG gels in vitro using 100 mM DTT and GSH individually. As indicated in Table 1, DTT degrades gels under 10 minutes whereas glutathione degrades in 1 to 3 days.

[0110] Characterization of rapid forming PEG gels. Effect of polymer weight percentage and polymer molecular weight on mechanical strength of hydrogels are studied in Fig. 2A. Formulations Fl to F5 have a storage modulus ranging from -400-800 Pa. Formulation 6 containing 20 kDa sized 2% w / v 4-arm PEG-SH and 2% w / v 4-arm PEG¬OPSS possessed a significantly higher storage modulus of 1712 ± 267 Pa, indicative of an increased elasticity in comparison to other formulations tested.' S! -

[0111] The pore size of each formulation was evaluated using particle tracking microrheology (FIG. 2B). It was hypothesized that increasing PEG-SH / PEG-OPSS concentrations or molecular weights would lead to decreases in pore size which may change release kinetics of encapsulated cargoes. Interestingly, only a weak dependence on total PEG concentration was found with a relatively small range of pore sizes observed across formulation conditions. Changes in pore size as a function of PEG MW were observed, but a consistent trend was not observed. Total available functional groups for crosslinking, flexibility of polymer chain to facilitate disulfide bond formation and steric hindrance due to bulkiness of OPSS reactive groups compared to SH could be reasons for inconsistent trends.

[0112] Swelling and degradation dictate the rate of release of therapeutics and biodegradability, which are vital for use in biomedical applications. FIG. 2C shows the equilibrium swelling ratio for each formulation. F6 with highest storage modulus also has the highest swelling ratio and is 2-fold higher than rest of the formulations. For degradation studies (FIG. 2D), PEG gels are immersed in PBS volume that is 50 times the volume of gel. An initial increase in gel mass was observed due to swelling followed by slow degradation. Gels made with lOkD polymer, Fl, F3, and F5 showed degradation up to 30% of initial swollen mass in 30 days whereas gels made of 20kD polymers, F2, F4, and F6 degraded up to 15% of initial swollen mass in the same time frame. Longer retention and slow degradation of PEG gels can be due to stability of ether linkages of PEG polymer and susceptibility to degradation due to auto-oxidation. FIG. 2E further illustrates the linkers within the PEG gels are reducible as immersion of the rhodamine-loaded PEG gel in PBS alone shows rhodamine release but no degradation of the gel. How ever, addition of DTT shows complete degradation of the gel within 10 minutes. Scanning electron microscope images (FIG. 2F and FIG. 2G) of freeze-dried hydrogels of formulation F6 made with 20kD polymers reveals porous structure of the hydrogel network. FTIR spectra, as shown in FIG. 13, confirms successful crosslinking between PEG-4SH and PEG-4OPSS. The spectra also demonstrate consistent peaks across hydrogels, regardless of the polymer molecular weight, indicating that crosslinking is independent of polymer size.

[0113] Self-healing of rapid in situ forming PEG hydrogels. To assess the functional resilience of the rapidly forming PEG hydrogels, their self-healing properties were investigated. When two gels of formulation F2 (1% w / v 4-arm PEG-SH, 20 kDa, crosslinked with 1.5% w / v 4-arm PEG-OPSS, 20 kDa) are brought into contact, they autonomously heal at the interface through disulfide bond reformation and hydrogen bonding as demonstrated in FIG. 3A. The self-healing behavior was further assessed via cyclic time sweep test withalternating low / high strains using formulation F6. Strain levels of 200%, 500%, and 1000% which are beyond the linear viscoelastic region shown in FIG. 3B were applied for 2 minutes to induce material failure, followed by a 2-minute recovery phase under a low strain of 1%. The hydrogels exhibited self-healing across all tested strains, with varying efficiencies. At 200% strain, the hydrogels achieved near-complete recovery7, regaining a substantial portion of their original mechanical strength (FIG. 3C). Furthermore, the responses were rapid and repeatable. Although self-healing persisted at 500% strain, recovery7efficiency declined, resulting in only partial restoration of mechanical properties (Fig. 3D). Under extreme deformation at 1000% strain, the hydrogels retained limited self-repair capacity, yvith markedly reduced mechanical recovery7(FIG. 3E). These findings underscore the ability of PEG hy drogels to autonomously repair damage, minimizing the risk of detachment at the application site and preventing therapeutic cargo leakage. This self-healing capability further highlights their potential for use in dynamic physiological environments, such as localized delivery7to the cardiac tissue, yvhere repeated deformation and recovery are critical for sustained functionality.

[0114] Mucoadhesive properties of rapid in situ forming PEG hydrogels. The ability of these PEG gels to adhere to mucosal tissues yvas evaluated next. Prior work has shown PEG based biomaterials can adhere to mucus-coated tissues through a combination of PEG- mucin entanglement and hydrogen bonding (FIG. 4A). It was hypothesized that these gels may also be able to form disulfide bonds with cysteine-rich domains of mucins via PEG- OPSS and / or PEG-SH leading to increases in mucoadhesion. To test this, PEG-4OPSS and PEG-4SH solutions were applied to the surface of pig intestine yvhere instantaneously formed a uniform layer of PEG gel (FIG. 4B). Pull-apart tests were then performed to measure their mucoadhesive strength immediately and 24 hours after application (FIG. 4C and FIG. 4D). Also included for comparison were tissues treated with 2% w / v 4-arm PEG-DBCO and 2 % w / v 4-arm PEG-azide as a control for PEG gels without OPSS / SH groups to form disulfide bonds and 4% w / v chitosan solution which is known to possess mucoadhesive properties. All formulations tested were found to adhere to pig intestine yvith mucoadhesive strengths >600 Pa. Although statistically non-significant, the 4% w / v chitosan possessed greater mucoadhesive strength than all PEG gel formulations likely due to the net-positively charged chitosan adhering to net-negatively charged mucin chains. However, PEG-DBCO and PEG- azide gels showed comparable mucoadhesive strength to PEG-OPSS and PEG-SH gels indicating the gels are not likely able to access the cysteines on the mucosal tissue. Therefore, adhesion is likely mediated by entanglement during sol-gel transition and hydrogen bonding.This is further confirmed through flow sweep measurements, which showed no change in viscosity when mucin solutions were mixed PEG-4SH and PEG-4OPSS indicating no covalent bond formation (FIG. 8C).

[0115] Release of cargo from rapid forming PEG gels. We characterized the release kinetics of different model cargos, including bovine serum albumin (BSA), immunoglobulin (IgG), and 40 nm nanoparticles (NP), from PEG gels over 24 hours. We found BSA and IgG were released in roughly 4 hours and 10 hours respectively whereas 40 nm NP were retained within the gel for at least 24 hours (FIG. 5). This is likely explained with the difference in cargo size with BSA and IgG being ~4-5 times smaller than 40 nm NP. These data indicate release of protein therapeutics would likely be driven by diffusion out of the gel whereas delivery of encapsulated nanoparticles is more likely to be driven by gel degradation. Further, effect of polymer weight percentage and swelling ratio has no effect on BSA release, minimal effect on IgG release and highest effect on 20 nm nanoparticles exhibiting increased release in formulation 6 due to higher swelling ratio (FIG. 11).

[0116] Biocompatibility of rapid forming PEG gels. To ensure the gel precursor components and the PEG gel itself are well-tolerated by mammalian cells, biocompatibility studies were conducted using HEK-293 cells (FIG. 6). First, we treated HEK-293 cells were treated with either 4-arm PEG-SH or PEG-OPSS at concentrations up to 4% w / v (FIG. 6A). It was found that 10 kDa 4-arm PEG-SH was well-tolerated at all concentrations tested. Although statistically significant differences were observed with other treatments, more than 80% cell viability was observed except for 10 kDa 4-arm PEG OPSS at 4% w / v concentration. HEK-293 cells w ere next treated with PEG gels and no toxicity w as observed for all lead formulations tested (FIG. 6B). These data suggest that PEG gels are generally safe for use as an injectable biomaterial. However, acute exposure to 10 kDa 4-arm PEG formulations may pose some toxicity concerns and thus, 20 kDa PEG formulation may be better suited for use in future drug delivery applications.

[0117] In vivo nasal retention of rapid in situ forming PEG gels. To evaluate the in vivo retention of the rapid-forming PEG hydrogel, its abi 1 i ty to prolong the residence time of a model cargo (nanoparticles) in the nasal cavity of mice was assessed. Materials deposited in the nasal mucosa are subject to rapid clearance every 15 to 20 minutes through mucociliary clearance, posing a significant challenge for effective nasal drug delivery. To test whether the hydrogel could enhance retention, near-infrared (NIR)-emitting 100 nm polystyrene nanoparticles (NPs) were encapsulated within the gel and administered them intranasally. Because polystyrene NPs are non-biodegradable, the presence of fluorescence signal directlycorrelates with gel retention. As shown in FIG. 7A and FIG. 7B, NPs administered in PBS (without hydrogel) were cleared from the nasal cavity within 5 hours and completely undetectable by 48 hours. In contrast, NPs delivered using the hydrogel vehicle exhibited near-complete retention up to 48 hours. By day 15, fluorescence signals returned to baseline levels, consistent with the complete degradation of the hydrogel and clearance of NPs. Additionally, body eight monitoring throughout the study (FIG. 7C) showed a steady increase, indicating no adverse effects from the hydrogel treatment. These results demonstrate that the rapid in situ forming PEG hydrogel significantly enhances cargo retention in the nasal cavity and provides the ideal formulation strategy for sustained drug release at mucosal sites.

[0118] Described herein is a rapid in situ forming PEG hydrogel capable of adhering to mucosal tissues. Unlike other chemistries used in previous work, bio-reducible disulfide linked PEG gels can be formed that are able to be degraded over time (days to weeks) upon administration. Both protein and nanoparticle-based therapeutics may be encapsulated into the gel for extended release at mucosal sites.

[0119] Although the present disclosure has been described with respect to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

CLAIMS:

1. A crosslinked hydrogel formed from a mixture of a multivalent thiol components and a multivalent disulfide components in an aqueous medium, wherein the multivalent thiol components and the multivalent disulfide components create a crosslinked network via disulfide exchange between a thiol of the multivalent thiol components and a disulfide of the multivalent disulfide components.

2. The crosslinked hydrogel according to claim 1, wherein the multivalent thiol component comprises two or more thiol groups.

3. The crosslinked hydrogel according to claim 1, wherein the multivalent thiol component comprises two to ten thiol groups.

4. The crosslinked hydrogel according to claim 1, wherein the multivalent thiol component comprises one or more polyethylene glycol groups.

5. The crosslinked hydrogel according to claim 1, wherein the multivalent thiol component comprises one or more of the following groups:wherein n is greater than or equal to 0.

6. The crosslinked hydrogel according to claim 1, wherein the multivalent thiol component has the following structure:wherein each n is greater than zero and each n is the same or different.

7. The crosslinked hydrogel according to claim I . wherein the multivalent thiol component has a concentration of 0. 1 to 99.9% w / v.

8. The crosslinked hydrogel according to claim 1, wherein the multivalent disulfide component comprises two or more disulfide groups.

9. The crosslinked hydrogel according to claim 1, wherein the multivalent disulfide component comprises two to ten disulfide groups.

10. A crosslinked hydrogel according to claim 1, wherein the multivalent disulfide component comprises one or polyethylene glycol groups.

11. The crosslinked hydrogel according to claim 1, wherein the multivalent disulfide component comprises one or more of the following groups:wherein n is greater than or equal to 0 and R is an aliphatic group, cyclic aliphatic group, aromatic groups, aryl group, heteroaromatic groups, or heteroaryl group.

12. The crosslinked hydrogel according to claim 11, wherein R is13. The crosslinked hydrogel according to claim 11, wherein the multivalent disulfide component is:wherein each n is greater than or equal to 0 and each n is the same or different.

14. The crosslinked hydrogel according to claim 13, wherein the multivalent disulfide component is:wherein each n is greater than or equal to 0 and each n is the same or different.

15. The crosslinked hydrogel according to claim 1, wherein the multivalent disulfide has a concentration of 0. 1 to 99.9% w / v.

16. The crosslinked hydrogel according to claim 1, wherein the aqueous medium is water.

17. The crosslinked hydrogel according to claim 1, wherein the aqueous medium is buffered water.

18. The crosslinked hydrogel according to claim 17, wherein the aqueous medium has a pH of 5 to 8.

19. The crosslinked hydrogel according to claim 17, wherein the buffered water is buffered by phosphate buffered saline, HEPES, MOPS, TRIS, citrate buffer, or MES hydrate.

20. The crosslinked hydrogel according to claim 1, wherein the hydrogel further comprises one or more cargo.

21. The crosslinked hydrogel according to claim 20, wherein the one or more cargo are drugs, small molecules, peptides, proteins, enzymes, antibodies, DNA, RNA, siRNA, nanoparticles, drug nanocrystals, liposomes, viral vectors, bacteriophages, mammalian and bacterial cell therapies, or any combination thereof.

22. A method for making a crosslinked hydrogel according to claim 1. wherein a first mixture comprising the multivalent thiol components in an aqueous medium is mixed with a second mixture comprising the multivalent disulfide components in an aqueous medium such that a disulfide exchanges occurs between the multivalent thiol components and the multivalent disulfide components such that the crosslinked hydrogel is formed.

23. The method according to claim 22, wherein the first mixture and second mixture are mixed in a ratio of 0.1 to 20 : 0.1 to 20 (first mixture : second mixture, v:v).

24. The method according to claim 22, wherein the multivalent thiol component of the first mixture has a concentration of 0. 1 to 99.9% w / v.

25. The method according to claim 22, wherein the multivalent thiol component comprises two or more disulfide bonds.

26. The method according to claim 22, wherein the multivalent disulfide component of the second mixture has a concentration of 0.1 to 99.9% w / v.

27. The method according to claim 22, wherein the aqueous medium of the first mixture is the same or different as the aqueous medium of the second mixture.

28. The method according to claim 27, wherein the aqueous medium is buffered water.

29. The method according to claim 28, wherein the buffered water is buffered with phosphate buffered saline, HEPES, MOPS, TRIS, citrate buffer, or MES hydrate.

30. The method according to claim 22, wherein the first mixture and / or the second mixture further comprises one or more cargo.

31. The method according to claim 30, wherein the one or more cargo are drugs, small molecules, peptides, proteins, enzymes, antibodies, DNA, RNA, siRNA, nanoparticles, drug nanocrystals, liposomes, viral vectors, bacteriophages, mammalian and bacterial cell therapies, or any combination thereof.

32. The method according to claim 22, further comprises heating a mixture comprising the first mixture and the second mixture to a temperature of 4 to 45 °C.

33. A method of delivering a therapeutic to an individual comprising contacting a surface of an individual with a crosslinked hydrogel according to claim 1, wherein the crosslinked hydrogel comprises one or more cargo.

34. The method according to claim 33, wherein the crosslinked hydrogel is formed on the surface of the individual.

35. The method according to claim 33, wherein the surface is a mucosal tissue.

36. The method according to claim 35, wherein the mucosal tissue is a mucosal tissue of an eye, gastrointestinal tract, nose, lung, vagina, uterus, buccal, or urinary bladder of the individual.

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