Adhesive hydrogels crosslinked by catechol-thiol bonds

Abstract

The present disclosure generally relates to hydrogels, e.g., hydrogels formed by crosslinking. Such hydrogels can provide robust gelation and adhesion, while retaining bioactivity. The hydrogel of the disclosure can be implemented in many applications, for non-limiting example, antifouling and lubricious coatings, tissue adhesives, and wound dressings. The hydrogel offers desirable biocompatibility, bioactivity, biodegradability, and mechanical properties.

Description

Adhesive Hydrogels Crosslinked by Catechol-Thiol Bonds RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 639,581, filed on April 26, 2024, and U.S. Provisional Application No.63 / 563,231, filed on March 8, 2024. The entire teachings of the above applications are incorporated herein by reference. GOVERNMENT SUPPORT

[0002] This invention was made with government support under ES007020 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0003] Mucus supports human health by hydrating, lubricating, and preventing infection of wet epithelial surfaces. The beneficial material properties and bioactivity of mucus stem from glycoproteins called mucins. However, the use of mucin-derived hydrogels for wound dressings and antifouling coatings requires robust gelation and adhesion to a wide range of substrates. SUMMARY

[0004] In the present disclosure, catechol-thiol bonding has been used to drive gelation of native mucin proteins, forming soft, adhesive hydrogels that can be coated on a diverse range of surfaces. The gelation dynamics and adhesive properties can be systematically tuned by varying the hydrogel composition, polymer architecture, and thiol availability, with gelation timescales adjustable from seconds to hours, and values of elastic modulus, failure stress, and debonding work spanning orders of magnitude. The present disclosure demonstrates the functionality of these gels in applications such as tissue adhesives, using porcine skin as a proxy for human skin, and as bioactive surface coatings to prevent bacterial colonization. The results highlight catechol-thiol crosslinking as a versatile platform for engineering multifunctional glycoprotein hydrogels with applications in wound repair and antimicrobial surface engineering.

[0005] The present disclosure generally relates to hydrogels formed by crosslinking. Such hydrogels can, in some embodiments, provide robust gelation and adhesion, while retaining - 1 - 4092104.v2bioactivity. The hydrogels of the disclosure can be implemented in many applications, for non- limiting example, antifouling and lubricious coatings, tissue adhesives, and wound dressings.

[0006] An embodiment disclosed herein is a hydrogel crosslinked by a thiol-containing agent and a catechol-containing agent, wherein the thiol-containing agent is a thiol-containing branched polymer or mucin.

[0007] Another embodiment disclosed herein is a method of modulating immune response, comprising administering a hydrogel of the present disclosure to a subject in need thereof, wherein the hydrogel further comprises an immunostimulant or an immunosuppressant.

[0008] Another embodiment disclosed herein is a bioadhesive, comprising a hydrogel of the present disclosure. In some aspects, the bioadhesive is a tissue adhesive.

[0009] Another embodiment disclosed herein is a shaving device, comprising a hydrogel of the present disclosure.

[0010] Another embodiment disclosed herein is a cartridge comprising a razor blade, said razor blade comprising a hydrogel of the present disclosure.

[0011] Another embodiment disclosed herein is an antifouling coating, comprising a hydrogel of the present disclosure.

[0012] Another embodiment disclosed herein is a method of reducing or suppressing biofilm formation on an article, the method comprising applying a hydrogel of the present disclosure to the article.

[0013] Another embodiment disclosed herein is a method of providing an article with a surface exhibiting a lubricious property, the method comprising applying a hydrogel of the present disclosure to the article.

[0014] Another embodiment disclosed herein is a medical device having a lubricious coating on at least a section of the device, the coating comprising a hydrogel of the present disclosure.

[0015] Another embodiment disclosed herein is a method of treating a wound in a subject in need thereof, said method comprising contacting the wound with a hydrogel of the present disclosure, under conditions to allow the hydrogel to adhere to tissue surrounding the wound.

[0016] Another embodiment disclosed herein is a method of making a hydrogel, comprising: combining a solution comprising mucin with a reducing agent to form reduced mucin; removing the reducing agent from the solution; and adding a catechol-containing agent and an oxidase - 2 - 4092104.v2(e.g., tyrosinase) to the solution, thereby forming the hydrogel, wherein the hydrogel is crosslinked by the reduced mucin and a catechol-containing agent.

[0017] Yet another embodiment disclosed herein is a kit comprising a hydrogel of the present disclosure.

[0018] Yet another embodiment disclosed herein is a polymer of formula (I): , wherein: R is a polyol coreinteger from about 3 to about 100.

[0019] The hydrogels of the present disclosure provide desirable biocompatibility, bioactivity, biodegradability, and mechanical properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] FIGs.1A-B: Schematic overview of polymers. FIG.1A: Thiol-functionalized polymers. FIG.1B: catechol-functionalized crosslinker. The average functionalization of thiol or catechol groups per molecule is stated in the image labels, in one embodiment.

[0022] FIG.1C: 1H NMR (500 MHz, CDCl3, δ (ppm)) of 8-arm PEG-catechol.

[0023] FIG.1D:1H NMR (500 MHz, CDCl3, δ (ppm)) of 4-arm PEG.

[0024] FIG.2:1H NMR (500 MHz, CDCl3, δ (ppm)) of 4-arm PEG mesylate.

[0025] FIG.3:1H NMR (700 MHz, CDCl3, δ (ppm)) of 4-arm PEG-thiol.

[0026] FIG.4A: Gelation of PEG-4SH, catecholic crosslinkers, and oxidizers, where the number indicates the approximate gelation time in days. Gray boxes (with dashes) indicate no gelation, and empty white boxes indicate that no measurement was made. Enzymatic oxidation involved 0.16 mg / ml tyrosinase for PEG-8C and PEG-1C. Chemical oxidation involved ratios of 1:1 periodate:catechol for PEG-8C / PEG-4SH, and 1:2 periodate:thiol for PEG-4SH alone. All - 3 - 4092104.v2experiments were conducted in a 50 mM phosphate buffer, pH 7, except compositions indicated as pH 9, which were conducted in a 50 mM carbonate buffer, pH 9.

[0027] FIG.4B: N-acetyl cysteine (NAC) prevents the gelation of 4% PEG-8C oxidized by 0.16 mg / ml tyrosinase.

[0028] FIGs.4C-G: Images of tube gelation taken within hours of mixing the solutions. Bolded text indicates a gel; non-bolded text indicates a liquid, with gelation determined by inspection. FIG.4C: 1% PEG-8C and tyrosinase with varying concentration of PEG-4SH. Every tube gelled within 2 days. FIG.4D: 1% PEG-8C and periodate with varying concentrations of PEG-4SH. FIG.4E: Varying concentrations of PEG-4SH with periodate. FIG.4F: 1% PEG-1C with varying concentrations of PEG-4SH. All tubes remained a liquid. FIG.4G: 4% PEG-8C and varying concentrations of N-acetyl cysteine (NAC) in the presence of tyrosinase. All but the 1:1 tube gelled within 1 day, noting that the image in the figure was taken before gelation in the 0.75:1 tube was observed.

[0029] FIGs.5A-E: UV-vis absorbance spectroscopy of catechol- and thiol-functionalized polymers. FIG.5A: Absorbance of individual components. FIG.5B: Time-dependent absorbance of 1% PEG-8C and 0.16 mg / ml tyrosinase, with the absorbance of 1% PEG-8C shown as a reference. FIG.5C: Time-dependent absorbance of 1% PEG-8C, 2% PEG-4SH, and 0.16 mg / ml tyrosinase, with the absorbance of 1% PEG-8C shown as a reference. FIG.5D: Time-dependent absorbance of 2% PEG-8C and 0.16 mg / ml tyrosinase, with the absorbance of 2% PEG-8C shown as a reference. FIG.5E: Time-dependent absorbance of 2% PEG-8C, 2% PEG-4SH, and 0.16 mg / ml tyrosinase, with the absorbance of 2% PEG-8C shown as a reference.

[0030] FIG.6A: Raman spectroscopy of catechol- and thiol-functionalized polymers using a 785 nm laser. FIG.6B: Raman spectroscopy of catechol- and thiol-functionalized polymers using a 532 nm laser. The following solutions were tested: 1% PEG-4SH, either alone (4SH), with 16 mg / ml tyrosinase (4SH-tyr), or with 0.05% hydrogen peroxide (4SH-H2O2); 1% PEG-8C, either alone (8C), with 0.16 mg / ml tyrosinase (8C-tyr), or with 1% PEG-4SH and 0.16 mg / ml tyrosinase (8C-4SH-tyr). Dashed lines in plots on left identify peaks measured previously for PEG.94,95Plots on right show enlarged views of the shaded regions, with dashed lines indicating peaks at 670 cm-1and 2570 cm-1previously attributed to -SH deformation and stretching, respectively, in PEG-thiol.90- 4 - 4092104.v2

[0031] FIG.7A: Gelation dynamics of catechol- and thiol-functionalized polymers. Evolution of the storage modulus Gʹ vs time t at fixed frequency ω = 5 rad / s and strain amplitude γ0= 0.1 for 1% PEG-8C, 0–1% PEG-4SH, 0.16 mg / ml tyrosinase. Dashed lines show the minimum measurable moduli in the rheometer for different parallel plate test fixtures (diameter d = 8 or 25 mm). The gelation timescale tgel where Gʹ = 1 Pa, fastest mutation timescale λ*, storage modulus Gλ(λ = 3 h), and final storage modulus Gfare indicated.

[0032] FIG.7B: Average values of tgel, λ*, and Gλ=3has a function of wt% PEG-4SH (N ≥ 3); error bars show the standard deviation.

[0033] FIG.7C: Sketches of the proposed crosslinking of PEG-4SH by PEG-8C in the presence of tyrosinase.8-spoked asterisks represent PEG-8C; triangles represent thiol groups in PEG-4SH.

[0034] FIG.7D: Example plot of linear viscoelastic storage and loss moduli (Gʹ and Gʺ) for an example hydrogel sample composed of 1% PEG-8C, 1% PEG-4SH, and 0.16 mg / ml tyrosinase.

[0035] FIG.8A: Adhesion of hydrogels formed from catechol- and thiol-functionalized polymers. Extensional engineering stress σEvs engineering strain ε measured immediately after the gelation measurements shown in FIG.7A.1% PEG-8C, 0–1% PEG-4SH, 0.16 mg / ml tyrosinase. The area under the curves gives the debonding work normalized by the initial gel volume W / V.

[0036] FIG.8B: Average values of failure stress σfand debonding work W / V vs concentration of PEG-4SH for PEG-8C (black circles).

[0037] FIGs.9A-D: Analysis of gelation and adhesion data from FIGs.7A-B, 8A-B, 10A-C, and 11A-C. FIG.9A: Longitudinal modulus M vs Gf. Solid line shows a theoretical prediction described in Example 4. FIG.9B: Failure stress stress σf vs final modulus Gf. Dashed line shows a fit to the results of PEG-4SH gels. FIG.9C: Failure stress ratio calculated by dividing the measured failure stress of each gel by the fitted failure stress of gels composed of PEG-4SH (dashed line). FIG.9D: Debonding work / initial gel volume W / V vs Gf.

[0038] FIGs.10A-C: Measurements of PEG-4SH hydrogels oxidized by H2O2 (0.4:1 H2O2:thiol). FIG.10A: Storage modulus Gʹ vs time t measured during gelation. FIG.10B: Tensile engineering stress σ vs engineering strain ε measured after gelation. FIG.10C: Average - 5 - 4092104.v284002 values of final modulus Gf, failure stress σf, and debonding work W / V vs percentage of PEG- 4SH.

[0039] FIG.11A: Gelation and adhesion of 1.4% or 2.8% (2x) mucin proteins crosslinked by 1% PEG-8C and 0.16 mg / ml tyrosinase. Mucins were either as-purified (MUC2), reduced (MUC2r), or alkylated (MUC2a). Storage modulus Gʹ vs time t. Dashed line shows the sensitivity limit of the parallel plate geometry (d = 8 mm).

[0040] FIG.11B: Engineering stress σEvs engineering strain ε of 1.4% or 2.8% (2x) mucin proteins crosslinked by 1% PEG-8C and 0.16 mg / ml tyrosinase, measured immediately after gelation.

[0041] FIG.11C: Average values of final modulus Gf, failure stress σf, and debonding work W / V of 1.4% or 2.8% (2x) mucin proteins crosslinked by 1% PEG-8C and 0.16 mg / ml tyrosinase.

[0042] FIG.11D: Sketches of the proposed crosslinking of different preparations of mucin proteins by PEG-8C in the presence of tyrosinase.8-spoked asterisks represent PEG-8C. Merged grey triangles represent disulfide bonds, grey triangles (in the middle image) represent thiol groups, and black triangles (in the right image) represent alkylated thiol groups.

[0043] FIG.12A: Gelation and adhesion measurements of 1.4% MUC2, 0.16 mg / ml tyrosinase (MUC2); 1.4% reduced MUC2, 0.16 mg / ml tyrosinase (MUC2r); 1.4% reduced BSM (Sigma), 1% PEG-8C, 0.16 mg / ml tyrosinase (BSMr / PEG-8C); 1% PEG-8C, 0.16 mg / ml tyrosinase (PEG-8C). Data for PEG-8C also appears in FIGs.7-8. Storage modulus Gʹ vs time t. Dashed lines show the sensitivity limits of the parallel plate geometries (d = 8 or 25 mm).

[0044] FIG.12B: Engineering stress σEvs engineering strain ε of 1.4% MUC2, 0.16 mg / ml tyrosinase (MUC2); 1.4% reduced MUC2, 0.16 mg / ml tyrosinase (MUC2r); 1.4% reduced BSM (Sigma), 1% PEG-8C, 0.16 mg / ml tyrosinase (BSMr / PEG-8C); 1% PEG-8C, 0.16 mg / ml tyrosinase (PEG-8C), measured immediately after gelation.

[0045] FIG.13A: Anti-biofouling of mucin-based hydrogels: box plot representation of biofilm volume on glass surfaces, either bare or hydrogel-coated, after incubation in Pseudomonas aeruginosa. Hydrogel films were composed of 1.4% reduced MUC5AC, 1% PEG- 8C, 0.16 mg / ml tyrosinase (MUC5ACr / PEG-8C). Open squares show the mean of each dataset. - 6 - 4092104.v284002

[0046] FIG.13B: Box plot representation of biofilm volume on bare or coated glass surfaces after incubation in Pseudomonas aeruginosa, for coatings composed of in-lab purified mucin (MUC2 and MUC5AC) and commercial bovine submaxillary mucin (BSM), each crosslinked by PEG-8C and tyrosinase. Open squares show the mean of each dataset. Representative fluorescence images are shown on right. Scale bars are 50 μm.

[0047] FIGs.14A-D: Control biofouling experiments. FIG.14A: Bacteria retain viability after exposure to hydrogel surfaces. FIG.14B: Images of bacteria on hydrogel surfaces. FIG. 14C: Glass surfaces incubated in solutions of mucin proteins and then washed following the same protocol used for hydrogel films show no reduction in biofilm volume. FIG.14D: Images show representative biofilms. Scale bars are 50 μm.

[0048] FIG.15A-B: Adhesion of mucin-based hydrogels between pigskin surfaces. FIG. 15A: Evolution of the storage modulus Gʹ during gelation of 1.4% reduced MUC2, 0.16 mg / ml tyrosinase, and either 1% or 2% PEG-8C. FIG.15B: Adhesion of the mucin-based hydrogels measured after gelation.

[0049] FIG.16: Profilometer scan of a mucin-based hydrogel film (1.4% MUC2, 1% PEG- 8C, 0.16 mg / ml tyrosinase) deposited via spin coating on stainless steel and table with height and average roughness measured in nanometers.

[0050] FIG.17A: Stribeck plot showing test friction measurements of different preparations of mucin proteins or control materials deposited on surfaces by adsorption from solution. HEPES is a buffer condition, sigma PGM is a commercially-available degraded porcine gastric mucin (PGM), PGM identifies an in-lab purified porcine gastric mucin, PEG is a polyethylene glycol control, and Apo PGM is a deglycosylated PGM.

[0051] FIG.17B: Schematic of a friction measurement performed with a three ball-on-flat test geometry.

[0052] FIG.18: Preliminary friction measurements of a hydrogel-coated surface in contact with a steel countersurface in aqueous conditions. Contact between a bare silica surface and steel in the same conditions is shown for comparison.

[0053] FIG.19: Mucin polymer coatings adhered to bare stainless steel. Adhesion measurement performed in a parallel plate rheometer. Inset summarizes the maximum adhesion stress σad. - 7 - 4092104.v2

[0054] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments. DETAILED DESCRIPTION

[0055] A description of example embodiments follows.

[0056] Protein-based materials such as silk, wool, glue, and leather have been used throughout human history.1–3Now, the demand for sustainable and multifunctional materials has led to a renewed interest in protein-based materials,3–5particularly for biomedical applications where proteins such as collagen, elastin, and fibrin offer desirable biocompatibility, biodegradability, and mechanical properties.6However, these proteins lack the bioactivity found in many biological niches in the body.

[0057] Wet epithelial surfaces are coated in a layer of mucus with bioactive properties that critically support human health, as the mucus layer hydrates, lubricates, and presents a physicochemical barrier protecting the underlying tissue. The primary solid component of mucus is a family of heavily glycosylated proteins called mucins which assemble into viscoelastic, disulfide linked networks with bioactive properties.7Mucins polymerize by disulfide bonding between cysteine residues to form dynamic hydrogel networks that that hydrate8and lubricate9tissue surfaces, filter pathogens10and regulate microbial composition and pathogenicity,11,12immune response,13,14and protein activity.15The possibility of recapitulating these properties using purified mucin proteins has driven recent research on mucin-based biomedical materials.16,17

[0058] Despite the potential advantages of mucin-based materials, many applications such as antifouling and lubricating coatings, tissue adhesives, and wound dressings require robust gelation and adhesion to tissue and implant materials in physiological environments, far beyond the transient crosslinking18and noncovalent adsorption19of native mucins. Enhanced crosslinking of mucin proteins has previously been achieved using disulfide bonding20or covalent modification21,22and adhesion of mucin monolayers has been achieved by multi-step covalent attachment to surfaces,23,24but there remains a need for strategies to simultaneously crosslink and adhere mucin- based hydrogels while preserving mucin bioactivity. To fully realize the potential of mucin-based - 8 - 4092104.v2materials, improved methods for achieving gelation and adhesion of mucin-based materials are needed.

[0059] Adhesion in physiological environments, where water undermines adhesive interactions, is a longstanding challenge. To overcome this challenge, researchers have taken inspiration from the robust adhesion of marine mussels. Marine mussels adhere to wet surfaces by fabricating a structure called a byssus, a collection of adhesive plaques and energy-dissipating threads composed of proteins rich in the amino acid 3,4-dihydroxyphenylalanine (Dopa).25Dopa and related catechol analogs can participate in a diverse range of intermolecular interactions that contribute to crosslinking and adhesion of the byssus, inspiring the design of catechol-based materials including self-healing gels,84biocompatible coatings,85biomedical adhesives.86An example of a bioadhesive hydrogel modified with Dopa can be found in US Patent App. Pub. No. US 2012 / 0156164 A1, the entire content of which is incorporated herein by reference. These diverse interactions also simultaneously enable plaque gelation and adhesion to surfaces.26,27The ability of Dopa and catechol analogs to drive gelation and adhesion in wet environments, and especially adhesion to medically-relevant surfaces such as tissue, implant materials, and mucus, has led to widespread research on catechol-based materials for biomedical applications.28Of the interactions formed by catechol analogs, covalent bonding between catechols and thiols is particularly relevant to mucin-based biomaterials. Catechol–thiol crosslinks occur rapidly in physiological environments by Michael addition of thiols to oxidized catechol,26and appear in diverse natural contexts including marine adhesion,29,30biological pigmentation,31neurodegeneration,32and the allergic reaction to plants such as poison ivy.33These interactions has also previously been shown to enable formation of gels37-41and adhesives.87-89Based on the high cysteine content of mucin proteins (~5% of the primary structure34), it is hypothesized that catecholic polymers could crosslink mucins by catechol–thiol bonding, while also enabling adhesion to surfaces. However, although catechol–thiol bonding has been used for polymerization35,36and gelation37–41of peptides and synthetic polymers, the relationships between the composition, gelation, and adhesion of hydrogels crosslinked by catechol–thiol bonds are not well understood, and catecholic polymers have not been used to crosslink and adhere mucin-based hydrogels. - 9 - 4092104.v284002

[0060] The present disclosure demonstrates that combinations of model catechol- and thiol- functionalized polymers form adhesive hydrogels crosslinked by catechol–thiol bonds. Thiol- functionalized polymers including 4-arm PEG-thiol (PEG-4SH) and native mucin proteins (MUC2 and MUC5AC) (FIG.1A) were combined with catechol-functionalized crosslinkers (e.g., 8-arm PEG-catechol (PEG-8C)) (FIG.1B). It is demonstrated herein that catechol–thiol bonding drives the formation of adhesive hydrogels, with gelation dynamics and adhesive properties tuned by the hydrogel composition, polymer architecture, and availability of thiol functionalities for crosslinking. With an understanding of catechol–thiol crosslinking in hand, the present disclosure describes the application of the crosslinking strategy to mucin proteins. Hydrogels of the present disclosure are demonstrated to adhere to tissue surfaces and discourage bacterial biofilm formation, highlighting the promise of catechol–thiol crosslinking for the design of biomedical materials with applications in wound repair and antimicrobial surface engineering. Hydrogel composition, polymer architecture, and thiol availability systematically tune gelation dynamics and adhesive properties, including gelation and mutation timescales, elastic modulus, and failure stress and debonding work. The hydrogels adhere to tissue surfaces and discourage bacterial biofilm formation, raising the possibility of developing biocompatible surgical adhesives and antifouling gel coatings. Collectively, the results suggest the promise of catechol–thiol crosslinking as a bio-inspired strategy for achieving adhesive hydrogels for biomedical applications.

[0061] The present disclosure generally relates to crosslinked hydrogels. As used herein, the term “hydrogel” refers to a class of materials that may be natural or synthetic, which have an affinity for an aqueous medium, and may absorb an amount of the aqueous medium, but which do not normally dissolve in the aqueous medium. In one embodiment, the hydrogel is an adhesive hydrogel. In one embodiment, the hydrogel is composed of (e.g., comprises) mucin proteins crosslinked by catechol-thiol bonds.

[0062] In one embodiment, the hydrogel of the present disclosure may be crosslinked by using, for non-limiting example, at least one, or one or more, types of hydrogel-forming agent, and setting or solidifying the one or more types of hydrogel-forming agent in an aqueous medium to form a three-dimensional network, wherein formation of the three-dimensional network may cause the one or more types of hydrogel-forming agent to gel so as to form the hydrogel. The term “hydrogel-forming agent” refers to any chemical compound or material, natural or synthetic, that may be used to make a hydrogel. As defined herein, a chemical - 10 - 4092104.v284002 compound may be a small molecule, a micromolecule, or a macromolecule, such as and not limited to, carbohydrates, proteins, peptides, polymers, micelles, nucleic acids, and lipids. Non- limiting examples of materials include nanotubes, microparticles, nanoparticles, or assemblies (e.g., supramolecular polymer assemblies).

[0063] In some embodiments, the hydrogel-forming agent is a catechol-containing agent or a thiol-containing agent. A catechol-containing agent is defined herein to be a chemical compound or material that contains a catechol functional group (e.g., non-substituted catechol, substituted catechols such as chlorocatechol, nitrocatechol, etc.). In some embodiments, the catechol- containing agent is an analog of the parent catechol. A thiol-containing agent is defined herein to be a chemical compound or material that contains a thiol functional group.

[0064] Crosslinking includes, and is not limited to, physical and chemical cross-linking. Physical cross-linking may take place via, for non-limiting example, complexation, hydrogen bonding, desolvation, van der Waals interactions, or ionic bonding. In various embodiments, a hydrogel may be formed by self-assembly of one or more types of hydrogel-forming agents in an aqueous medium. The term “self-assembly” refers to a process of spontaneous organization of components of a higher order structure by reliance on the attraction of the components for each other, and without chemical bond formation between the components. For example, polymer chains may interact with each other via, for non-limiting example, any one of hydrophobic forces, hydrogen bonding, Van der Waals interaction, electrostatic forces, or polymer chain entanglement, induced on the polymer chains, such that the polymer chains aggregate or coagulate in an aqueous medium to form a three-dimensional network, thereby entrapping molecules of water to form a hydrogel. Examples of physically cross-linkable polymers, synthetic or natural, that may be used include, but are not limited to, gelatin, alginate, pectin, furcellaran, carageenan, chitosan, derivatives thereof, copolymers thereof, and mixtures thereof.

[0065] Chemical crosslinking may take place via, for example, chain reaction (addition) polymerization, and step reaction (condensation) polymerization. Chemical crosslinking in hydrogels, as used herein, refers to an interconnection between hydrogel-forming agents via chemical bonding, such as, but not limited to, covalent bonding, ionic bonding, or affinity interactions (for non-limiting example, ligand / receptor interactions, antibody / antigen interactions, etc.). Examples of chemically cross-linkable polymer, synthetic or natural, that may - 11 - 4092104.v284002 be used include, but are not limited to, agarose, collagen, chitosan, alginate, pectin, starch, gellan gum, dextran, hyaluronic acid, polyethylene oxides, polyphosphazenes, derivatives thereof, copolymers thereof, and mixtures thereof. Chemically cross-linkable polymer polymers may be functionalized with any chemical functional group. In some embodiments, the functional group is a thiol or catechol group.

[0066] Chemical cross-linking may take place in the presence of a chemical cross-linking agent. The term “chemical cross-linking agent” refers to an agent which induces chemical cross- linking. The chemical cross-linking agent may be any agent that is capable of inducing a chemical bond between adjacent polymeric chains. For non-limiting example, the chemical cross-linking agent may be a chemical compound. In another non-limiting example, the chemical cross-linking agent may be an enzyme, such as polyphenol oxidase (e.g. tyrosinase, catechol oxidase) and peroxidase (e.g., horseradish peroxidase). Examples of chemical compounds that may act as cross-linking agent include, but are not limited to, 1-ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride (EDC), vinylamine, 2-aminoethyl methacrylate, 3-aminopropyl methacrylamide, ethylene diamine, ethylene glycol dimethacrylate, methymethacrylate, N,N′-methylene-bisacrylamide, N,N′-methylene-bis-methacrylamide, diallyltartardiamide, allyl(meth)acrylate, lower alkylene glycol di(meth)acrylate, poly lower alkylene glycol di(meth)acrylate, lower alkylene di(meth)acrylate, divinyl ether, divinyl sulfone, di- or trivinylbenzene, trimethylolpropane tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, bisphenol A di(meth)acrylate, methylenebis(meth)acrylamide, triallyl phthalate, diallyl phthalate, transglutaminase, derivatives thereof or mixtures thereof. In one embodiment, the chemical cross-linking agent is an oxidase. In some embodiments, the oxidase is a tyrosinase enzyme.

[0067] Also provided herein, in some embodiments, is a method of making a hydrogel of the present disclosure, comprising combining a catechol-containing agent with a thiol-containing agent. In some embodiments, the method further comprises a cross-linking agent. An example method of making the hydrogel of the present disclosure is provided in Example 2.

[0068] In some embodiments, the hydrogel-forming agents are themselves capable of chemical or physical cross-linking without use of a cross-linking agent.

[0069] In some embodiments, the selective oxidation of catechols, while avoiding thiol oxidation, is necessary for catechol-thiol crosslinking. - 12 - 4092104.v284002

[0070] In some embodiments, two thiols may bind a single catechol ring. The binding process may result in slower gelation, but ultimately yield a higher density of crosslinks and stiffer gels.

[0071] The hydrogel of the present disclosure may be applied to a surface in a single step via various coating methods such as and not limited to spin, spray, or dip coating, by relying on the adsorption of mucin to the surface. The hydrogel of the present disclosure may also be applied to a surface via a multi-step approach.

[0072] In some embodiments, the hydrogels of the present disclosure have a thickness of from about 0 nm to about 1000 nm (e.g., about 10 nm to about 900 nm, about 10 nm to about 800 nm, about 10 nm to about 700 nm, about 10 nm to about 600 nm, about 10 nm to about 500 nm, about 50 nm to about 500 nm, about 100 nm to about 500 nm, about 200 nm to about 500 nm, about 300 nm to about 500 nm, about 400 nm to about 500 nm, about 450 nm to about 500 nm, about 10 nm to about 100 nm, about 10 nm to about 50 nm, about 10 nm to about 40 nm, about 10 nm to about 30 nm, about 10 nm to about 20 nm, etc.). In one embodiment, the hydrogel has a thickness of less than about 500 nm. In one embodiment, the hydrogel has a thickness of from about 10 nm to about 50 nm.

[0073] In some embodiments, the thiol-containing agent is a thiol-containing polymer. As used herein, a thiol-containing polymer is any polymer, synthetic or natural, that contains a thiol functional group. In some embodiments, the thiol-containing polymer is a linear or branched polymer. In some embodiments, the thiol-containing polymer may have one branch point or multiple branch points. As defined here, “branch point” refers to a bifurcation point comprising one or more atoms at which a polymer splits or branches from a linear structure into one or more additional polymer arms. In some embodiments, the thiol-containing polymer is a dendrimer. In other embodiments, the branches are not regular repeats. In other embodiments, the thiol- containing polymer is a star-shaped polymer, e.g., a star-shaped polymer functionalized with a thiol at the terminus of each branch. For non-limiting example, the thiol-containing polymer contains branches in an integer amount from about 3 to about 100, about 3 to about 90, about 3 to about 80, about 3 to about 70, about 3 to about 60, about 3 to about 50, about 3 to about 40, about 3 to about 30, 3 to about 20, about 3 to about 15, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, or about 8 to about 10. In some - 13 - 4092104.v284002 embodiments, the number of branches of the thiol-containing polymer is about 3, about 4, about 6, or about 8.

[0074] In some embodiments, the thiol-containing agent is a thiol-containing macromolecule. As used herein, a thiol-containing macromolecule is any molecule greater than about one kilodalton (> 1 kDa), synthetic or natural, such as and not limited to, polysaccharides, peptides, proteins, nucleic acids, that contains a thiol functional group. In some embodiments, the thiol-containing macromolecule is a mucin, or a mucin-inspired polymer. Mucin-inspired polymers, as used herein, refers to synthetic polymers with high molecular weight (>10 kDa), thiol functionalities, and carbohydrate or anionic functionalities to mimic mucin glycans. The thiol-containing agent, for non-limiting example, mucin, may be reduced, non-reduced, or oxidized. In some embodiments, the mucin is present in an amount of about 2.8 wt% in the hydrogel of the present disclosure. In other embodiments, the mucin is reduced mucin.

[0075] In some embodiments, the thiol-containing branched polymer is , wherein n is an integer. For non-limiting example, n is an integerabout 1 to about 5000, about 1 to about 4000, about 1 to about 3000, about 1 to about 2000, about 1 to about 1000, or about 1 to about 500. An example method of making the thiol-containing agent of the present disclosure is provided in Example 1.

[0076] In some embodiments, the thiol-containing agent is present in an amount of about 0.1 wt% to about 20 wt%, about 0.1 wt% to about 15 wt%, about 0.1 wt% to about 10 wt%, about 0.1 wt% to about 9 wt%, about 0.1 wt% to about 8 wt%, about 0.1 wt% to about 7 wt%, about 0.1 wt% to about 6 wt%, about 0.1 wt% to about 5 wt%, about 0.1 wt% to about 4 wt%, about 0.1 wt% to about 3 wt%, about 0.5 wt% to about 3 wt%, about 1 wt% to about 3 wt%, about 1 wt% to about 2 wt%, about 2 wt% to about 3 wt% about 1 wt% to about 1.5 wt%, or about 2.5 - 14 - 4092104.v284002 wt% to about 3 wt%. In some embodiments, the thiol-containing agent is present in an amount of about 1 wt% to about 10 wt%. In some embodiments, the thiol-containing agent is present in an amount of about 1.4 wt%. In other embodiments, the thiol-containing agent is present in an amount of about 2.8 wt% in the hydrogel of the present disclosure.

[0077] In some embodiments, the catechol-containing agent is a catecholic polymer. As used herein, a catecholic polymer is any polymer, synthetic or natural, that contains at least one catechol functional group. In some embodiments, the catecholic polymer is a linear or branched polymer. In some embodiments, the catecholic polymer may have one branch point or multiple branch points. In other embodiments, the catecholic polymer is a star-shaped polymer, e.g., a star-shaped polymer functionalized with a catechol at the terminus of each branch. For non- limiting example, the catecholic polymer contains branches in an integer amount from about 3 to about 100, about 3 to about 90, about 3 to about 80, about 3 to about 70, about 3 to about 60, about 3 to about 50, about 3 to about 40, about 3 to about 30, 3 to about 20, about 3 to about 15, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, or about 8 to about 10. In some embodiments, the number of branches of the catecholic polymer is about 3, about 4, about 6, or about 8.

[0078] In some embodiments, the catecholic polymer is a polymer of formula (I): , wherein:R is a polyol core; n is an integer from about 1 to about 1000; and A is an integer from about 3 to about 100.

[0079] For non-limiting example, in formula (I), n is an integer from about 1 to about 10000, about 1 to about 5000, about 1 to about 4000, about 1 to about 3000, about 1 to about 2000, about 1 to about 1000, or about 1 to about 500. For non-limiting example, A is an integer from about 3 to about 100, about 3 to about 90, about 3 to about 80, about 3 to about 70, about 3 to about 60, about 3 to about 50, about 3 to about 40, about 3 to about 30, about 3 to about 25, - 15 - 4092104.v284002 about 3 to about 20, about 3 to about 15, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, or about 8 to about 10. In some embodiments, A is about 3, about 4, about 6, about 8, about 30, or about 100. In some embodiments, A is less than or equal to about 100. In other embodiments, A is less than or equal to about 30.

[0080] In some embodiments, a branched polymer is a derivative of a polyol. As defined herein, a polyol core refers to the any compound containing two or more hydroxyl groups, that forms the branched structure of a branched polymer. In some embodiments, the polyol is a multi- arm polyethylene glycol. For non-limiting example, the multi-arm polyethyleneglycol is, and is not limited to, 3-arm-polyethyleneglycol, 4-arm-polyethyleneglycol, 6-arm-polyethyleneglycol or 8-arm-polyethyleneglycol. In some embodiments, the polyol is a glycol, trimethylolpropane, pentaerythritol, hexaglycerol, or tripentaerythritol. An example method of making the catechol- containing agent of the present disclosure is provided in Example 1.

[0081] In some embodiments, the catecholic polymer, wherein n is an integer5000, about 1 to about 4000, about 1 to about 3000, about 1 to about 2000, about 1 to about 1000, about 1 to about 500, etc.). As illustrated herein, PEG-8C has a tripentaerythritol core.

[0082] A non-limiting example of preparing a catechol-containing agent (e.g., PEG-8C) is as follows: For every 1g of 8-arm PEG-SG with a molecular mass of 20 kDa, add 152 mg of dopamine HCl. 1. Argon / vacuum cycling. Cycle 3 times. 2. Open flask, add solid reactants (PEG-SG and Dopamine HCl) and stirring bar, while flowing argon, and close flask again. 3. When collecting DMF and TEA, add an equal volume of argon gas. 4. Dissolve solid in enough DMF – about 12 ml per gram of polymer. - 16 - 4092104.v284002 5. Add TEA – about 111 μL per gram of polymer (2 mol equivalent to PEG-SG). 6. Move flask into silicon oil bath, heat to 55°C in an oil bath on a hot plate. 7. Turn off vacuum and argon. 8. Allow reaction to proceed overnight.

[0083] In some embodiments, the catechol-containing agent is an oxidation-resistant catechol analog (e.g., nitrocatechol).

[0084] In some embodiments, the catechol-containing agent is present in an amount of about 0.1 wt% to about 20 wt%, about 0.1 wt% to about 15 wt%, about 0.1 wt% to about 10 wt%, about 0.1 wt% to about 9 wt%, about 0.1 wt% to about 8 wt%, about 0.1 wt% to about 7 wt%, about 0.1 wt% to about 6 wt%, about 0.1 wt% to about 5 wt%, about 0.5 wt% to about 5 wt%, about 0.5 wt% to about 4 wt%, about 0.5 wt% to about 3 wt%, about 0.5 wt% to about 2 wt%, about 0.5 wt% to about 1.5 wt%, about 0.5 wt% to about 1 wt%, or about 1 wt% to about 1.5 wt%. In some embodiments, the catechol-containing agent is present in an amount of about 0.5 wt% to about 5 wt%. In some embodiments, the catechol-containing agent is present in an amount of about 1 wt%. In certain embodiments, PEG-8C is present in an amount of about 1 wt% in the hydrogel of the present disclosure.

[0085] In some embodiments, the catechol-containing agent comprises catechol groups which are physically crosslinked to a thiol-containing agent, chemically crosslinked to a thiol- containing agent, or a combination thereof. In some embodiments, catechol-thiol covalent bonds are formed between the catechol groups of catechol-containing agent and the thiol groups of thiol-containing agent, thereby producing a chemically crosslinked hydrogel.

[0086] In some embodiments, the hydrogel is crosslinked in the presence of an enzyme (e.g., an oxidase), for non-limiting example, tyrosinase. In some embodiments, the enzyme is present in an amount of about 0.01 mg / ml to about 10.0 mg / ml, about 0.01 mg / ml to about 5.0 mg / ml, about 0.01 mg / ml to about 4.0 mg / ml, about 0.01 mg / ml to about 3.0 mg / ml, about 0.01 mg / ml to about 2.0 mg / ml, about 0.01 mg / ml to about 1.0 mg / ml, about 0.05 mg / ml to about 1.0 mg / ml, about 0.05 mg / ml to about 0.9 mg / ml, about 0.05 mg / ml to about 0.8 mg / ml, about 0.05 mg / ml to about 0.7 mg / ml, about 0.1 mg / ml to about 0.7 mg / ml, about 0.15 mg / ml to about 0.65 mg / ml, about 0.16 mg / ml to about 0.64 mg / ml, about 0.15 mg / ml to about 0.35 mg / ml, about 0.35 mg / ml to about 0.65 mg / ml, about 0.15 mg / ml to about 0.2 mg / ml, about 0.3 mg / ml to about 0.35 - 17 - 4092104.v284002 mg / ml, or about 0.6 mg / ml to about 0.65 mg / ml. In some embodiments, the enzyme is present in an amount of about 0.16 mg / ml. In some embodiments, the enzyme is present in an amount of about 0.32 mg / ml. In other embodiments, the enzyme is present in an amount of about 0.64 mg / ml. In one embodiment, the enzyme is an oxidase (e.g., tyrosinase) and it is present in an amount of less than or equal to (not more than) about 2 mg / ml.

[0087] In accordance with other embodiments thereof, the present disclosure provides a hydrogel further comprising a therapeutic agent. The therapeutic agent includes and is not limited to, a peptide, a protein, an anti-bacterial agent, an anti-cancer agent, an anti-inflammatory agent, an anti-viral agent, or a combination thereof. In some embodiments, the therapeutic agent is a peptide drug, a protein drug, an anti-bacterial agent, an anti-cancer agent, an anti- inflammatory agent, an anti-viral agent, an analgesic or anesthetic agent, an immunomodulator (e.g., immunostimulant, immunosuppressant), an antifungal agent, a hemostasis agent, or a combination thereof.

[0088] Non-limiting examples of peptide or protein drugs include agonists or antagonists of: fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), bone morphogenetic protein (BMP), human growth hormone (hGH), pig growth hormone (pGH), granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF, e.g., TNFα or TNFβ), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), interferon-α,β,γ, interleukin-2 (IL-2), calcitonin, nerve growth factor (NGF), growth hormone releasing hormone, angiotensin, luteinizing hormone releasing hormone (LHRH), or thyrotropin-releasing hormone (TRH). In some embodiments, the peptide or protein drugs are luteinizing hormone releasing hormone agonist (LHRH agonist), insulin, angiostatin, endostatin, somatostatin, glucagon, endorphine, bacitracin, mergain, colistin, antibodies (e.g., monoclonal antibodies), and vaccines.

[0089] In other embodiments, the peptide or protein drugs are recombinant proteins. Non- limiting examples of recombinant proteins include, but are not limited to, insulin, lepirudin, somatropin, aldesleukin, interferon gamma 1b, anakinra, interferon alpha 2b, interferon beta 1b, interferon beta 1a, PEG interferon alpha 2a, filgrastim, pegfilgrastim, oprelvekin, reteplase, denileukin diftitox, follitropin alfa, recFSH, thyrotropin alfa, imiglucerase, becaplermin, sargramostim, darbepoetin, erythropoietin, DNAse, Factor VIIa, Factor IX, Factor XIII, - 18 - 4092104.v284002 drotrecogin, alteplase, tenecteplase, moroctocog alfa (BDDrFVIII), Factor VIII-2, Factor VIII, peginteferon, ribavarin, clostridial collagenese, alglucosidase alpha2, incobotulinumtoxina, pegloticase, palifermin, galsulfase, idursulfase.

[0090] In certain embodiments, the protein drug is an antibody or variant thereof. As used herein, unless otherwise indicated, “antibody” means an intact antibody or antigen-binding fragment of an antibody, including an intact antibody or antigen-binding fragment that has been modified or engineered, or that is a human antibody. Examples of antibodies that have been modified or engineered are chimeric antibodies, humanized antibodies, multiparatopic antibodies (e.g., biparatopic antibodies), and multispecific antibodies (e.g., bispecific antibodies). Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, single chain antibodies (e.g., scFv), double-stranded Fv (dsFv), minibodies and diabodies. The antibodies disclosed herein can be whole antibodies or antibody fragments provided that the antibody or antibody fragment is able to recognize and bind to its specific antigen in vitro or in vivo. Such antibodies may inhibit (e.g., reduce, block, interfere with) one or more activities, thereby leading to target neutralization.

[0091] Examples of antibodies include, but are not limited to, etanercept, abciximab, gemtuzumab, rituximab, adalimumab, palivizumab, trastuzumab, bevacizumab, natalizumab, omalizumab, infliximab, alemtuzumab, efalizumab, cetuximab, golimumab, abobotulinumtoxina, canakinumab, ustekinumab, ofatumumab, certolizumab pegol, tocilizumab, denosumab, abatacept, ranibizumab, panitumumab, eculizumab, brentixumab, iplimumab, belimumab, and rilonacept.

[0092] Non-limiting examples of an anti-bacterial agent include minocycline, tetracycline, ofloxacin, fosfomycin, mergain, profloxacin, ampicillin, penicillin, doxycycline, thienamycin, cephalosporin, nocardicin, gentamicin, neomycin, kanamycin, paromomycin, micronomicin, amikacin, tobramycin, dibekacin, cefotaxime, cefaclor, erythromycine, ciprofloxacin, levofloxacin, enoxacin, vancomycin, imipenem, and fusidic acid.

[0093] Non-limiting examples of an anti-cancer agent include paclitaxel, taxotere, adriamycin, endostatin, angiostatin, mitomycin, bleomycin, cisplatin, carboplatin, doxorubicin, daunorubicin, idarubicin, 5-fluorouracil, methotrexate, and actinomycin-D. - 19 - 4092104.v284002

[0094] Non-limiting examples of an anti-viral agent include a nucleoside reverse transcriptase inhibitor, a non-nucleoside reverse transcriptase inhibitor, a fusion inhibitor, an integrase inhibitor, a nucleoside analog, a protease inhibitor, a reverse transcriptase inhibitor. Examples of antiviral agents include, but are not limited to, abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, boceprevir, cidofovir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, oseltamivir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, stavudine, tea tree oil, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir (Valtrex), valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

[0095] Non-limiting examples of an analgesic or anesthetic agent include paracetamol, an opiate, diproqualone, phenazone, cocaine, or lidocaine. In certain embodiments, the opioid is a natural opium alkaloid, phenylpiperidine derivative, diphenylpropylamine derivative, benzomorphan derivative, oripavin derivative, or morphinan derivative. In some embodiments, the analgesic is a salicylic acid derivative, pyrazolone, or anilide. In other embodiments, the analgesic is an ergot alkaloid, corticosteroid derivative, or selective serotonin (5HT1) agonist. Examples of local anesthetics include, but are not limited to, Esters of aminobenzoic acid like metabutethamine, procaine, tetracaine, chloroprocaine, benzocaine; Amides like bupivacaine, lidocaine, mepivacaine, prilocaine, butanilicaine, cinchocaine, etidocaine, articaine, ropivacaine, levobupivacaine, tetracaine, chloroprocaine, benzocaine; Esters of benzoic acid like cocaine; Other local anesthetics like ethyl chloride, dyclonine, phenol, capsaicin.

[0096] Non-limiting examples of an antifungal agent include a polyene antifungal, an imidazole, triazole, or thiazole antifungal, a triazole antifungal, a thiazole antifungal, an allylamine derivative, or an echinocandin derivative. Examples of antifungal agents include, but are not limited to, polyene derivatives like natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin, hamycin; imidazole derivatives like miconazole, ketoconazole, clotrimazole, - 20 - 4092104.v284002 econazole, omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole; tetrazole derivatives like fluconazole, itraconazole, isavuconazole, posaconazole, voriconzaole, terconazole, albaconazole; thiazole derivatives like abafungin; allylamine derivative like terbifine, naftifine, butenafine; echinocandin derivatives like anidulafungin, caspofungin, micafungin; other antifungals like polygodial, benzoic acid, ciclopirox, tonaftate, undecylenic acid, flycytosine, griseofulvin, haloprogin, sodium bicarbonate, pirctone olamine, zinc pyrithione, selenium sulfide, tar, and tea tree oil.

[0097] Non-limiting examples of a hemostasis agent or antihemorrhagic agent include an antifibrinolytic (amino acid or proteinase inhibitor), a vitamin K, fibrinogen, a local hemostatic, and a blood coagulation factor. Examples of hemostasis agents include, but are not limited to, amino acids like aminocaproic acid, tranexamic acid, aminomethylbenzoic acid; proteinase inhibitors like aprotinin, alfa1 antitrypsin, C1-inhibitor, camostat; vitamin K like phytomenadione, menadione; fibrinogen like Human fibrinogen; local hemostatics like absorbable gelatin sponge, oxidized cellulose, tetragalacturonic acid hydroxymethylester, adrenalone, thrombin, collagen, calcium alginate, epinephrine, human fibrinogen; blood coagulation factors like coagulation factor IX, II, VII and X in combination, coagulation factor VIII, factor VIII inhibitor bypassing activity, coagulation factor IX, coagulation factor VII, von Willebrand factor and coagulation factor VIII in combination, coagulation factor XIII, eptacog alfa, nonacog alfa, thrombin; and other systemic hemostatics like etamsylate, carbazochrome, batroxobin, romiplostim, eltrombopag.

[0098] In some embodiments, the present disclosure provides a hydrogel comprising an immunostimulant or an immunosuppressant. In other embodiments, disclosed herein is a method of modulating immune response, comprising administering a hydrogel to a subject in need thereof, wherein the hydrogel further comprises an immunostimulant or an immunosuppressant. As used herein, modulating immune response refers to altering, suppressing, or stimulating the immune system.

[0099] The term "immunostimulant" as used herein refers to a molecule that stimulates the immune system by acting as an agonist of an immunostimulatory molecule, or as an antagonist of an immunosuppressive molecule. The immunostimulant may be biological, such as an antibody or antibody fragment, other protein or vaccine, or may be a small molecule drug. Non- - 21 - 4092104.v284002 limiting examples of an immunostimulant include ligands of OX40 (e.g., OX40L), ligands of CD-28 (e.g., CD80, CD86), ligands of ICOS (e.g., B7RP1), ligands of 4- IBB (e.g., 4-1BBL, Ultra4-1BBL), ligands of CD27 (e.g., CD70), ligands of CD40 (e.g., CD40L), and ligands of TCR (e.g., MHC class I or class II molecules, IMCgplOO).

[0100] The term "immunosuppressant" as used herein refers to a molecule that suppresses the immune system by acting as an agonist of an immunosuppressing molecule, or as an antagonist of an immunostimulant molecule. The immunosuppressant may be biological, such as an antibody or antibody fragment, other protein or vaccine, or may be a small molecule drug. Non-limiting examples of an immunosuppressant include calcineurin inhibitors like ciclosporin, tacrolimus; mTOR inhibitors like sirolimus, everolimus; anti-proliferatives like azathioprine, mycophenolic acid; corticosteroids like prednisolone, hydrocortisone; monoclonal anti-IL-2Ra receptor antibodies like basiliximab, daclizumab; polyclonal anti-T-cell antibodies like antithymocyte globulin (ATG), anti-lymphocyte globulin (ALG); monoclonal anti-CD20 antibodies like rituximab; interleukin inhibitors like daclizumab, basiliximab, anakinra, rilonacept, ustekinumab, mepolizumab, tocilizumab, canakinumab, briakinumab; tumor necrosis factor alpha (TNF-α) inhibitors like etanercept, infliximab, afelimomab, adalimumab, certolizumab pegol, golimumab; selective immunosuppressants like muromonab-CD3, antilymphocyte immunoglobulin (horse), antithymocyte immunoglobulin (rabbit), mycophenolic acid, sirolimus, leflunomide, alefacept, everolimus, gusperimus, efalizumab, abetimus, natalizumab, abatacept, eculizumab, belimumab, fingolimod, belatacept; or other immunosuppressants like azathioprine, thalidomide, methotrexate, and lenalidomide. Delivery of therapeutic agent

[0101] The administration of a hydrogel may be parenteral or non-parenteral, including intravenous, intra-arterial, intraperitoneal, intramuscular, intracavity, subcutaneous, intradermal, topical, inhalation, transmucosal, rectal or transdermal. In some embodiments, the administration routes is oral, rectal, subcutaneous, transdermal, ocular, and intraperitoneal. In other embodiments, the hydrogel may be administered ex vivo. For example, the hydrogel may serve as an ex vivo drug delivery and / or testing platform for patient-derived organoids. - 22 - 4092104.v284002

[0102] In some embodiments, the hydrogel delivers a therapeutic agent to a target site by diffusion and / or osmosis over time ranging from hours to days. In certain embodiments, the drug is delivered directly to the target site. In some embodiments, the procedure of delivering a hydrogel comprising a therapeutic agent to a target site is repeated several times, if needed. In other embodiments, the therapeutic agent is released from the hydrogel through biodegradation of the hydrogel. In some embodiments, the therapeutic agent is released through a combination of diffusion, osmosis, and / or hydrogel degradation mechanisms.

[0103] In some embodiments, the therapeutic agent is released from the hydrogel though diffusion or osmosis. In certain embodiments, the therapeutic agent is substantially released from the hydrogel within about 180 days, about 150 days, about 120 days, about 90 days, about 80 days, about 70 days, about 60 days, about 50 days, about 40 days, about 35 days, about 30 days, about 28 days, about 21 days, about 14 days, about 10 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, about 0.5 day, about 6 hours, about 4 hours, about 2 hours, about or 1 hour.

[0104] In some embodiments, the release of a therapeutic agent from the hydrogel is controlled by the composition of the hydrogel. In some embodiments, the pore size of the hydrogel is small enough to prevent the early phase release of the therapeutic agent. In certain embodiments, the pore size of the hydrogel is large enough to allow the early phase release of the therapeutic agent. In some embodiments, the ratio of the pore size of the hydrogel to the size of the therapeutic agent determines the release rate of the therapeutic agent. Bioadhesive

[0105] In accordance with some embodiments thereof, the present disclosure provides a bioadhesive comprising a hydrogel of the present disclosure. In some embodiments, the bioadhesive is a tissue adhesive or hemostat. For non-limiting example, the hemostat may be a topical hemostat further comprising an astringent or a coagulant. For non-limiting example, the bioadhesive can be applied to various medical situations, such as surgery, for example, cerebral nervous system surgery including vascular surgery, orthopedic surgery including bone bonding, hemostasis in patients with lacerations, closure of the femoral artery, closure after incision of an eye affected with cataract, the healing of cartilage, e.g., articular cartilage, dermal adhesion, - 23 - 4092104.v284002 hemostasis at incised portions in organs / secretory glands, anastomosis of organs such as gastrointestinal organs, healing of ligaments and tendons, etc. The bioadhesive can also be used for wound adhesion and surgical anastomoses as an adhesive of the skin, gastrointestinal tract, blood vessels, trachea, and other such soft tissues.

[0106] Furthermore, the bioadhesive comprising the hydrogel of the present disclosure may be used to adhere tissues to a sensor electrode, stimulation electrode, implant, or other such medical member. The bioadhesive may also be used as a suitable adhesive for immobilizing dental and bone implants. Pigments, drugs, and other such optional additives may be added as needed when used as a bioadhesive.

[0107] For non-limiting example, the performance of the hydrogel as a bioadhesive may be determined by the failure stress of the hydrogel. An example method of measuring the failure stress is provided in Example 2. For non-limiting example, the failure stress of the hydrogels of the present disclosure is present in an amount of from about 1 kPa to about 500 kPa, from about 1 kPa to about 400 kPa, from about 1 kPa to about 300 kPa, from about 1 kPa to about 200 kPa, from about 1 kPa to about 100 kPa, from about 1 kPa to about 90 kPa, from about 1 kPa to about 80 kPa, from about 1 kPa to about 70 kPa, from about 1 kPa to about 60 kPa, from about 1 kPa to about 50 kPa, from about 1 kPa to about 40 kPa, from about 1 kPa to about 30 kPa, from about 1 kPa to about 20 kPa, from about 1 kPa to about 15 kPa, from about 1 kPa to about 14 kPa, from about 1 kPa to about 13 kPa, from about 1 kPa to about 12 kPa, from about 1 kPa to about 11 kPa, from about 1 kPa to about 10 kPa, from about 2 kPa to about 9 kPa, or from about 3 kPa to about 8 kPa. In other embodiments, the failure stress is about 5 kPa.

[0108] In some embodiments, the bioadhesive is partially or completely transparent, allowing for ease of monitoring the surface below or the device encapsulated within. In some embodiments, the bioadhesive is translucent or opaque. Implant material

[0109] In some embodiments, the present disclosure provides an implant material comprising a hydrogel of the disclosure, e.g., for tissue regeneration and / or augmentation. For non-limiting example, the implant material can be used in cartilage regeneration, bone regeneration, periodontal regeneration, skin regeneration, cardiac tissue regeneration, artificial intraocular lens, - 24 - 4092104.v284002 spinal cord regeneration, cranial regeneration, vocal regeneration and augmentation, adhesion barrier, urinary incontinence treatment, wrinkle removal, wound dressing, tissue augmentation or intervertebral disc treatment. The hydrogel may be designed to replace cartilage for any joint surface in the body. The hydrogel may be formed in a defect site, which may be similar to the natural extracellular matrix (ECM) of cartilage, and stimulate adhesion and spreading of chondrocyte cells. Shaving device

[0110] This present disclosure further relates to a shaving device comprising a hydrogel of the disclosure. The shaving device may be powered or non-powered. The surface may include a non-cutting or cutting component. For non-limiting example, the shaving device may be a shaving razor. The shaving razor may include a cartridge, which may be configured to be selectively coupled a handle. The cartridge may or may not be disposable. The cartridge may include one or more razor blades disposed on a cutting surface of the cartridge. Once the razor blade(s) are dull, the user may disconnect the cartridge from the handle and reconnect a razor cartridge which may be the same cartridge prior to disconnecting, or a different cartridge. In some embodiments, the disclosure provides a cartridge comprising a razor blade, said razor blade comprising the hydrogel of the disclosure. The resultant surface or blade may or may not be a hybrid type of surface, having porous and solid characteristics. The resultant surface or blade may be smooth, and to some extent, slippery and / or wet. In some embodiments, the hydrogel is a coating or a film on a surface of a shaving device.

[0111] In some embodiments, the low friction properties of the hydrogel applied on the surface(s) of the shaving device or used as a component of the razor blade, may provide significant benefits, e.g., the hydrogel may elevate performance attributes such as comfort and glide.

[0112] It is further contemplated in the present disclosure that a shaving device component may in and of itself be fully or partially comprised of the hydrogel via the application of multiple layers, e.g., one on top of each other. This may potentially simplify the manufacturing process of the shaving device components by eliminating process steps or materials. - 25 - 4092104.v284002

[0113] The hydrogel of the present disclosure may be applied to almost any type of shaving device component material, inclusive but not limited to, polymers, metals, ceramics, or glasses. In some embodiments, the hydrogel is a coating on a shaving component, such as a razor blade. In some embodiments, the coating has a friction coefficient comparable to PTFE, e.g., when against wet skin.

[0114] Various techniques may be employed to deposit the hydrogel (e.g., mucin-based films) on the surface of the shaving device. Such techniques include but are not limited to spin coating and spray coating. Hydrogel coatings of varying thicknesses (e.g., from about 10 nm to about 500 nm, from about 10 nm to about 50 nm etc.) may be deposited onto shaving devices. The shaving devices may comprise a metal oxide surface (e.g., TiO2, Cr2O3, combinations thereof, etc.). The properties of the resulting hydrogel coatings may depend on the operating parameters (e.g., spin rate, spin time, injection volume, etc.) and the viscosity of the precursor solution. For non-limiting example, mucin or mucin-inspired polymer concentration in the precursor solutions may be altered and changes in viscosity (e.g., shear viscosity, extensional viscosity) quantified (e.g., using shear or extensional rheometry). In some embodiments, for metal oxide surface preparation, a layer of metal (e.g., titanium, chromium) is sputter-deposited onto wafers (e.g., stainless steel wafers), followed by oxidation to form metal oxide (e.g., TiO2, Cr2O3) surfaces. In some embodiments, the thickness and roughness of the coatings are measured with a surface profilometer (e.g., Dektak 150 surface profilometer, (Bruker, Berlin, Germany)). and / or film uniformity assessed using a non-contact optical profilometer.

[0115] The spray coating technique may involve parameters such as but not limited to spray velocity, spray angle, nozzle dimensions, and spray patterns, such as circular, linear, or raster patterns, for precise control over the coating coverage. In some embodiments, solvent-based and / or solvent-free spray coating method is employed to deposit the hydrogel on the surface of the shaving device. Solvent-based spray coating involves the dissolution of hydrogel precursors in a suitable solvent, which is then atomized and sprayed onto the substrate. Other solvent-free techniques may be employed for deposition, such as but not limited to airless or air-assisted spray coating. Other parameters such as hydrogel concentration and viscoelasticity may be varied to tune final coating properties. - 26 - 4092104.v284002

[0116] In some embodiments, while also providing lubricity, a hydrogel applied to a shaving device surface or razor blade may assist in preventing bacteria from growing on the surface and / or blade, thereby providing a consumer benefit protection to the skin by not transmitting and / or infecting the skin with bacteria generally found on a shaving device surface or razor blade, particularly after many uses. As a non-limiting example, razor handles are often used for many years in warm wet environments, and cartridges contain blood and skin and hair fragments creating a breeding ground for a host of bacteria which may also be found in organizer trays and the cartridge dispensers. Additionally, the hydrogel may assist in preventing calcium carbonate deposits.

[0117] In some embodiments, the hydrogel of the present disclosure provides enhanced glide and / or wear resistance. The hydrogel may also be applied to skin management areas such as the guard or cap area of the shaving device which may result in further optimized cartridge performance such as stretch and / or glide during shaving. Antifouling

[0118] In some embodiments, the hydrogel of the present disclosure can be used as an antifouling coating. In some embodiments, the antifouling coating prevents proteins and cells from adhering to devices, e.g., implantable medical devices such as catheters, stents, and cardiac pacemakers. For non-limiting example, urinary tract infections triggered by microorganisms forming colonies in catheters are a problem as a frequent nosocomial infection. The adhesion of microorganisms to these medical devices can be prevented by coating them with the hydrogel. Pigments, antimicrobials, and other such optional additives may be added as needed when the hydrogel is used as a coating material for antifouling.

[0119] The hydrogel of the present disclosure can also be used as a ship bottom antifouling coating. The common method for preventing biofouling is to affix an antifouling material to a surface. Since the hydrogel of the present disclosure can adhere firmly to metals such as iron, the ship's bottom can be coated with hydrogel. Coating the bottom of the ship with hydrogel can prevent sea creatures such as barnacles from adhering to the bottom of the ship. Organic antifouling components, plasticizers, inorganic dehydrating agents (stabilizers), antisagging agents, antisettling agents, coloring pigments, rust inhibitors, and the - 27 - 4092104.v284002 like may be added as needed when the hydrogel of the present disclosure is used as a ship bottom antifouling coating. In some embodiments, the hydrogel is co-applied with one or more of them. In some embodiments, the hydrogel comprises one or more of them.

[0120] For non-limiting example, the performance of the hydrogel used as an antifouling coating may be determined by the ability to reduce biofilm formation. An example method of measuring changes in biofilm formation is provided in Example 6.

[0121] The present disclosure further provides a method of reducing or suppressing biofilm formation, e.g., on an article or object, the method comprising applying a hydrogel of the disclosure. In some embodiments, the article or object according to the method of the present disclosure may be, without being limited to, an object designed for functioning in water, e.g., the hull of a boat, a pipe, a filter, a pump, or a heat-exchanger; a medical implant such as a stent; a medical device such as a catheter; or a biomedical pad such as an adhesive bandage. Lubricity

[0122] Also described herein is a method of providing an article or object with a surface exhibiting a lubricious property, the method comprising applying the hydrogel of the disclosure. The article or object may be, for non-limiting example, a medical device. In some embodiments, the article or object is a medical device having a lubricious coating on at least a section of the device, the coating comprising the hydrogel of the disclosure. When used herein, the term "lubricious" describes a surface which is slippery after wetting. For non-limiting example, a coated surface is considered to be "lubricious" if the friction force is smaller than the original friction force for the non-coated surface. In certain embodiments, a surface coated with the hydrogel has a higher surface lubricity than the lubricity of a non-coated surface. In another non- limiting example, if A has a smaller coefficient of friction than B, A is more lubricious than B.

[0123] In some embodiments, a surface coated with hydrogels of the present disclosure is a lubricious surface.

[0124] In some embodiments, the coefficient of friction of a surface coated with hydrogels of the present disclosure is from about 0 to about 0.5 (e.g., about 0 to about 0.4, about 0 to about 0.35, about 0 to about 0.3, about 0 to about 0.25, about 0 to about 0.2, about 0 to about 0.15, about 0 to about 0.1, etc.).Ex - 28 - 4092104.v284002 Target site treatment and delivery

[0125] The invention provides, in other embodiments, methods of treating a target (e.g., a wound) in a subject in need thereof, said method comprising contacting the target with a hydrogel of the disclosure, under conditions to allow the hydrogel to adhere to tissue surrounding the target.

[0126] For non-limiting example, a wound dressing comprising the hydrogel of the disclosure is used to contact the wound. In some embodiments, the hydrogel may be held in place by tape or other secondary support methods or devices. The hydrogel may be polymer- reinforced, e.g., by a moisture barrier to prevent dehydration of the hydrogel. When a hydrogel of this invention is placed in contact with a wound and is held in place by an adhesive around the wound site, the hydrogel may be placed topically prior to dressing the wound, or incorporated into a film, e.g., during manufacture. The hydrogel contacting the wound may further comprise a therapeutic agent, such as those described herein.

[0127] Other embodiments of the present disclosure include: hydrogel-based dressings, e.g., dressings that contain heparin and / or other materials to provide optimal biocompatibility; hydrogel-based dressings free of stimulants and adhesive groups that have minimal protein absorption / activation and have minimal ‘sting’ to the wound; hydrogel-based dressings that have high water content and low modulus, both of which act to provide wound comfort; and hydrogel- based dressings that have multi-layer structure, with each layer designed for optimized multifunctional wound care.

[0128] In some embodiments, the hydrogel-forming agents, for non-limiting example a catecholic-containing agent and a thiol-containing agent, undergo polymerization at a target site inside a body, or on the surface of a body, e.g., a mammalian body. In some embodiments, the target site is inside a human being. In certain embodiments, the target site is on the human body. For non-limiting example, the target site is a wound, an organ, or a tumor. In some embodiments, the target site is accessible through surgery. In certain embodiments, the target site is accessible through minimally invasive surgery. In some embodiments, the target site is accessible through an endoscopic device. - 29 - 4092104.v284002

[0129] In some embodiments, the hydrogel-forming agents are delivered as an in vivo pre- formulation to a target site by a device to form a hydrogel at the target site. In certain embodiments, the device comprises a catheter or a needle. The hydrogel according to the present disclosure can be cross-linked in situ with, for non-limiting example, the aid of a dual syringe kit or can be sprayed using a nozzle-mounted dual syringe kit. Bioabsorbance of the Hydrogel

[0130] In some embodiments, the hydrogel is a bioabsorbable hydrogel. In certain embodiments, the hydrogel is bioabsorbed within about 365 days, 180 days, about 150 days, about 120 days, about 90 days, about 80 days, about 70 days, about 60 days, about 50 days, about 40 days, about 35 days, about 30 days, about 28 days, about 21 days, about 14 days, about 10 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, or about 1 day. In certain embodiments the hydrogel is bioabsorbed within less than 365 days, 180 days, less than 150 days, less than 120 days, less than 90 days, less than 80 days, less than 70 days, less than 60 days, less than 50 days, less than 40 days, less than 35 days, less than 30 days, less than 28 days, less than 21 days, less than 14 days, less than 10 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days, or less than 1 day. In some embodiments the hydrogel is bioabsorbed within more than 365 days, 180 days, more than 150 days, more than 120 days, more than 90 days, more than 80 days, more than 70 days, more than 60 days, more than 50 days, more than 40 days, more than 35 days, more than 30 days, more than 28 days, more than 21 days, more than 14 days, more than 10 days, more than 7 days, more than 6 days, more than 5 days, more than 4 days, more than 3 days, more than 2 days, or more than 1 day. In some embodiments, the hydrogel is substantially non- bioabsorbable. Kit

[0131] Also disclosed herein is a kit comprising a hydrogel or pre-formulation of the present disclosure. For non-limiting example, a kit includes substrates (for non-limiting example a catechol-containing agent and a thiol-containing agent) and / or one or more crosslinkers. The substrate and crosslinker components may be present in separate containers in the kit, for non- - 30 - 4092104.v284002 limiting example, where the substrate is present in a first container and the crosslinker is present in a second container, where the container(s) may or may not be present in a combined configuration. The requisite buffer solutions for the substrate and crosslinker compositions may be provided in additional, separate, containers. A container is understood to refer to any structure that may hold or surround the components of a hydrogel of the disclosure; example containers include syringes, vials, pouches, capsules, carpules, ampules, cartridges, and the like. The containers may be shielded from visible, ultraviolet, or infrared radiation through the use of additional components (for non-limiting example, a foil pouch surrounding a syringe) or through selection of the material properties of the container itself (for non-limiting example, an amber glass vial or opaque syringe).

[0132] The kit may also include a mixing device, for mixing the substrates and crosslinker together. The kit may also include a delivery device (which may or may not include a mixing element), such as a catheter device (for non-limiting example, tubes with one or more lumens of identical or differing sizes and shapes with exit points of varying geometries, dimensions, and positions), syringe(s) of similar or different diameters and volumes, spray elements, check valves, stopcocks, Y-connectors, air bleeder elements (for non-limiting example, a membrane that permits the removal of air from a liquid solution prior to delivery to the patient), inlet ports or chambers for the introduction of a forced air stream, disposable cartridges that allow for prolonged deposition of the hydrogel composition, applicators or spreaders, assemblies for realizing a mechanical advantage in delivering the composition of the invention, housings or casings to protect and contain the above mentioned components, and the like.

[0133] The kit may further include other components, for non-limiting example, desiccants or other means of maintaining control over water content in the kit, oxygen scrubbers or other means of maintaining control over oxygen content within the kit, an inert gas atmosphere (for non-limiting example, nitrogen or argon), indicators, e.g., to convey the maximum temperature experienced by the kit, indicators to convey exposure to sterilizing radiation, ethylene oxide, autoclave conditions, and the like, retaining or positioning structures to prevent damage to the components (for non-limiting example, trays or packaging cards) that are required to maintain the product in good condition during transport and storage. A kit of the disclosure may further include instructions for using the kit. - 31 - 4092104.v284002 Methods

[0134] In some embodiments, the present disclosure relates to methods of making a hydrogel, comprising one or more of: combining a solution comprising mucin with a reducing agent to form reduced mucin; removing the reducing agent from the solution; and adding a catechol-containing agent and an oxidase (e.g., tyrosinase) to the solution; thereby forming the hydrogel, wherein the hydrogel is crosslinked by the reduced mucin and a catechol-containing agent.

[0135] In some embodiments, an oxidase (e.g., tyrosinase) is added to a solution comprising mucin before the addition of a catechol-containing agent. In some embodiments, an oxidase is added to a solution comprising mucin after the addition of a catechol-containing agent. In some embodiments, an oxidase and a catechol-containing agent are added to a solution comprising mucin simultaneously.

[0136] In some embodiments, the present disclosure relates to methods of making a hydrogel, comprising: combining a solution comprising mucin with a reducing agent to form reduced mucin; removing the reducing agent from the solution; and adding a catechol-containing agent and an oxidase (e.g., tyrosinase) to the solution; thereby forming the hydrogel, wherein the hydrogel is crosslinked by the reduced mucin and a catechol-containing agent.

[0137] In some embodiments, a solution comprising mucin at a concentration of from about 0.1 mg / ml to about 10 mg / ml (e.g., about 0.1 mg / ml to about 10 mg / ml, about 0.5 mg / ml to about 10 mg / ml, about 1 mg / ml to about 10 mg / ml, about 1 mg / ml to about 9 mg / ml, about 1 mg / ml to about 8 mg / ml, about 1 mg / ml to about 7 mg / ml, about 1 mg / ml to about 6 mg / ml, about 1 mg / ml to about 5 mg / ml, etc.) is combined with a reducing agent. In some embodiments, a solution comprising mucin at a concentration of from about 1 mg / ml to about 5 mg / ml is combined with a reducing agent. In some embodiments, the reducing agent has a concentration of from about 0.1 mM to about 100 mM (e.g., about 0.1 mM to about 90 mM, about 0.1 mM to - 32 - 4092104.v284002 about 80 mM, about 0.1 mM to about 70 mM, about 0.1 mM to about 60 mM, about 0.1 mM to about 50 mM, about 0.5 mM to about 50 mM, about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 20 mM, etc.). In some embodiments, the reducing agent has a concentration of about 10 mM.

[0138] In some embodiments, the reducing agent is one or more of: dithiothreitol (DTT), 2- mercaptoethanol, tris(2-carboxyethyl)phosphine (TCEP), mercaptoethane sulfonate (MES), cysteine, glutathione, sodium borohydride, sodium dithionite, 1,4-dithiothreitol, thioglycolic acid, trisodium phosphate, formic acid, ascorbic acid, sodium bisulfite, phenylarsine oxide, thiourea, potassium cyanide, sodium thiosulfate, sodium sulfite, sodium acetate, sodium formate, mercaptoacetic acid, thioacetamide, N-acetylcysteine, thiodiglycol, dimedone, sodium hydrosulfite, dithionite, or thiol-containing peptides. In some embodiments, the reducing agent is dithiothreitol.

[0139] In some embodiments, combining a solution comprising mucin with a reducing agent comprises contacting the mucin with the reducing agent for at least about 0.1 hour (e.g., at least about 0.1 hour, at least about 0.5 hour, at least about 1 hour, etc.). In some embodiments, combining a solution comprising mucin with a reducing agent comprises contacting the mucin with the reducing agent for from about 0.1 hour to about 5 hours (e.g., about 0.5 hour to about 5 hours, about 0.5 hour to about 4 hours, about 0.5 hour to about 3 hours, about 0.5 hour to about 2 hours, etc.). In some embodiments, combining a solution comprising mucin with a reducing agent comprises contacting the mucin with the reducing agent for at least about 0.1 hour at a temperature of from about 20°C to about 25°C. In some embodiments, combining a solution comprising mucin with a reducing agent comprises contacting the mucin with the reducing agent for about 1 hour at a temperature of from about 20°C to about 25°C. In some embodiments, the mucin is contacted with the reducing agent at a pH of about 7.5 to about 8.5. In some embodiments, the mucin is contacted with the reducing agent at a pH of about 8.

[0140] In some embodiments, removing a reducing agent from a solution comprising reduced mucin comprises centrifuging the solution through a filter. In some embodiments, removing a reducing agent from a solution comprising reduced mucin comprises centrifuging the solution through a filter to a reducing agent concentration of no greater than about 10 μM (e.g., - 33 - 4092104.v284002 about 9 μM, about 8 μM, about 7 μM, about 6 μM, about 5 μM¸ about 4 μM, about 3 μM, about 2 μM, about 1 μM, etc.).

[0141] In some embodiments, methods of the present disclosure further comprises adding an oxidizing agent (e.g., oxygen gas (O₂), hydrogen peroxide (H₂O₂), peracetic acid, nitrogen dioxide (NO₂), organic peroxides (e.g., tert-butyl hydroperoxide), etc.) to the solution prior to adding an oxidase (e.g., tyrosinase) to the solution. In some embodiments, methods of the present disclosure further comprises adding oxygen gas to the solution prior to adding the oxidase to the solution.

[0142] In some embodiments, the catechol-containing agent is present in an amount of about 0.1 wt% to about 20 wt%, about 0.1 wt% to about 15 wt%, about 0.1 wt% to about 10 wt%, about 0.1 wt% to about 9 wt%, about 0.1 wt% to about 8 wt%, about 0.1 wt% to about 7 wt%, about 0.1 wt% to about 6 wt%, about 0.1 wt% to about 5 wt%, about 0.5 wt% to about 5 wt%, about 0.5 wt% to about 4 wt%, about 0.5 wt% to about 3 wt%, about 0.5 wt% to about 2 wt%, about 0.5 wt% to about 1.5 wt%, about 0.5 wt% to about 1 wt%, or about 1 wt% to about 1.5 wt%. In some embodiments, the catechol-containing agent is present in an amount of about 0.5 wt% to about 5 wt%. In some embodiments, the catechol-containing agent is present in an amount of about 1 wt%. In certain embodiments, PEG-8C is present in an amount of about 1 wt% in the hydrogel of the present disclosure. In some embodiments, the thiol-containing agent is present in an amount of about 1 wt% to about 10 wt%, and the catechol-containing agent is present in an amount of about 0.5 wt% to about 5 wt%.

[0143] In some embodiments, the catechol-containing agent is a catecholic polymer. In some embodiments, the catecholic polymer is a linear or branched polymer. In some embodiments, the catecholic polymer may have one branch point or multiple branch points. In other embodiments, the catecholic polymer is a star-shaped polymer, e.g., a star-shaped polymer functionalized with a catechol at the terminus of each branch. For non-limiting example, the catecholic polymer contains branches in an integer amount from about 3 to about 100, about 3 to about 90, about 3 to about 80, about 3 to about 70, about 3 to about 60, about 3 to about 50, about 3 to about 40, about 3 to about 30, 3 to about 20, about 3 to about 15, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, or about 8 to about 10. In some - 34 - 4092104.v284002 embodiments, the number of branches of the catecholic polymer is about 3, about 4, about 6, or about 8.

[0144] In some embodiments, the catecholic polymer is a polymer of formula (I): , wherein:R is a polyol core; n is an integer from about 1 to about 1000; and A is an integer from about 3 to about 100.

[0145] For non-limiting example, in formula (I), n is an integer from about 1 to about 10000, about 1 to about 5000, about 1 to about 4000, about 1 to about 3000, about 1 to about 2000, about 1 to about 1000, or about 1 to about 500. For non-limiting example, A is an integer from about 3 to about 100, about 3 to about 90, about 3 to about 80, about 3 to about 70, about 3 to about 60, about 3 to about 50, about 3 to about 40, about 3 to about 30, about 3 to about 25, about 3 to about 20, about 3 to about 15, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, or about 8 to about 10. In some embodiments, A is about 3, about 4, about 6, about 8, about 30, or about 100. In some embodiments, A is less than or equal to about 100. In other embodiments, A is less than or equal to about 30.

[0146] In some embodiments, the branched polymer is , wherein n is an integer, andOther aspects

[0147] In some embodiments, further (additional) agents may be added to the pre- formulation of the hydrogel. For non-limiting example, agents may be added to a pre- formulation comprising the catechol-containing agent and thiol-containing agent, in order to - 35 - 4092104.v284002 increase the viscosity of the pre-formulation, preventing the pre-formulation from spreading and allowing a pre-formulation to stay at a target site. Such agents include, but are not limited to, acacia, agar, alginic acid, bentonite, carbomers, carboxymethylcellulose calcium, carboxymethylcellulose sodium, carrageenan, ceratonia, cetostearyl alcohol, chitosan, colloidal silicon dioxide, cyclomethicone, ethylcellulose, gelatin, glycerin, glyceryl behenate, guar gum, hectorite, hydrogenated vegetable oil type I, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, hypromellose, magnesium aluminum silicate, maltodextrin, methylcellulose, polydextrose, polyethylene glycol, poly(methylvinyl ether / maleic anhydride), polyvinyl acetate phthalate, polyvinyl alcohol, potassium chloride, povidone, propylene glycol alginate, saponite, sodium alginate, sodium chloride, stearyl alcohol, sucrose, sulfobutylether β-cyclodextrin, tragacanth, xanthan gum, and derivatives and mixtures thereof.

[0148] Thickening agents, surfactants, antioxidants, light resistance stabilizers, defoaming agents, plasticizers, pigments, and other such coloring agents may also be added as needed to alter the process suitability and properties of the hydrogel. Other aqueous dispersions, for example, vinyl acetate-based, ethylene-vinyl acetate-based, acrylic, acrylic-styrene-based, and other such emulsions; styrene-butadiene-based, acrylonitrile-butadiene-based, acrylic-butadiene- based, and other such latexes; polyethylene-based, polyolefin-based, and other such ionomers; polyurethane, polyester, polyamide, epoxy-based resins, and the like may also be added.

[0149] In other embodiments, the hydrogel may be dissolvable or nondissolvable. The hydrogel of the present disclosure can be customized in terms of physicochemical properties including, for non-limiting example, gelation time, hydrogel stability (time taken to degrade), mechanical strength and water content. In addition, the physicochemical properties of the hydrogel may be controlled with the molecular weight of the agent to be used.

[0150] In some embodiments, the hydrogel of the present disclosure provides multifunctional properties. In some applications, antifouling properties and tissue adhesion are desirable in combination, such as for surgical adhesives that prevent infection, suggesting catechol–thiol crosslinking as a strategy for multifunctional materials. - 36 - 4092104.v284002 DEFINITIONS

[0151] It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0152] Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present disclosure, example materials and methods are described herein.

[0153] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0154] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0155] Unless the context requires otherwise, throughout the specification and claims that follow, the word “comprise” and synonyms and variants thereof such as “have” and “include”, as well as variations thereof, such as “comprises” and “comprising”, are to be construed in an open, inclusive sense, e.g., “including, but not limited to.” The transitional terms “comprising,” “consisting essentially of,” and “consisting of” are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of” excludes any element - 37 - 4092104.v284002 or step not specified in the claim; and (iii) “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention and disclosure. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of” and “consisting essentially of.”

[0156] “About” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the disclosure, claims, result or embodiment, “about” means within one standard deviation per the practice in the art, or can mean a range of ± 20%, ± 10%, ± 5%, ±4, ±3, ±2 or ± 1% of a given value. It is to be understood that the term “about” can precede any particular value specified herein, except for particular values used in the Examples.

[0157] All percents are intended to be weight percent unless otherwise specified. The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, other various embodiments of and modifications to the present disclosure, in addition to those described herein, will be apparent to those of ordinary skill in the art from the foregoing description and accompanying drawings. Thus, such other embodiments and modifications are intended to fall within the scope of the present disclosure. Further, although the present disclosure has been described herein in the context of a particular implementation in a particular environment for a particular purpose, those of ordinary skill in the art will recognize that its usefulness is not limited thereto and that the present disclosure may be beneficially implemented in any number of environments for any number of purposes. Accordingly, the claims set forth below should be construed in view of the full breadth and spirit of the present disclosure as described herein.

[0158] “PEG” or “polyethylene glycol” as used herein, is meant to encompass any water- soluble polyethylene oxide. Typically, PEGs for use in the present invention will comprise one of the two following structures: “—(CH2CH2O)n—” or “—(CH2CH2O)n-1CH2CH2—,” depending upon whether or not the terminal oxygen(s) has been displaced, e.g., during a synthetic transformation, or, e.g., the identity of adjacent functional groups. The variable (n) - 38 - 4092104.v284002 may, for non-limiting example, range from 1 to about 5000, and the terminal groups and architecture of the overall PEG may vary.

[0159] “Molecular mass” in the context of a polymer, refers to the nominal average molecular mass of a polymer, typically determined by size exclusion chromatography, light scattering techniques, or intrinsic viscosity determination in water or organic solvents. Molecular weight in the context of a polymer can be expressed as either a number-average molecular weight or a weight-average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the number-average molecular weight. Both molecular weight determinations, number-average and weight-average, can be measured using gel permeation chromatographic techniques. Other methods for measuring molecular weight values can also be used, such as the use of end-group analysis or the measurement of colligative properties (e.g., freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number- average molecular weight or the use of light scattering techniques, ultracentrifugation or viscometry to determine weight-average molecular weight.

[0160] As used herein, “treat”, “treating” or “treatment” means inhibiting or relieving a disease or disorder, including a wound or a tumor. For example, treatment can include a postponement of development of the symptoms associated with disease or disorder, and / or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated.

[0161] As used herein, “subject” refers to a mammal (e.g., human, non-human primate, cow, sheep, goat, horse, swine, dog, cat, rabbit, guinea pig, rat, mouse). In a particular embodiment, the subject is a human. A “subject in need thereof” refers to a subject (e.g., patient) who has, or is at risk for developing, a disease or condition that can be treated (e.g., improved, ameliorated, prevented). - 39 - 4092104.v2EXAMPLES

[0162] It is understood that the skilled artisan may modify any of the examples, protocols and procedures in order to generate and / or test suitable compounds as described herein. Example 1: Preparation of thiol-containing and catechol-containing agents

[0163] Tyrosinase (6,540 units / mg, Sigma Aldrich) was prepared at 1.6 mg / ml in 40 mM phosphate buffer, pH 7, 20% glycerol, flash frozen and stored in aliquots at -80 °C.8-arm polyethylene glycol (20 kDa, tripentaerythritol core) functionalized with succinimidyl glutarate (PEG-8SG) was obtained from JenKem Technology USA. Catechol groups were appended to each arm by a published procedure (see Scheme 1).42Briefly, solid dopamine HCl and solid 8- PEG-8SG were combined in a 2:1 molar ratio of dopamine:succinimidyl glutarate under argon or nitrogen gas and then dissolved in DMF. TEA was added in a 2:1 molar ratio of TEA:SG. Reaction was kept at 55 °C overnight. Product was extracted with chloroform, which was then dried with Na2SO4. Solution was concentration by rotary evaporation, precipitated in diethyl ether, dried under vacuum, re-dissolved in water, and then flash frozen and lyophilized, yielding a white powder which was stored at -20 °C. An average catechol content of 5.8 catechols / molecule was measured by absorbance at 280 nm, in good agreement with the value of 6.1 catechols / molecule from 1H NMR (500MHz; CDCl3): δ = 6.7–6.8 (3H, catechol group); 3.4–3.7 (4H, PEG backbone) ppm (FIG.1C). Scheme 1. Preparation of PEG-8C - 40 - 4092104.v2, n is an integer.

[0164] The coupling procedure between PEG-SG and dopamine is as follows: For every 1g of 8-arm PEG-SG with a molecular mass of 20 kDa, add 152 mg of dopamine HCl. 1. Argon / vacuum cycling. Cycle 3 times. 2. Open flask, add solid reactants (PEG-SG and Dopamine HCl) and stirring bar, while flowing argon, and close flask again. 3. When collecting DMF and TEA, add an equal volume of argon gas. 4. Dissolve solid in enough DMF – about 12 ml per gram of polymer. 5. Add TEA – about 111 μL per gram of polymer (2 mol equivalent to PEG-SG). 6. Move flask into silicon oil bath, heat to 55°C in an oil bath on a hot plate. 7. Turn off vacuum and argon. 8. Allow reaction to proceed overnight.

[0165] Purification – Filtration. 1. Pour product into separatory funnel, which is washed carefully and is kept in a giant beaker as a placeholder, without getting the stirring bar mixed in. 2. Prepare 2 beakers (one for water and one for chloroform) of about 300 mL each. 3. Rinse flask with chloroform and pour it into separatory funnel. 4. Add water to the funnel, mix, add chloroform to the funnel, and shake vigorously. - 41 - 4092104.v284002 5. Extract the layer containing chloroform and product carefully. 6. Repeat mix of water and chloroform several times until chloroform layer is clear.

[0166] Purification - Drying. 1. Add Na2SO4 into the flask. 2. Decant the solution into rotary evaporator round flask. 3. Use rotary evaporator to remove chloroform. 4. Collect product and pour it into centrifuge tubes. Add diethyl ether to the tubes and place in -20°C freezer for a few hours. 5. Centrifuge and remove solvent. 6. Vacuum dry the rest under the hood.

[0167] Synthesis of 4-arm PEG-thiol (PEG-4SH, 10 kDa, 3.7 thiols / molecule) is described as follows.

[0168] Materials and Methods: Diethyl ether, 100% (VWR chemicals), potassium hydroxide, 99.5%, and dichloromethane (DCM), 99% (Fischer Scientific), 4-arm PEG 10 kDa (JenKem Technology USA Inc.), tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (TCI Deutschland GmbH). Other chemicals were purchased from Merck KGaA, Darmstadt, Germany and used without purification. NMR spectra (1H and13C) were measured at 300 K using a Jeol Eclipse 500 MHz (Tokyo, Japan) or a Bruker AVANCE III 700 MHz spectrometer (Billerica, MA, USA). Chemical shifts δ were reported in ppm with the deuterated solvent peak used as a standard. Thiol content of PEG-4SH was verified using the Ellman’s test, with absorbance measured on a Tecan Spark microplate reader.

[0169] Synthesis of 4-arm PEG mesylate.

[0170] 10 kDa dried 4-arm PEG OH (FIG.1D, 7 g, 0.7 mmol, 1 eq.) was dissolved in anhydrous dichloromethane (DCM, 50 mL) and combined with TEA (0.97 mL, 7 mmol, 10 eq.) in a dry reaction flask. The mixture was cooled in an ice bath, and methanesulfonyl chloride (0.43 mL, 5.6 mmol, 8 eq.) was added by dropwise addition. The reaction was run for 1 day. The resulting crude product was washed three times with brine. with the organic phase was dried over Na2SO4, and the solution was then concentrated by rotary evaporation. The concentrated product was precipitated in cooled diethyl ether, collected, and dried overnight under a vacuum. The - 42 - 4092104.v284002 precipitate product was obtained as a white powder with an 85% isolated yield.1H NMR (500 MHz, CDCl3, δ (ppm)): 3.08 (3H, s), 3.40 - 3.78 (m), 4.37-4.38 (2H, t) (FIG.2).

[0171] Synthesis of 4-arm PEG thiol (PEG-4SH). Scheme 2. Synthesis of 4-arm PEG-thiol O SH

[0172] 4-arm PEG mesylate (4.33 g, 0.43 mmol, 1 eq.) and thiourea (0.66 g, 8.7 mmol, 20 eq.) were combined with 1-propanol (10 mL) in a reaction flask. The reaction was conducted at 80 °C (sealed by septum with pressure released by a needle) for 1 d to obtain 4-arm PEG isothiouronium intermediate. After removing 1-propanol, KOH (0.024 g, 0.43 mmol, 4 eq.) and water (40 mL) were added to the reaction flask and the solution was then heated to 80 °C for 1 d. Afterward, TCEP (0.5 g, 1.7 mmol, 4 eq.) was added to the crude mixture which was stirred for 2 h. To purify the product, the crude mixture was saturated with NaCl by addition of solid NaCl. The product was then extracted with DCM three times, and water was removed with Na2SO4. The DCM layer was concentrated and them precipitated in cooled diethyl ether. Dried 4-arm PEG thiol as a pale yellowish powder was obtained with a 90% isolated yield.1H NMR (700 MHz, CDCl3, δ (ppm)): 1.58-1.60 (1H, J = 8.2 Hz, t), 2.68-2.71 (2H, J = 6.4 and 8.1 Hz, q), 3.41 - 3.74 (m) (FIG.3). The thiol content of 4-arm PEG thiol was characterized by the Ellman’s test using a cysteine standard calibration curve, which showed an average of 3.7 thiol groups per PEG-4SH molecule.

[0173] Synthesis of 8-arm PEG-catechol (PEG-8C).

[0174] 8-arm polyethylene glycol (20 kDa, tripentaerythritol core) functionalized with succinimidyl glutarate (PEG-8SG) was obtained from JenKem Technology USA. Catechol groups were appended to each arm by a published procedure.42Briefly, solid dopamine HCl and solid 8-PEG-8SG were combined in a 2:1 molar ratio of dopamine:succinimidyl glutarate under argon or nitrogen gas and then dissolved in DMF. TEA was added in a 2:1 molar ratio of - 43 - 4092104.v284002 TEA:SG. Reaction was kept at 55 °C overnight. Product was extracted with chloroform, which was then dried with Na2SO4. Solution was concentration by rotary evaporation, precipitated in diethyl ether, dried under vacuum, re-dissolved in water, and then flash frozen and lyophilized, yielding a white powder which was stored at -20 °C. An average catechol content of 5.8 catechols / molecule was measured by absorbance at 280 nm, in good agreement with the value of 6.1 catechols / molecule from 1H NMR (500MHz; CDCl3): δ = 6.7–6.8 (3H, catechol group); 3.4–3.7 (4H, PEG backbone) ppm (FIG.1C).

[0175] Preparation of mucin.

[0176] Mucin proteins were obtained from porcine intestines and stomachs (largely MUC2 and MUC5AC, respectively43) following established protocols,44lyophilized, and stored at -20 °C until use. Lyophilized mucin was solubilized at the desired concentration in ultrapure water, shaking at 4 °C overnight. Commercial bovine submaxillary mucin (BSM, Sigma-Aldrich) was solubilized and used without further purification. To reduce thiol group in mucin, mucin at 1–5 mg / ml were incubated for 1h at room temperature in 10 mM dithiothreitol (DTT), 200 mM ammonium bicarbonate buffer, pH 8. Excess DTT was then removed by centrifugal filtration (Amicon Ultra, Millipore, 100 kDa cutoff, 2 mL maximum volume) to a residual DTT concentration of ~1 μM. After filtration, the concentrated mucin solution was brought to 20 or 40 mg / ml by the addition of ultrapure water. To alkylate thiol groups, iodoacetamide was added at 30–50 mM after reduction but before filtration. The solution was incubated in the dark for 1h, and then filtered and aliquoted according to the same procedure used for reduced mucin. As- purified mucin was similarly washed by centrifugation. Aliquots of the mucin preparations were flash frozen and stored at -80 °C. The Ellman’s test confirmed the presence of free thiols in reduced mucin. Assuming 50–80 % mucin glycosylation yielded 100–400 thiols / molecule, consistent with the primary structure of MUC2 (215 thiols / molecule).34Free thiols were not measurable in mucin before reduction and after alkylation. Example 2: Preparation and characterization of hydrogels

[0177] Hydrogel precursor solutions were prepared by combining stock solutions of polymers (multi-arm PEG, mucin) and buffer (final buffer conditions 50 mM phosphate, pH 7). The polymer solution was bubbled briefly with oxygen gas produced by the decomposition of - 44 - 4092104.v284002 hydrogen peroxide by catalase. Catechols were oxidized by tyrosinase (0.16–0.64 mg / ml), periodate (1:1 catechol: periodate), or auto-oxidation (pH 9, 50 mM carbonate buffer). For enzymatic oxidation or auto-oxidation, the polymer solution was briefly bubbled with oxygen gas produced by catalase-induced decomposition of hydrogen peroxide. For control measurements of PEG-4SH gels, thiols were oxidized to disulfide bonds by hydrogen peroxide (0.4–4 H2O2:thiol).

[0178] Gelation was measured using a strain-controlled rheometer (ARES-G2, TA Instruments, New Castle, DE) in a stainless steel parallel plate geometry (diameter d, separation distance h0). Two sizes of plates were used: d = 8 mm (h0= 200 μm) and d = 25 mm, h0= 250 μm. In each gelation experiment, gel precursor solution was injected between the plates, excess solution was trimmed, and mineral oil was placed along the rim to prevent evaporation. The storage and loss moduli Gʹ(ω, t) and Gʺ(ω, t) were measured as functions of time t by small- amplitude oscillatory shear at a fixed frequency and amplitude (ω = 5 rad / s, γ0= 0.1, 10% strain), with minimum measurable moduli of 1 Pa (d = 8 mm) and 0.03 Pa (d = 25 mm).45The gelation time tgel was defined as the time at which the storage modulus exceeded 1 Pa. Themutation time λ was calculated as 1 / ^^ = ^^ ln ^^ᇱ / ^^^^, where the minimum mutation time λminduring gelation corresponded to the maximum relative growth rate of Gʹ. In some cases, the storage modulus did not reach a plateau during the time frame of the experiments. The storage modulus Gλ corresponding to λ = 3 h was reported. The final storage modulus measured in a gelation experiment was named Gf.

[0179] Immediately after gelation, adhesion of the gels to the steel rheometer plates wasmeasured by separating the plates at a constant velocity (ℎ^ = 100 μm / s) while measuring thenormal force F and separation distance h, used to calculate the extensional engineering stress^^ா = 4^^ / ^^^^ଶ and engineering strain ^^ = (ℎ − ℎ^) / ℎ^. The maximum stress measured duringseparation was named the failure stress σf, and occurred at the failure strain εf. The area under the stress–strain curve gave the debonding work normalized by the initial gel volume W / V, a measure of the energy dissipation for tack-style tests.46To measure adhesion of the gels to tissue, circular sections of pig skin (8 mm diameter) were attached to the rheometer plates with cyanoacrylate glue (Krazy Glue). Control gels (4% PEG-4SH, 4:1 H2O2:thiol) were cured for at least 5 minutes before measuring adhesion. Hydrogel precursor solution was injected into the gap - 45 - 4092104.v284002 between the pigskin surfaces (~ 1 mm). Several minutes after injection, the sample area was flooded with water to prevent dehydration. Example 3: Tube gelation experiments

[0180] To investigate the crosslinking of thiolated polymers by catechol–thiol bonding, reaction conditions enabling gelation of a model thiolated polymer, 4-arm polyethylene glycol- thiol (PEG-4SH), crosslinked by 8-arm PEG-catechol (PEG-8C) (see FIGs.1A-B), were identified.

[0181] To identify polymer concentrations resulting in gelation, and to confirm that gelation resulted from crosslinking between catechols and thiols, a series of tube gelation experiments were performed. In each experiment, gel precursor solutions were combined in microcentrifuge tubes. Gelation was indicated by stability of the material in response to flicking the tube. This method yields the approximate time at which the hydrogel storage modulus exceeds ~1 Pa. The following experiments were performed.

[0182] 1. Combining catecholic polymers, PEG-4SH, and tyrosinase over a range of polymer concentrations showed gelation at concentrations as low as 0.5% PEG-8C / 0.25% PEG-4SH, whereas individual polymers in oxidizing conditions required higher concentrations to form gels (2% PEG-8C, or 2% PEG-4SH) (FIG.4A, 4C).

[0183] 2. When combinations of PEG-8C and PEG-4SH were oxidized with periodate rather than tyrosinase, gelation did not occur below 2 wt% total polymer (1% PEG-8C, 1% PEG-4SH) (FIG.4A, 4D). Upon exposure to periodate, thiols are expected to oxidize to disulfides, thereby discouraging catechol–thiol crosslinking. This result suggests that selective oxidation of catechols while avoiding thiol oxidation promotes catechol–thiol bonding.

[0184] 3. To rule out contributions from disulfide bonding, a solution of 16% PEG-4SH at the experimental pH 7 was prepared. Gelation was observed after 1 month, whereas periodate oxidation (1:2 periodate:thiol) resulted in immediate gelation of PEG-4SH (FIG.4E), suggesting that auto-oxidation of PEG-4SH is too slow for disulfide bonding to contribute to gelation in the experiments. Moreover, reduced, mucin-inspired polymers at concentrations as high as 2.8% showed no gelation in harsh (periodate) or mild (hydrogen peroxide) oxidizing conditions. - 46 - 4092104.v284002

[0185] 4. To confirm that monofunctional thiols or catechols would not contribute to a load- bearing polymer network, combining PEG-4SH with monofunctional catechol (PEG-1C) and tyrosinase did not result in gelation (FIG.4F). Furthermore, the rapid (within minutes) gelation of 4% PEG-8C and tyrosinase was delayed or prevented by the addition of N-acetyl cysteine (NAC), a monofunctional thiol (FIG.4G). 5. To test whether the presence of polymers could induce gelation of 1% PEG-8C by a crowding effect, control experiments of 1% PEG-8C, 0.16 mg / ml tyrosinase, and the following polymers at 2.8%: polyethylene glycol (MW = 35 kDa), and carboxymethylcellulose (MW = 90 kDa, DS = 0.7) were prepared. The solutions showed no observable gelation over 12 hours. UV-Vis Absorbance Spectroscopy

[0186] UV-vis absorbance spectroscopy was conducted with a Nanodrop One spectrophotometer (Thermo Fisher Scientific). FIG.5A shows the absorbance of individual components, including 1 and 2% PEG-8C, 2% PEG-4SH, and 0.16 mg / ml tyrosinase, with PEG- 8C showing the expected maximum at 280 nm typical of the catechol functionality. FIG.5B shows the absorbance of 1% PEG-8C and 0.16 mg / ml tyrosinase at two times: 1 min after adding tyrosinase, and 5 h after adding tyrosinase. The plots show the development of a shoulder above 300 nm that increases from 1 min to 5 h, which is attributed to the complex products of catechol oxidation and subsequent crosslinking. Addition of 2% PEG-4SH to 1% PEG-8C and 0.16 mg / ml tyrosinase greatly reduces the shoulder in the absorption curve (FIG.5C). FIG.5D-E show similar behavior for an increased concentration of PEG-8C (2%). Because 2% PEG-8C and tyrosinase formed a gel after approximately 20 min, the absorbance is measured at 1 and 10 min. The suppression of the absorbance shoulder above 300 nm upon addition of PEG-4SH to PEG- 8C and tyrosinase suggests that PEG-4SH prevents crosslinking between catechol functionalities. The results are consistent with the formation of catechol–thiol crosslinks, which are known to absorb at a similar wavelength to catechol. For example, various forms of cysteinyldopa show maximum absorbance values between 290-300 nm.47Raman Spectroscopy

[0187] Raman spectroscopy was performed with a WITec alpha300 apyron Confocal Raman (Oxford Instruments) with 532 (1 mW) and 785 nm (80 mW) excitation lasers, recording 10 x 10 - 47 - 4092104.v284002 s integration. Two aspects of the Raman intensity profiles shown in FIGs.6A-B support the formation of catechol–thiol bonds.

[0188] 1. FIG.6A shows a peak at 670 cm-1for PEG-4SH, consistent with -SH deformation.90As expected, the peak disappeared when PEG-4SH was treated with hydrogen peroxide (H2O2) to induce disulfide formation. The peak was enhanced when PEG-4SH was combined with tyrosinase, likely a result of the glycerol in the tyrosinase stock solutions (final glycerol concentration 2%), which shows a Raman peak at 675 cm-1according to a database of Raman spectra.91Consistent with this interpretation, PEG-8C showed no peak at 670 cm-1, whereas PEG-8C and tyrosinase showed a slight peak. The combination of PEG-8C, PEG-4SH, and tyrosinase also showed a slight peak at 670 cm-1of similar size to the peak seen for PEG-8C and tyrosinase, and smaller than the peak seen for PEG-4SH and tyrosinase, consistent with the formation of catechol–thiol bonds that consume the available free thiols. A peak was also observed near 2570 cm-1, attributed to -SH stretching,17and absent in PEG-4SH treated with hydrogen peroxide, or PEG-8C alone (FIG.6B). However, background fluorescence of tyrosinase prevented tracking of this peak as an indicator of catechol–thiol bonds.

[0189] 2. FIG.6B shows prominent background fluorescence for PEG-8C and tyrosinase excited with a 532 nm laser, presumably due to crosslinked oxidation products of catechols. Upon addition of PEG-4SH to PEG-8C and tyrosinase, much less background signal was observed, consistent with the reaction of thiols with quinones, preventing further crosslinking. Peaks in the range 1450–1570 cm-1previously attributed to the catechol ring of PEG-catechol41were not resolved, nor were peaks in the range of 640–820 cm-1attributed to catechol–thiol bonding between polymers,41likely due to overlap with the much stronger PEG peaks.

[0190] In summary, catechol–thiol bonding occurs by oxidation of a catechol group to a quinone, followed by Michael addition of a thiol group to the quinone ring.70Three methods of catechol oxidation were evaluated: enzymatic oxidation by tyrosinase,47chemical oxidation by periodate,71and auto-oxidation at pH 9.37For each oxidation method, solutions of PEG-4SH and PEG-8C were combined across a range of concentrations, and crosslinks in the resulting gels were identified (FIGs.4A-G, 5A-E, 6A-B). Specific oxidation of catechol groups by tyrosinase drove rapid gelation, which was attributed to intermolecular catechol–thiol crosslinking. By - 48 - 4092104.v284002 contrast, nonspecific oxidation by periodate or alkalinity slowed or prevented gelation, indicating the formation of intramolecular disulfide and di-catechol bonds that do not contribute to gelation. Gelation of catechol- and thiol-functionalized polymers

[0191] To test whether crosslinking between catechol and thiol functionalities would drive the formation of adhesive hydrogels, the gelation and adhesion of three thiolated polymers were investigated: 4-arm PEG-thiol, and purified mucin proteins, combined with 8-arm polyethylene glycol-catechol (PEG-8C). To selectively oxidize catechols, mushroom tyrosinase, an enzyme previously used to enable catechol–thiol crosslinking47and for biomedical materials,48was used.

[0192] Gelation of a model thiolated polymer, 4-arm PEG-thiol (PEG-4SH), crosslinked by catecholic polymer PEG-8C, was investigated. Because catecholic polymers can form gels by di- catechol bonding,49tube gelation experiments were performed to identify concentrations of PEG- 8C (1%) that did not form gels alone (FIG.4A). PEG-8C was combined with varying concentrations of PEG-4SH in the presence of tyrosinase, and the viscoelastic moduli by small amplitude oscillatory shear rheometry was monitored. Plots of storage modulus Gʹ vs time t are shown for PEG-8C / PEG-4SH in FIG.7A. The loss modulus Gʺ was close to or below the sensitivity limit of the experimental configuration and was omitted for clarity. Without PEG- 4SH, PEG-8C showed weak or unmeasurable gelation (Gʹ < 0.1 Pa). Addition of PEG-4SH to the catecholic crosslinkers resulted in pronounced gelation, with the gelation time tgel, minimum mutation time λmin, and storage modulus Gλ, presented in FIG.7B for PEG-8C (black circles) as a function of the concentration of PEG-4SH. The plots show that gelation occurs most quickly at intermediate concentrations of PEG-4SH, where tgeland λminare minimized. Increasing the concentration of PEG-4SH slows gelation, but yields stiffer gels (higher Gλ).

[0193] The gelation results may be interpreted by considering the expected type, formation rate, and distribution of crosslinks. In the system of catechol- and thiol-functionalized polymers, possible crosslinking interactions include di-catechol, disulfide, or catechol–thiol bonds. The lack of gelation of catecholic polymers alone in FIG.7A-B indicates that di-catechol bonds cannot account for the observed gelation upon addition of PEG-4SH. Moreover, tube gelation experiments show that disulfide bonding occurs too slowly to influence measurements (FIG. - 49 - 4092104.v284002 4A). These results indicate that catechol–thiol crosslinking contributes to the measured gelation, a claim supported by tube gelation experiments.

[0194] In summary, to quantitively evaluate gelation by catechol–thiol crosslinking, solutions of PEG-4SH were combined with PEG-8C in the presence of tyrosinase, and the temporal evolution of the linear viscoelastic storage modulus Gʹ(ω, t) and loss modulus Gʹʹ(ω, t) were tracked by small amplitude oscillatory shear rheometry at a fixed frequency ω = 5 rad / s. FIGs.7A–B shows the evolution of Gʹ as a function of time t for different concentrations of PEG-4SH crosslinked by PEG-8C. The plot shows that Gʹ increases steadily with time as gelation occurred; Gʹʹ remained near the sensitivity limit of the 8 mm diameter rheometer fixture (Gʹʹ ≤ 1 Pa, see FIG.7D) and is omitted for clarity. FIG.7D shows that Gʺ (depicted as hollow circles) remained below the sensitivity limit of the parallel plate test fixture (8 mm diameter). The uncertainty in the value of Gʺ motivated the definition of the gelation timescale as the as the time at which Gʹ first exceeds 1 Pa, rather than the intersection of Gʹ and Gʺ or parallelism of Gʹ and Gʺ when plotted vs oscillation frequency.96Plots of mucin-inspired polymer or native mucin proteins crosslinked by 1% PEG-8C and tyrosinase showed similarly low values of Gʺ. To characterize the dynamics of the gelation process, the gelation timescale tgelis defined as the time at which Gʹ first exceeds 1 Pa, noting that weak gels may not surpass this threshold. A mutation timescale λ(t) is calculated from the relative growth rate of Gʹ through the relationship ^^(^^) = (^^ ln ^^′ / ^^t)−1. As gelation proceeds, the minimum value of the mutation timescale λ* (1 / λmin) occurs when the gelation rate is fastest. The stiffness of the gels was characterized by the storage modulus Gλ measured when the mutation timescale first exceeded 3 h. Gλ is reported rather than the final measured storage modulus Gfto facilitate comparison between experiments in which Gʹ did not reach a plateau. FIG.7B shows the average values of tgel, λ*, and Gλas functions of the molar ratio of thiols to catechols. For each catecholic crosslinker, the gelation dynamics depended systematically on the composition of the hydrogels. Intermediate thiol:catechol ratios resulted in rapid gelation and minimum values of tgeland λ*. Higher thiol:catechol ratios slowed gelation, with tgel and λ* increasing as the thiol:catechol ratio approached 1:1, but ultimately resulted in stiffer gels, with Gλ increasing by up to four orders of magnitude (0.1 Pa to 1 kPa).

[0195] The formation of catechol–thiol crosslinks is consistent with the dependence of gelation dynamics on hydrogel composition. Catechol-thiol crosslinks are known to form more - 50 - 4092104.v284002 quickly than di-catechol crosslinks.50At low ratios of thiol to catechol, individual PEG-4SH molecules will quickly crosslink multiple catecholic polymers, thereby accelerating gelation. Based on the composition-dependent gelation dynamics, the following example crosslinking behavior is proposed, as illustrated in FIG.7C. Without PEG-4SH, the catecholic polymers are sufficiently dilute that no gelation occurs. Upon addition of PEG-4SH at a low thiol:catechol ratio, individual PEG-4SH molecules bind to multiple catecholic polymers and rapidly form a sparsely crosslinked polymer network, resulting in short gelation timescales and weak gels. As the thiol:catechol ratio increases, thiols in unbound PEG-4SH molecules compete for the available catechols and delay the formation of a sample-spanning network. Gelation occurs as remaining catechols and thiols form crosslinks, with a second thiol potentially binding to each catechol ring,51as occurs naturally in melanin52and in the eyes of the alligator gar,51resulting in slower gelation but ultimately greater crosslinking density and gel stiffness. With a mechanistic understanding of gelation by thiol–catechol bonding in hand, the adhesive properties of the hydrogels was next investigated. Example Proposed Gelation Mechanisms

[0196] Based on the results, the following example crosslinking behavior of thiol- and catechol functionalized polymers in the presence of tyrosinase is proposed. With tyrosinase as a specific oxidizer of catechols, disulfide bonds do not form. Because catechol–thiol bonds form more quickly than di-catechol bonds,50thiols quickly consume the available quinones, resulting in gelation primarily due to catechol–thiol crosslinking, with a secondary contribution from di- catechol bonding,49and negligible influence of disulfide bonding. By contrast, in the presence of periodate or alkalinity, both catechols and thiols are expected to oxidize, forming intra- and intermolecular di-catechol and disulfide crosslinks, and potentially intermolecular catechol–thiol crosslinks. Of these, only intermolecular crosslinks contribute to gelation, whereas intramolecular crosslinks form loop defects in the polymer network.92Polymers incapable of forming intramolecular bonds are expected to be less sensitive to the oxidation method. For example, peptides bearing one catechol and one thiol formed intramolecular catechol–thiol bonds in the presence of periodate.71In that case, intramolecular di-catechol and disulfide bonding was impossible, and intramolecular catechol–thiol bonding was presumably limited by steric - 51 - 4092104.v284002 constraints. Mucin proteins contain thiols capable of intramolecular disulfide bonding.93Tyrosinase was chosen as the oxidation method for further study.

[0197] It is proposed that selective catechol oxidation promotes the formation of catechol– thiol crosslinks, increases the ratio of intermolecular to intramolecular crosslinks, and decreases the polymer concentration required for gelation, making tyrosinase a more efficient catechol oxidation method than periodate or alkalinity for achieving gelation of multifunctional catechol- and thiol-functionalized polymers. Example 4: Adhesion of model hydrogels

[0198] In addition to gelation, adhesion is an important property of hydrogels for biomedical applications such as surgical adhesives and coatings for catheters and stents.23,53The stainless- steel plates of the rheometer test fixtures possess a metal oxide passivating layer similar to implant materials, making them useful model surfaces for adhesion studies. Based on the ability of catechols to bind to chromium oxide,54it is hypothesized that the model hydrogels described in FIGs.7A-D would adhere to steel. To quantitatively assess the adhesive properties of the catechol–thiol hydrogels, the normal force F(t) and the separation distance h(t) between the two rheometer plates while separating the plates at a constant velocity (^^ℎ / ^^t = 100 μm / s) were measured. Plots of extensional engineering stress σE(^^E=4^^ / ^^^^2) vs engineering strain ε (^^=(ℎ−ℎ0) / ℎ0) measured immediately after the gelation are shown for PEG-8C / PEG-4SH gels in FIG.8A. The stress–strain curves are characterized by the failure stress σf(FIG.8B) which occurs at strain εf, equal to the largest tensile engineering stress measured during separation, and the debonding work W / V, calculated as the integrated area under the stress–strain curve normalized by the initial gel volume. Like the gelation dynamics, the adhesive properties of the hydrogels depended systematically on the gel composition.FIG.8B summarizes the failure stress and the debonding work vs concentration of PEG-4SH for PEG-8C (black circles). As shown in FIG.8B, the average values of failure stress and debonding work were positively correlated with thiol:catechol ratio, as well as with the final modulus Gf of the gels (FIGs.9B, 9D). The failure stress and the debonding work increased with increasing concentrations of PEG-4SH, demonstrating that hydrogel adhesion can be tuned by composition. The failure stress greatly - 52 - 4092104.v284002 exceeded Gf due to the highly confined geometry, characterized by the ratio of the rheometer plate diameter to the initial hydrogel thickness (d / h0 = 40), see below.

[0199] The experimental configuration involved confined hydrogels (diameter / initial gel thickness ^^^^ / ℎ0=40 for d = 8 mm). Due to the confinement, the initial slope of the tensile stress–strain curve gave the longitudinal modulus M, related to the shear modulus G by97^^=3^^(1+^^2 / 8ℎ02)≈600^^

[0200] This relationship is validated in FIG.9A, where the final storage modulus Gf was much greater than the loss modulus and was independent of oscillation frequency, making it approximately equivalent to the shear modulus G. A consequence of the confinement is that the hydrogels support tensile stresses up to three orders of magnitude higher than their shear modulus with modest deformation (strain ε < 1) before failure. Hydrogel failure can begin by cavitation, in which defects in the gel expand elastically, or by fracture, in which defects grow by crack propagation. The cavitation stress can be modeled as ^^^^av=2^^ / ^^+5^^ / 2, where γ is the surface energy, a is the defect size of the hydrogel, and G is the shear modulus.98According to linear elastic fracture mechanics, the fracture stress can be modeled as ^^^^rac~(^^^^ / ^^)1 / 2, where ^^ is the energy release rate.75Cavitation and fracture are not attempted to be distinguished in the measurements, which remains a significant scientific challenge,98but the large disparity between failure stress and shear modulus of the hydrogels suggests that the defect sizes are sufficiently small to suppress failure at stresses comparable to the shear modulus. Moreover, both models predict a positive correlation between failure stress and shear modulus. Therefore, by demonstrating that catechol–thiol bonds contribute to the gelation (as shown in Example 3), the results also indicate that catechols improve the cohesion of the hydrogels disclosed herein.

[0201] Additional contributions of catechols to adhesion were assessed, beyond the effect due to the hydrogel modulus. FIG.9B plots the failure stress σ vs final storage modulus G for catechol- based gels (FIGs.7A-7D, 8A-B, 11A-D) and gels composed of PEG-4SH alone (FIGs.10A-C), omitting data measured with the 25 mm diameter parallel plate due to the increased influence of viscous effects during separation.99To enable comparison between catechol-based gels and PEG- 4SH gels of different stiffnesses, the data for PEG-4SH is fitted with an empirical equation of theAvrami form100^^^^=^^(1−exp(−^^^^^^))- 53 - 4092104.v284002 shown as a dashed line in FIG.9B. The fit of the PEG-4SH data was used to normalize the measured failure stresses for hydrogels of different compositions, yielding the failure stress ratios shown in FIG.9C. The results show that, for a given modulus, hydrogels composed of PEG-4SH and PEG-8C showed modest enhancements in failure stress relative to hydrogels composed of PEG-4SH alone, whereas hydrogels composed of mucin crosslinked by PEG-8C showed 3 to 6-fold enhancements in failure stress. The fit overpredicts the failure stress of PEG-4SH gels at low moduli, and therefore the true enhancement at low moduli may be higher than reported. The enhancement in failure stress may result from binding of catechol groups to the chromium oxide surface of the stainless steel rheometer test fixtures,54with possible additional contribution from adsorption of mucin, both of which might discourage adhesive failure. However, more experiments, including direct measurement of the failure mode, are needed to test this hypothesis, as well as to explain the increase in debonding work with modulus (FIG.9D).

[0202] To interpret the relationships between hydrogel composition, modulus, and adhesion, possible failure modes of the hydrogels during separation of the surfaces were considered. Failure can occur within the bulk of the gel (cohesive failure) or at the gel–substrate interface (adhesive failure).75As discussed above and in FIGs.9A-D and 10A-C, the contribution of catechol–thiol crosslinks to the gel modulus establishes that catechols strengthen cohesion. Moreover, catechols enhance the hydrogel failure stress by up to 50% relative to control gels composed of PEG-4SH alone, possibly by forming bidentate hydrogen bonds or coordination bonds27at the gel–steel interface that discourage adhesive failure, indicating that the catechols provide multiple contributions to adhesion. Example 5: Mucin-based hydrogels

[0203] Having demonstrated gelation and adhesion of model thiolated polymers, catechol– thiol crosslinking was used to create adhesive hydrogels from mucin proteins. However, catechol–thiol crosslinking requires thiol groups in the reduced state, whereas mucin thiols are known to form disulfide bonds18and native mucins polymerize by disulfide bonding.67To test whether reduction of mucin thiols would enable catechol–thiol crosslinking, mucin, predominantly MUC2 from porcine intestines, was first purified. In-lab purification of mucin is critical to avoid the degradation associated with the harsh processing of commercially available mucins.55The mucin, either as-purified or reduced, was combined with 1% PEG-8C and - 54 - 4092104.v284002 tyrosinase, and gelation and adhesion were measured in the rheometer. FIG.11A plots storage modulus Gʹ vs time t for each mucin preparation during gelation; FIG.11B plots extensional engineering stress σEvs engineering strain ε measured during separation of the surfaces. Average values of final modulus, failure stress, and debonding work are shown in FIG.11C. Sketches of the proposed crosslinking mechanisms for different preparations of mucin are shown in FIG. 11D, emphasizing the dependence of gelation on the availability of mucin thiols for crosslinking. Crosslinking between catechol groups and either amine, imidazole, or other catechol groups is proposed to contribute to gelation regardless of mucin preparation, whereas catechol–thiol crosslinking requires reduced thiol groups, and is prevented by disulfide bonding or thiol alkylation. FIGs.11A-C shows that combining as-purified or reduced mucin with 1% PEG-8C and tyrosinase resulted in the formation of hydrogels with different properties. As-purified mucin and PEG-8C required hours for gelation and adhesion (MUC2), whereas reduced mucin and PEG-8C showed formation in minutes of stiffer, more adhesive gels (MUC2re). Relative to gels composed of as-purified mucin (MUC2), gels composed of reduced mucin (MUC2r) formed more quickly (ten-fold lower values of tgel) and showed five-fold increases in final modulus and failure stress, as well as a slightly higher value of debonding work. The modulus and adhesive properties were further increased by doubling the concentration of reduced mucin (2x MUC2r). Doubling the concentration of reduced mucin further increased the gelation rate, gel stiffness, and adhesion (2x MUC2r). To confirm the involvement of thiols in crosslinking, alkylation of mucin thiols weakened gelation (MUC2a, triangles, FIG.11A), presumably by preventing catechol–thiol bonding. Gelation and adhesion were greatly weakened by alkylation of mucin thiols (FIGs.11A-B, MUC2a) or omission of PEG-8C from mixtures of mucin and tyrosinase (FIGs.12A-B), consistent with the role of catechols in crosslinking.. Although the gelation results indicate that reduced mucin proteins can be crosslinked by catechol–thiol bonds, the gelation of as-purified and alkylated mucin suggests that PEG-8C can form additional crosslinks with mucin. Catechol–amine, catechol–imidazole, or di-catechol crosslinks likely contribute to gelation, given that lysine, histidine, and tyrosine each constitute ~2% of the mucin primary structure,34and that tyrosine can be oxidized to Dopa by tyrosinase.26- 55 - 4092104.v284002 Table 1. Possible crosslinks and final storage modulus Gf for various hydrogels. Where present, the concentrations of each component were 1.4% mucin (as-purified, reduced, or alkylated), 1% PEG-8C, and 0.16 mg / ml tyrosinase enzyme. Catechol–X indicates crosslinks between catechols and either amines, imidazoles, or other catechols. Assuming proportionality between the modulus and the crosslinking density as given by the affine network model of polymer gelation,77it is estimated that mucin–mucin and catechol–X crosslinks each account for about 10% of the modulus measured for MUC2r, PEG-8C, and tyrosinase, with the remaining 80% attributed to catechol–thiol bonds. Hydrogel Possible Final Modulus Gf[Pa] Composition Crosslinks

[0204] Based on the moduli of the hydrogels shown in Table 1, and the proportionality between modulus and crosslinking density predicted by the affine network model of polymer gelation,77it is estimated that catechol–thiol bonds account for approximately 80% of the crosslinks in gels composed of 1.4% reduced MUC2 and 1% PEG-8C, with other types of bonds accounting for the remainder of the crosslinks. Sketches of the proposed example crosslinking mechanisms for different preparations of mucin are shown in FIG.11D, emphasizing the dependence of gelation on the availability of mucin thiols for crosslinking.

[0205] The generality of the crosslinking approach was tested using mucin purified from porcine stomachs (mainly MUC5AC, also a major component of pulmonary mucus), noting that mucins from different biological niches can exhibit different physical and biochemical properties.7Combining reduced MUC5AC with PEG-8C and tyrosinase resulted in gelation in - 56 - 4092104.v284002 minutes (FIG.12A), albeit to a lower final modulus than the values measured for reduced MUC2 and PEG-8C (FIGs.11A, C). By contrast, commercial bovine submaxillary mucin (BSM) (Sigma) was not observed to form gels under the same crosslinking conditions (FIGs.12A-B), highlighting the importance of in-lab mucin purification to avoid the harsh processing conditions that degrade commercial mucin (43). Example 6: Antifouling properties of mucin-based polymer-based hydrogel

[0206] In addition to tissue adhesion, the materials design strategy disclosed herein offers the possibility of reproducing the bioactive properties of mucus such as the ability to discourage biofouling.23,24An advantage of mucin-based materials is the potential to recapitulate the bioactive properties of native mucins. For non-limiting example, the ability of mucin MUC5AC to dismantle existing biofilms57is desirable for antifouling coatings to reduce infections related to medical devices.58

[0207] To assess anti-biofouling properties of the hydrogels, an overnight culture of Pseudomonas aeruginosa WT strain PAO1, carrying pBBR1(MCS5)-pLac-GFP for constitutive green fluorescent protein (GFP) expression,57was prepared in Luria Broth (LB, Difco) supplemented with 30 μg / mL gentamicin to maintain the plasmid, with shaking at 37 °C. Wells of a glass-bottom 96-well plate (Celvis) were coated with 20 μL of 1% PEG-8C, 0.16 mg / ml tyrosinase, and a reduced polymer, either MUC2 (0.7%), MUC5AC (1.4%), or BSM (1.4%). Plates were centrifuged at 100 x g for 5 min immediately after addition of the hydrogel precursor solution to obtain a uniform coating. The plate was soaked in water for 20 min, followed by removal of the water and sterilization by ultraviolet (UV) light radiation for 1h. Overnight cultures were diluted in fresh LB medium to an initial OD600 of 0.01, added to the plate, and incubated for 24 hours at 37 °C. Viable dispersed cells were quantified via counts of the colony forming units (CFU) on LB agar plates. Supernatant containing non-adherent cells was removed from the plate, and the biofilm remaining in each well was washed at least three times with fresh LB. Adhered cells were imaged by confocal microscopy. Image acquisition was performed using a confocal laser scanning microscope (LSM 800; Zeiss) equipped with a 20× / 0.5 NA dry objective with 2x digital zoom at an excitation wavelength of 488 nm. Images were analyzed - 57 - 4092104.v284002 with the Zeiss ZEN 2.1 imaging software (Thornwoods, NY, USA). Biofilm quantification was performed using BiofilmQ.59

[0208] To test the antifouling properties of the materials described herein, glass surfaces were coated with mucin-based hydrogels composed of either MUC2 or MUC5AC, each crosslinked by PEG-8C and tyrosinase. For comparison, coatings composed of commercial bovine submaxillary mucin (BSM) were also tested under the same crosslinking conditions, noting that BSM was not observed to form gels under these conditions (FIGs.12A-B). The coated surfaces were incubated overnight in Pseudomonas aeruginosa, a biofilm-forming pathogen associated with medical device infections,81and the volume of biofilm on the surfaces were then measured by confocal microscopy and calculated with BiofilmQ software.59FIG.13A compares the biofilm volume on bare glass and on glass coated in mucin hydrogel, and shows that the mucin hydrogel greatly reduced biofilm formation. FIG.13B shows that the gel coatings composed of native MUC2 and MUC5AC reduced the median biofilm volume by 97% and 98%, respectively, relative to the biofilm volume measured on bare glass. Lesser reductions in biofilm volume were observed for coatings composed of BSM (88%). The decreases in biofilm volume could not be attributed to toxicity of the coatings, since the coatings do not alter P. aeruginosa viability relative to medium alone (FIGs.14A-B). No reduction in biofilm volume was observed after incubating the surfaces in solutions of native mucin without PEG-8C (FIGs.14C-D), indicating that the PEG-8C enables retention of the mucin on the glass surface. Moreover, the results for BSM indicate that PEG-8C can retain mucin on glass even in the absence of gelation. Nevertheless, the gel coatings composed of native MUC2 and MUC5AC showed superior antifouling performance to the coatings composed of commercial BSM, highlighting the potent bioactivity of native mucin proteins. The results are particularly promising, given that previous mucin-based coatings that repel other bacteria have failed to reduce biofilm formation of P. aeruginosa.24, 82Moreover, existing methods for depositing mucin-based coatings involve multiple steps23or are limited to hydrophobic substrates,16whereas the gel coatings disclosed herein can be applied in a single step, and are expected to be compatible with both hydrophobic and hydrophilic substrates based on the broad adhesive properties of catechols.27The results for mucin proteins are particularly promising, given that existing methods for creating mucin coatings involve multi-step approaches23or rely on adsorption of mucin to specific surfaces,16whereas the method presented here can be applied in a - 58 - 4092104.v284002 single step via various coating methods such as spin, spray, or dip coating on a variety of surfaces. Example 7: Adhesion of mucin-based polymer-based hydrogels

[0209] An advantage of the materials design strategy is the potential to develop multifunctional hydrogels that combine the adhesive properties of catechols28with the tunable properties of mucin-inspired polymers56or the bioactivity of mucins68,78for applications such as tissue adhesives (e.g., surgical adhesives28) and antifouling coatings. To assess the capacity of the materials to bind to tissue, the adhesion of mucin-based hydrogels to moist pigskin surfaces was measured. Hydrogel precursor solutions composed of 1.4% reduced MUC2, 0.16 mg / ml tyrosinase, and either 1% or 2% PEG-8C were deposited between moistened pigskin disks glued to the upper and lower parallel plate test fixtures of the rheometer. After curing the gels for 35 min (FIG.15A), the surfaces were separated axially to measure the adhesion. FIG.15B shows a plot of normal stress σn vs strain ε, with failure stresses σf of 1-2 kPa, within the range previously reported for catechol-based tissue adhesives.60The failure stress and the debonding work increased with increasing concentrations of PEG-8C, indicating the formation of stiffer, more adhesive gels for 2% PEG-8C with reduced mucin (FIG.15B). For both compositions, the hydrogels failed cohesively, indicating that the adhesive performance of mucin-based gels could be improved by strengthening the cohesion of the gels, for example by incorporating energy dissipating crosslinks or hierarchical structures,79as has been achieved for other protein-based hydrogels.80

[0210] By demonstrating adhesion to tissue surfaces in minutes, the results serve as a proof- of-concept for the design of tissue adhesives crosslinked by catechol–thiol bonds. It is noted that antifouling properties and tissue adhesion are desirable in combination, such as for surgical adhesives that prevent infection, suggesting catechol–thiol crosslinking as a strategy for multifunctional materials.

[0211] Adhesion of hydrogels comprising mucin or mucin-inspired polymers to stainless steel was quantified using a commercial rheometer, measuring the normal force while separating the rheometer plates. Adhesion of hydrogels can be modulated by altering hydrogel composition and pre-treatment of mucin or mucin-inspired polymer. In a trial spin coating deposition of a - 59 - 4092104.v284002 mucin-based hydrogel film comprising 1.4% mucin type MUC2, 1% PEG-8C, and 0.16 mg / ml tyrosinase on stainless steel, a hydrogel thickness of 484.3 nm was achieved (FIG.16). Example 8: Lubricity of mucin-based coatings

[0212] Test friction measurements were performed using the following lubricants: HEPES buffer, sigma PGM (a commercially-available degraded pig gastric mucin), PGM (an in-lab purified porcine gastric mucin), PEG (polyethylene glycol control), and Apo PGM (a deglycosylated PGM). FIG.17A demonstrates that PGM provides the smallest coefficient of friction with respect to lubricant entrainment speed, thus indicating that a PGM-coated surface has the highest lubricity among the lubricants tested. FIG.17B presents a schematic of a friction measurement performed with a three ball-on-flat test geometry (FIG.17B is a possible experimental configuration that was not tested).

[0213] Trial friction measurements were performed using a commercial strain-controlled rheometer. An 8 mm plate was brought into contact with a mucin-based hydrogel film comprising 1.4% mucin type MUC2, 1% PEG-8C, and 0.16 mg / ml tyrosinase deposited on a silica substrate. The plate was rotated at a constant angular velocity while maintaining a constant normal load. Estimated friction coefficients were compared to the values measured for contact between silica and steel in purified water. An approximate 3-fold reduction in the friction coefficient was observed (FIG.18). Measurement of Mucin-Based Coating Tribology

[0214] Tribological assessment of mucin-based hydrogels will be performed using a strain- controlled rheometer.63Equipped with a tribology test geometry (FIG.17B), the rheometer provides advanced torque and normal force sensitivity for measuring the coefficient of sliding friction in a thin film. Three point contact fixtures (either a single ball-on-three plates or three balls-on-a flat plate test geometry as shown in the sketch) will be employed. In each configuration, the upper surface may consist of steel, while the lower surface may be mucin- coated metal oxide surfaces. To simulate shaving conditions, the contact region may be flooded with water or aqueous solutions of surfactants relevant to shaving, such as but not limited to - 60 - 4092104.v284002 sodium lauryl sulfate. Additionally, the contact region may be lubricated with lubricants such as but not limited to commercial shaving creams. Friction coefficients will be measured under a constant axial load and across a range of sliding speeds that are relevant to shaving. Manufacturers often apply chemical coatings, such as polyfluoroalkyl substances (PFAS), including PTFE (Teflon), to lubricate and reduce friction. The obtained results will be compared with friction coefficients measured with polytetrafluoroethylene (PTFE) surfaces prepared by dip or spin coating64an aqueous dispersion of PTFE followed by thermal annealing. Regions of boundary, mixed, and hydrodynamic lubrication will be identified. Model Skin-on-Blade Tribological Configuration

[0215] To address the influence of the counter surface on friction measurements, a tribological fixture using poly(dimethylsiloxane) (PDMS) will be constructed in a three- hemisphere-on-flat configuration. PDMS is employed as a tissue substitute in tribology experiments and offers tunable viscoelasticity and hydrophobicity.65PDMS hemispheres will be fabricated using a silicone elastomer kit, casting in molds obtained through various sources (e.g., 3D printing, commercial sources). PDMS hydrophobicity will be tuned by plasma treatment.65Three PDMS hemispheres will be arranged in a three ball-on-plate configuration, similar to the commercial test geometries described earlier. The friction measurements will be conducted against the hydrogel-coated metal oxide surfaces under shaving conditions. Durability Assessment

[0216] To assess durability, the friction coefficient of hydrogel films comprising mucin or mucin-inspired polymers at a constant sliding speed will be measured for long exposure times. Changes in friction coefficient signal degradation of the films. Friction measurements will be conducted over repeated dry / wet cycles by allowing the films to fully dry between repeated measurements. Damage of the films will be confirmed with atomic force microscopy (AFM) and scanning electron microscopy (SEM) imaging. Damage of the films will be correlated to the yield stress of the hydrated mucin films measured in a rheometer and AFM-based nanoscratch tests to identify compositions required for sufficient durability. - 61 - 4092104.v284002 Storage Assessment

[0217] To test the ability of the materials of the present disclosure to retain function after periods of storage, mucin-based hydrogel films will be kept in a desiccator for extended periods of time (up to months), followed by tribological assessment in shaving conditions. In addition, mucin-based hydrogel-coated razors and metal oxide surfaces will be subjected to accelerated aging tests, including exposure to UV radiation, thermal cycling, and humidity variations. Effects of environmental exposure on the coating's chemical composition, interfacial morphology and chemical composition will be analyzed using techniques such as SEM and X-ray photoelectron spectroscopy (XPS). Coated razors and metal oxide surfaces will be exposed to repetitive cycles of hydration and dehydration and track any changes to the coating via SEM, AFM, and profilometry.

[0218] Conclusion: The gelation and adhesion of hydrogels composed of model thiolated polymers and purified mucin proteins, crosslinked by catechol-functionalized crosslinkers, were investigated. Gelation was shown to result from catechol–thiol crosslinking, with gelation dynamics, gel properties, and adhesion tuned by gel composition. The crosslinking strategy disclosed herein enables fast gelation (<1 min) and adhesion to metal oxide and tissue surfaces in physiologically-relevant conditions. REFERENCES

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[0319] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0320] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims. - 71 - 4092104.v2

Claims

84002 CLAIMS What is claimed is:

1. A hydrogel, wherein the hydrogel is crosslinked by a thiol-containing agent and a catechol-containing agent, and wherein the thiol-containing agent is a thiol-containing branched polymer or mucin.

2. The hydrogel of claim 1, wherein the thiol-containing agent is present in an amount of about 0.1 wt% to about 10 wt%.

3. The hydrogel of claim 1 or claim 2, wherein the thiol-containing agent is present in an amount of about 1 wt% to about 3 wt%.

4. The hydrogel of any one of claims 1-3, wherein the thiol-containing agent is a thiol- containing branched polymer.

5. The hydrogel of claim 4, wherein the thiol-containing branched polymer contains branches in an integer amount from about 3 to about 100.

6. The hydrogel of claim 4, wherein the thiol-containing branched polymer is , and wherein n is an integer from about 1 to about 1000.

7. The hydrogel of any one of claims 1-6, wherein the thiol-containing agent is mucin which is a reduced mucin. - 72 - 4092104.v284002 8. The hydrogel of any one of claims 1-7, wherein the thiol-containing agent is a mucin which is present in an amount of about 2.8 wt%.

9. The hydrogel of any one of claims 1-8, wherein the catechol-containing agent is present in an amount of about 0.5 wt% to about 5 wt%.

10. The hydrogel of any one of claims 1-9, wherein the catechol-containing agent is present in an amount of about 1 wt%.

11. The hydrogel of any one of claims 1-10, wherein the catechol-containing agent is a catecholic polymer.

12. The hydrogel of claim 11, wherein the catecholic polymer is a linear or branched polymer.

13. The hydrogel of claim 11 or 12, wherein the catecholic polymer is a branched, star- shaped polymer functionalized with a catechol at a terminus of each branch.

14. The hydrogel of claim 13, wherein the catecholic polymer contains branches in an integer amount from about 3 to about 100.

15. The hydrogel of any one of claims 12 to 14, wherein the branched polymer is a polymer of formula (I): , and wherein:R is a polyol core; n is an integer from about 1 to about 1000; and A is an integer from about 3 to about 100. - 73 - 4092104.v284002 16. The hydrogel of claim 15, wherein the branched polymer is . 17.agent is an oxidation-resistant catechol analog.

18. The hydrogel of any one of claims 1-17, wherein the hydrogel is crosslinked in the presence of an oxidase, optionally wherein the oxidase is tyrosinase.

19. The hydrogel of claim 18, wherein the oxidase is present in an amount of about 0.16 mg / ml to about 0.64 mg / ml.

20. The hydrogel of any one of claims 1-19, further comprising a therapeutic agent.

21. The hydrogel according to claim 20, wherein the therapeutic agent is a peptide drug, a protein drug, an anti-bacterial agent, an anti-cancer agent, an anti-inflammatory agent, an anti-viral agent, an analgesic or anesthetic agent, an immunomodulator, an antifungal agent, a hemostasis agent, or a combination thereof.

22. A method of modulating immune response, comprising administering the hydrogel of any one of claims 1-19 to a subject in need thereof, wherein the hydrogel further comprises an immunostimulant or an immunosuppressant.

23. A bioadhesive, comprising the hydrogel of any one of claims 1-19.

24. The bioadhesive of claim 23, wherein the bioadhesive is a tissue adhesive.

25. The hydrogel of any one of claims 1-19 or the bioadhesive of claim 23 or 24, wherein the thiol-containing agent is present in an amount of about 1 wt% to about 10 wt%, and wherein the catechol-containing agent is present in an amount of about 0.5 wt% to about 5 wt%. - 74 - 4092104.v284002 26. The hydrogel of any one of claims 1-19 or the bioadhesive of claim 25, wherein the catechol-containing agent is PEG-8C in an amount of about 1 wt%.

27. A shaving device comprising the hydrogel of any one of claims 1-19.

28. The shaving device of claim 27, wherein the hydrogel has a thickness of from about 10 nm to about 500 nm, optionally from about 10 nm to about 50 nm.

29. The shaving device of claim 27 or 28, wherein the shaving device is a shaving razor.

30. A cartridge comprising a razor blade, said razor blade comprising the hydrogel of any one of claims 1-19.

31. An antifouling coating, comprising the hydrogel of any one of claims 1-19.

32. A method of reducing or suppressing biofilm formation on an article, the method comprising applying the hydrogel of any one of claims 1-19 to the article.

33. A method of providing an article with a lubricious surface, the method comprising applying the hydrogel of any one of claims 1-19 to the surface.

34. The shaving device of any one of claims 27-29 or the method of claim 33, wherein the surface has a coefficient of friction of from about 0 to about 0.

3.

35. The shaving device of any one of claims 27-29 or the method of claim 33 or 34, wherein the hydrogel has a thickness of from about 10 nm to about 500 nm, optionally from about 10 nm to about 50 nm.

36. A medical device having a lubricious coating on at least a section of the device, the coating comprising the hydrogel of any one of claims 1-21.

37. The medical device of claim 36, wherein the hydrogel has a thickness of from about 10 nm to about 500 nm. - 75 - 4092104.v284002 38. The medical device of claim 36 or 37, wherein the hydrogel has a thickness of from about 10 nm to about 50 nm.

39. A method of treating a wound in a subject in need thereof, said method comprising contacting the wound with the hydrogel of any one of claims 1-21, under conditions to allow the hydrogel to adhere to tissue surrounding the wound.

40. A kit comprising the hydrogel of any one of claims 1-21.

41. A polymer of formula (I): , wherein:n is an integer from about 1 to about 1000; and A is an integer from about 3 to about 100.

42. A method of making a hydrogel, comprising: combining a solution comprising mucin with a reducing agent to form reduced mucin; removing the reducing agent from the solution; and adding a catechol-containing agent and an oxidase to the solution; thereby forming the hydrogel, wherein the hydrogel is crosslinked by the reduced mucin and a catechol-containing agent.

43. The method of claim 42, wherein the solution which is combined with the reducing agent comprises mucin at a concentration of from about 1 mg / ml to about 5 mg / ml. - 76 - 4092104.v284002 44. The method of claim 42 or 43, wherein the solution comprising mucin is combined with a reducing agent at a concentration of about 10 mM.

45. The method of any one of claims 42-44, wherein the reducing agent is dithiothreitol.

46. The method of any one of claims 42-45, wherein the combining comprises contacting the mucin with the reducing agent for at least about 0.1 hour at a temperature of from about 20°C to about 25°C.

47. The method of claim 46, wherein the mucin is contacted with the reducing agent at a pH of about 8.

48. The method of any one of claims 42-47, wherein the removing comprises centrifuging the solution through a filter.

49. The method of any one of claims 42-48, wherein the method further comprises adding oxygen gas to the solution prior to adding the oxidase to the solution.

50. The method of any one of claims 42-49, wherein the catechol-containing agent is present in an amount of about 0.5 wt% to about 5 wt%.

51. The method of any one of claims 42-50, wherein the catechol-containing agent is present in an amount of about 1 wt%.

52. The method of any one of claims 42-51, wherein the catechol-containing agent is a catecholic polymer.

53. The method of claim 52, wherein the catecholic polymer is a linear or branched polymer.

54. The method of claim 53, wherein the catecholic polymer is a branched, star-shaped polymer functionalized with a catechol at a terminus of each branch.

55. The method of claim 54, wherein the branched polymer contains branches in an integer amount from about 3 to about 100. - 77 - 4092104.v284002 56. The method of any one of claims 53 to 55, wherein the branched polymer is a polymer of formula (I): , and wherein:n is an integer from about 1 to about 1000; and A is an integer from about 3 to about 100.

57. The method of claim 56, wherein the branched polymer is . 58.- 78 - 4092104.v2