Hemostatic and sealant compositions

Abstract

The present disclosure describes hemostatic and sealant compositions glycidyl methacrylated gelatin (GelMAG); methacrylated dopamine (DMA); poly(diallyldimethylammonium chloride) (pDDA); and a visible light-activated photoinitiator. The present disclosure also describes methods of sealing a wound in a tissue of a subject. These methods comprise contacting the wound of the subject with a therapeutically effective amount of the hemostatic and sealant hydrogel precursor composition; and photo-crosslinking the hemostatic and sealant hydrogel precursor composition by exposing the hemostatic and sealant hydrogel precursor composition to a visible light, thereby forming a hemostatic and sealant hydrogel. Methods of preparing hemostatic and sealant hydrogels are also described herein.

Description

[0001] Attorney Docket No. 50835-0006W01

[0002] HEMOSTATIC AND SEALANT COMPOSITIONS

[0003] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under EB023052 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] TECHNICAL FIELD

[0006] The present disclosure describes hemostatic compositions comprising photocrosslinkable gelatin-based composite adhesive hydrogels incorporating methacrylated dopamine (DMA) and poly(diallyldimethylammonium chloride) (pDDA). The disclosure also describes methods of sealing a wound in a tissue of a subject in need thereof using the hemostatic compositions and methods of preparing hemostatic hydrogels.

[0007] BACKGROUND

[0008] Trauma is one of the top contributors to the global burden of disease, causing more than five million fatalities per year and depleting hundreds of billions of dollars from the world economy. Even in non-fatal injuries, post-traumatic complications (PTCs) are common and awaken serious concerns including coagulopathy, thromboembolism, infection, sepsis, organ failure, and even stroke. Even though medical response to traumatic injuries has improved, numerous obstacles such as excessive blood loss, bacterial infection, and ineffective wound closure hinder patient survival and physiological recovery.

[0009] Uncontrollable hemorrhage is a leading contributor to preventable mortality after trauma and therefore requires urgent preventative investigation. Hemostasis has traditionally been achieved by compression with gauze or intravenous delivery of blood products, which have their own limitations. For example, gauze provides poor wound treatment due to weak tissue adhesion, high blood adsorption before hemostasis, and fiber shredding into the wound. Secondary bleeding is also common upon clot-filled gauze removal. Even commercial gauzes infused with hemostatic agents such as chitosan (ChitoGauze PRO®), kaolin (QuikClot Combat Gauze®), and zeolites (QuiCover™) have limited tissue adhesion and may cause thrombosis and damage to crucial organs upon extended exposure. Meanwhile, hemostats derived from blood products (e.g., platelets, fibrinogen concentrate) incur risk of thromboembolism and disease transmission. They can also be expensive, have a short shelf life, and involve complex preparation before transfusion. Overall, there are few efficient hemostatic agents currently used to control hemorrhage. Attorney Docket No. 50835-0006W01

[0010] Hemostatic biomaterials such as sprays, sponges, and foams have therefore been developed using natural or synthetic polymers reinforced by blood coagulating agents (e.g., tannic acid, graphene oxide, silica particles). These biomaterials topically conform to injuries and trigger blood coagulation, but most lack the mechanical strength and tissue adhesion required for their clinical applications. Thus, there is an urgent medical need to develop hemostatic biomaterials suited for multi-dimensional wound sealing and repair on elastic organs.

[0011] SUMMARY

[0012] Certain aspects of the present disclosure are directed to a hemostatic and sealant composition comprising: glycidyl methacrylated gelatin (GelMAG); methacrylated dopamine (DMA); poly(diallyldimethylammonium chloride) (pDDA); and a visible light-activated photoinitiator.

[0013] In some embodiments, the DMA is conjugated to a backbone of the GelMAG. In some embodiments, the GelMAG is present at a concentration of 20% (w / v). In some embodiments, the GelMAG is present at a concentration ranging from 15% (w / v) to about 25% (w / v). In some embodiments, the DMA is present at a concentration ranging from about 0.05% (w / v) to about 0.15% (w / v). In some embodiments, the DMA is present at a concentration of about 0.1% (w / v). In some embodiments, the pDDA is present at a concentration ranging from about 0.5% (v / v) to about 25% (v / v). In some embodiments, the pDDA is present at a concentration of about 2% (v / v). In some embodiments, the GelMAG has a degree of methacryloyl substitution of about 50%.

[0014] In some embodiments, the GelMAG has a degree of methacryloyl substitution between about 40% and about 60%. In some embodiments, the photoinitiator comprises Eosin Y, triethanolamine (TEA), N-vinyl-s-caprolactam (VC), or any combination thereof. In some embodiments, the composition is in a form of a solution or a hydrogel. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the hemostatic and sealant composition is formulated for topical use. In some embodiments, the visible light-activated photoinitiator is activated upon exposure of light having a wavelength between about 450 nanometers (nm) to about 550 nm. In some embodiments, the hemostatic and sealant composition is ionically conductive, biocompatible, biodegradable, adhesive, and antibacterial. Attorney Docket No. 50835-0006W01

[0015] In some embodiments, a hemostatic and sealant hydrogel precursor composition comprises any of the previously mentioned hemostatic and sealant compositions. In some embodiments, the disclosure provides a hemostatic and sealant hydrogel formed by photocrosslinking the hemostatic and the aforementioned sealant hydrogel precursor composition.

[0016] In some embodiments, the hemostatic hydrogel has: (i) a Young’s modulus ranging from about 30 kilopascals (kPa) to about 200 kPa; (ii) an ultimate strength ranging from about 60 kPa to about 150 kPa; (iii) a stretchability ranging from about 80% to about 220%; (iv) a toughness of about 20 kilojoules per cubic meter (kJ / m3) to about 105 kJ / m3; (v) a compression modulus of ranging from about 30 kPa to about 80 kPa; (vi) an energy loss of about 10% to about 30%; (vii) an adhesion strength ranging from about 20kPa to about 50 kPa; (viii) an adhesion energy ranging from about 10 joules per cubic meter (J / m3) to about 50 J / m3; (ix) a burst pressure ranging from about 20 kPa to about 60 kPa; (x) a conductivity ranging from about 0.1 Siemens per meter (S / m) to about 1 S / m; or (xi) any combination of (i)-(iv).

[0017] Certain aspects of the present disclosure are directed to a method of sealing a wound in a tissue of a subject, the method comprising contacting the wound of the subject with a therapeutically effective amount of the hemostatic and sealant hydrogel precursor composition of claim 17; and photo-crosslinking the hemostatic hydrogel precursor composition by exposing the hemostatic hydrogel precursor composition to a visible light, thereby forming a hemostatic hydrogel.

[0018] In some aspects, the present disclosure provides a method of preparing a hemostatic and sealant hydrogel using any of the hemostatic and sealant compositions previously mentioned, the method comprising dissolving the GelMAG in a solution comprising the visible light-activated photoinitiator; mixing the DMA with the solution comprising the visible light-activated photoinitiator and the dissolved GelMAG; incubating the solution comprising the visible light-activated photoinitiator, the dissolved GelMAG, and the DMA for at least twenty four hours; mixing the pDDA with the solution comprising the visible light-activated photoinitiator, the dissolved GelMAG, and the DMA; and photo-crosslinking the solution by exposing the solution to a visible light after mixing the pDDA with the solution, thereby forming the hemostatic and sealant hydrogel.

[0019] The terms “subject” or “patient” as used herein refer to any mammal (e.g., a human or a veterinary subject, e.g., a dog, cat, horse, cow, goat, sheep, mouse, rat, or rabbit) to which a composition or method of the present disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. The subject may seek or need Attorney Docket No. 50835-0006W01 treatment, require treatment, is receiving treatment, will receive treatment, or is under care by a trained professional for a particular disease or condition.

[0020] As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0021] As used herein, the expression “pharmaceutically acceptable” applies to a composition which contains composition ingredients that are compatible with other ingredients of the composition as well as physiologically acceptable to the recipient (e.g., a mammal such as a human) without the resulting production of excessive undesirable and unacceptable physiological effects or a deleterious impact on the mammal being administered the pharmaceutical composition. A composition as described herein can comprise one or more carriers, useful excipients, and / or diluents.

[0022] As used herein, the term “hydrogel” refers to a broad class of polymeric materials that may be natural or synthetic, have an affinity for an aqueous medium, and are able to absorb large amounts of the aqueous medium, but which do not normally dissolve in the aqueous medium.

[0023] As used herein, the term “aqueous medium” as used herein refers to water or a solution based primarily on water such as phosphate-buffered saline (PBS), or water containing one or more salts dissolved therein.

[0024] As used herein, the term “photo-crosslink” refers to an interconnection between polymer chains via chemical bonding, such as, but not limited to, covalent bonding, ionic bonding, or affinity interactions that are caused by exposure to a light source. The chemical cross-linking can be carried out by reactions, such as any one of free radical polymerization, condensation polymerization, anionic or cationic polymerization, or step growth polymerization.

[0025] As used herein, the term “biodegradable” refers to a substance which may be broken down by microorganisms, or which spontaneously breaks down over time (e.g., within about 14 days to about 6 months) when exposed to environmental conditions commonly found in nature. For example, the compositions described herein may be degraded by enzymes that are present in the body (e.g., in a tissue environment).

[0026] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood Attorney Docket No. 50835-0006W01 that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Furthermore, the use of the term “about,” as used herein, refers to an amount that is near the stated amount by about 10%, 5%, or 1%, including increments therein. For example, “about” can mean a range including the particular value and ranging from 10% below that particular value and spanning to 10% above that particular value.

[0027] As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[0028] Where values are described in the present disclosure in terms of ranges, endpoints are included. Furthermore, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.

[0029] Other features and advantages of the present disclosure will be apparent from the following detailed description and figures, and from the claims.

[0030] Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur according to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, Attorney Docket No. 50835-0006W01 including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0032] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0033] DESCRIPTION OF DRAWINGS

[0034] FIGs. 1A-1F show the synthesis and characterization of GDP. FIG. 1A is a chemical structure of (i) GelMAG synthesized through a one-step conjugation reaction using glycidyl methacrylate, (ii) DMA synthesized by covalent attachment of methacrylic anhydride, and (iii) pDDA. FIG. IB is a schematic of the hydrogel preparation by physical mixing of GelMAG, DMA, and pDDA in Eosin Y / TEA / VC photoinitiator solution, application of thermosensitive prepolymer solution to elastic organ injury, and in situ polymerization with visible blue light to form GPD hydrogel. FIG. 1C is a graph showing the Zeta potential of GDP prepolymer with varying amounts of pDDA and DMA prepared in Eosin Y / TEA / VC photoinitiator solution. FIG. ID are schematics of various chemical interactions within the crosslinked GDP composite. FIG. IE is anJH NMR analysis of (i) GDP prepolymer and hydrogel depicting aromatic protons of DMA, methyl protons of pDDA, and (ii) methacryloyl protons of GelMAG at 5.70 ppm and 6.08 ppm. FIG. IF is a table showing the degree of crosslinking within hydrogels made of pure GelMAG, GelMAG with 2% (v / v) pDDA (GP), GelMAG with 0.1% (w / v) DMA (GD), and GelMAG with 0.1% (w / v) DMA and 2% (v / v) pDDA (GDP). Data are represented as mean ± SD (*P < 0.05, **P < 0.01, ***P

[0035] < 0.001, ****P < 0.0001, N=4).

[0036] FIGs. 2A-2G are graphs showing the mechanical characterization of GDP adhesive hydrogels. Effect of varying pDDA and DMA concentrations on tensile properties of GDP hydrogels are shown: (FIG. 2A) Young’s modulus, (FIG. 2B) ultimate strength, (FIG. 2C) stretchability with (D) representative images of the optimal GDP adhesive before and after stretching to 210% , and (FIG. 2E) toughness after hydrogels were stretched to failure at a rate of 1 mm / min. Effect of varying pDDA and DMA concentrations on compression properties of GDP hydrogels: (FIG. 2F) compression modulus after hydrogels were compressed at 1 mm / min, and (FIG. 2G) energy loss after 12 cycles of cyclic compression to 50% at a rate of 1 mm / min. Data are represented as mean ± SD (*P < 0.05, **P < 0.01, ***P

[0037] < 0.001, ****P < 0.0001, N=4). Attorney Docket No. 50835-0006W01

[0038] FIGs. 3A-3H show the in vitro adhesion and conductivity characterization of GDP adhesive. FIG. 3A is an illustration of wound closure setup depicting hydrogel linking two pieces of glass-fixed porcine tissue that were pulled apart by universal tester. The effect of varying pDDA and DMA concentrations in GDP hydrogels on (FIG. 3B) adhesion strength and (FIG. 3C) adhesion energy while also compared to commercially available lung sealants CoSeal™ and Progel™ is shown. FIG. 3D is a schematic of various chemical interactions between GDP hydrogel and the tissue surface. FIG. 3E is an illustration of burst pressure setup showing hydrogel sealing a punctured collagen sheet within an air-tight chamber that was linked to a pump and pressure sensor. Effect of varying pDDA and DMA concentrations in GDP hydrogels on (FIG. 3F) burst pressure and (FIG. 3G) ionic conductivity. FIG. 3H is a schematic of GDP hydrogel interactions with water depicting the mechanism of ionic conductivity in an aqueous environment. Data are represented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, N=4).

[0039] FIGs. 4A-4F show ex vivo adhesion of GDP adhesives to lung injuries of variable sizes. FIG. 4A is an image showing the setup for ex vivo air leak sealant test by (i) attaching an unpunctured pig lung to a ventilator, (ii) creating a 10 mm wide pleural defect, (iii) applying the prepolymer solution, and (iv) photocrosslinking with visible light to form the adhesive hydrogel. FIG. 4B are representative graphs showing the gradual increase in ventilation pressure applied to the injured lung that was sealed with GelMAG or GDP adhesive. Lungs were cyclically expanded at the same pressure before pressure was increased. FIG. 4C is a graph showing the burst pressure of GelMAG, GDP, and CoSeal™ after sealing a shallow pleural defect (10 mm diameter and 0.5 mm depth). FIG. 4D is an image showing a large laceration (20 mm length and 2.5 mm depth) created and sealed with GDP adhesive. FIG. 4E is an image showing a large puncture (20 mm diameter and 1.5 mm depth) created and sealed with GDP adhesive. FIG. 4F is a graph showing the burst pressure of GDP adhesive after sealing lacerations and punctures of various dimensions. Data are represented as mean ± SD (*P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001, N=4).

[0040] FIGs. 5A-5H show the antibacterial activity of GDP adhesives. FIG. 5A is a graph showing the viability measured by optical density (OD) at 625 nm of P. aeruginosa during 6 days of culture with GelMAG, GelMAG and 0.1% (w / v) DMA (GD), GelMAG and 2% (v / v) pDDA, and GelMAG with 2% (v / v) pDDA and 0.1% (w / v) DMA (GDP). FIG. 5B is a graph showing the colony forming units (CFU) of P. aeruginosa after 6 days of hydrogel treatments. FIG. 5C are representative SEM images of GelMAG and GDP surface after 6 days of bacterial inoculation. FIG. 5D are images of the zone of inhibition (ZOI) formed by Attorney Docket No. 50835-0006W01

[0041] GelMAG or GDP against P. aeruginosa. FIG. 5E is a graph showing the viability of MRS A during 6 days of culture with GelMAG, GP, GD, or GDP. FIG. 5F is a graph showing the CFU of MRS A after 6 days of hydrogel treatments. FIG. 5G are representative SEM images of GelMAG and GDP surface after 6 days of bacterial inoculation. FIG. 5H are images of the ZOI formed by GelMAG or GDP against MRSA. Negative control represented bacteria without hydrogel treatment. Data are represented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, N=4).

[0042] FIGs. 6A-6E show rapid in vitro hemostatic performance of GDP adhesives. FIG. 6A is a table showing results of a hemostatic blood clotting assay performed on citrate- activated whole blood treated with GelMAG, GelMAG and 0.1% (w / v) DMA (GD), GelMAG and 2% (v / v) pDDA, and GelMAG with 2% (v / v) pDDA and 0.1% (w / v) DMA (GDP). Whole blood without any hydrogel treatment was used as a control. FIG. 6B is a graph showing the qualitative evaluation of complete blood clotting time taken by quenching clot formation with saline at specific time points. FIG. 6C is a graph showing the quantitative analysis of blood clotting time performed by measuring hemoglobin absorbance at 540 nm. FIG. 6D is a graph showing the clot weight for all samples measured at 30 min after treatment of activated blood with hemostatic hydrogels. FIG. 6E is a graph showing the blood clotting index of whole blood without hydrogel treatment compared to GDP. Data are represented as mean ± SD (*P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001, N=4).

[0043] FIGs. 7A-7E show the in vivo hemostatic efficacy of GDP adhesives using rat tail amputation and liver puncture models. FIG. 7A show a tail amputation model that was performed where (i) 6 cm of the rat tail was amputated, (ii) prepolymer solution was applied and photocrosslinked to form a hemostatic hydrogel, and (iii) blood loss was collected on filter paper and (iv) compared to commercial cellulose-based surgical hemostat Surgicel®. FIG. 7B is a liver puncture model that was performed where (i) the upper lobe of the rat liver was punctured 2 mm deep using a marked scalpel, (ii) prepolymer solution was applied and photocrosslinked to form a hemostatic hydrogel, and (iii) blood loss was collected on filter paper and (iv) compared to commercial cellulose-based surgical hemostat Surgicel®. Injuries without hemostatic biomaterial treatment were used as controls for both models. FIG. 7C are histology images of GelMAG or GDP adhesive with surrounding liver tissue stained with H&E after 14 days post-operation. Immunostaining of GelMAG or GDP adhesive and surrounding tissue after 14 days showing (FIG. 7D) lymphocytes (CD3) and (FIG. 7E) macrophages (CD68). Green colors in (FIG. 7D) represent lymphocytes, red colors in (FIG. Attorney Docket No. 50835-0006W01

[0044] 7E) represent macrophages, and blue colors represent cell nuclei (DAP I). Data are represented as mean ± SD (*P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001, N=3).

[0045] FIGs. 8A-8G show in vitro and in vivo biocompatibility of GDP adhesive. FIG. 8A are representative images of (i) live / dead and (i) actin / DAPI staining of 3T3 cells cultured with GelMAG or GDP adhesive for 5 days. Cells without hydrogel treatment were used as a control. FIG. 8B is a graph showing the viability and FIG. 8C is a graph showing the relative fluorescence unit (RFU) of 3T3 cells cultured with GelMAG or GDP for 1, 3, and 5 days FIG. 8D are histology images of GDP adhesive and surrounding tissue stained with H&E after post-operation days 7 and 28. FIG. 8E is a graph showing the biodegradation profile of subcutaneously implanted GelMAG and GDP adhesive. Immunostaining of GDP and surrounding tissue after 7 and 28 days showing (FIG. 8F) hematopoietic cells (CD45) and (FIG. 8G) macrophages (CD68). Green colors in (FIG. 8F) represent hematopoietic cells, red colors in (FIG. 8G) represent macrophages, and blue colors represent cell nuclei (DAPI). Data are represented as mean ± SD (*P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001, N=3).

[0046] FIGs. 9A-9H show in vivo hemostatic, adhesion, and biocompatibility performance of GDP on pig lung laceration model. FIGs. 9A are representative images of pig lung injury showing (i) the creation of a 15 mm and deep laceration on the pig lung following a lateral thoracotomy, (ii) observation of an air leak, (iii) application of prepolymer solution, (iv) photocrosslinking to form the hydrogel, and (v) confirmation of air-leak sealing by submerging injury in saline. FIGs. 9B are representative ultrasound images after the hydrogel was used to seal the air leak. H&E staining of the lung tissue that was sealed with either (FIG. 9C) GDP or (FIG. 9D) TISSEEL®. FIG. 9E shows hemostatic testing with (i) representative images and (ii) quantitative measurements of blood loss after a 1 min test. FIG. 9F shows adhesion testing with (i) increasing ventilator pressure to the lungs, (ii) pressure measurements depicting what the hydrogel-sealed lungs could withstand, and (iii) representative images of a water leak test to determine the air leak sealing. Immunostaining of tissue sealed with either GDP or TISSEEL® for (FIG. 9G) lymphocytes (CD3) or (FIG. 9H) pan-macrophages (CD68) and activated macrophages (CD80) along with cell nuclei (DAPI). Data are represented as mean ± SD (*P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001, N=3).

[0047] FIG. 10 is an1H NMR analysis of gelatin and GelMAG prepolymer.

[0048] FIG. 11 is an1H NMR analysis of dopamine and DMA.

[0049] FIG. 12 is an1H NMR analysis of GelMAG prepolymer and hydrogel. Attorney Docket No. 50835-0006W01

[0050] FIG. 13 is an1H NMR analysis of GelMAG with 2% (v / v) pDDA prepolymer and hydrogel.

[0051] FIG. 14 is an1H NMR analysis of GelMAG with 0.1% (w / v) DMA prepolymer and hydrogel.

[0052] FIG. 15 is a graph showing Zeta potential measurements taken in DI water of pDDA and GDP prepolymer solution containing 20% GelMAG, 0.1% DMA, and varying concentration of pDDA.

[0053] FIGs. 16A-16B are graphs showing the bacterial viability of (FIG. 16A) P. aeruginosa and (FIG. 16B) MRSA after hydrogel treatment. The bacterial viability was measured by optical density measurements after 1, 3, and 6 days of incubation with GDP hydrogels containing various concentrations of pDDA and DMA.

[0054] FIG. 17 is a table showing representative images of in vitro hemostatic test performed on citrated whole blood, measuring the clotting time of GDP hydrogel with various concentrations of pDDA and DMA.

[0055] FIG. 18 is a graph showing absorbance values of hemoglobin during in vitro hemostatic test on all hydrogel samples.

[0056] FIG. 19 is a graph showing the swelling ratio of GDP hydrogels with various concentrations of pDDA and DMA while incubated in PBS solution at 37°C.

[0057] FIGs. 20A-20F are representative images of live / dead and actin / DAPI staining of 3T3 cells after one day of incubation with GelMAG or GDP. FIGs. 20A-20C are representative images of live / dead staining of (FIG. 20A) a control group of 3T3 cells, (FIG. 20B) 3T3 cells after one day of incubation with GelMAG, and (FIG. 20C) 3T3 cells after one day of incubation with GDP. FIGs. 20D-20F are representative images of actin / DAPI staining of (FIG. 20D) a control group of 3T3 cells, (FIG. 20E) 3T3 cells after one day of incubation with GelMAG, and (FIG. 20F) 3T3 cells after one day of incubation with GDP.

[0058] FIG. 21 is a graph showing number of 3T3 cells cultured with GelMAG or GDP after 1, 3, and 5 days of incubation.

[0059] FIGs. 22A-22B are images of H&E staining of GelMAG hydrogel and tissue interface on days (FIG. 22A) 7 and (FIG. 22B) 28 after subcutaneous implantation.

[0060] FIGs. 23A-23D are immunostaining images of tissue surrounding GelMAG. Hematopoietic cells (CD45) (green) and cell nuclei (DAPI) (blue) are shown on days (FIG. 23A) 7 and (FIG. 23B) 28 after subcutaneous implantation. Macrophages (CD68) (red) and cell nuclei (DAPI) (blue) are shown on days (FIG. 23C) 7 and (FIG. 23D) 28 after subcutaneous implantation. Attorney Docket No. 50835-0006W01

[0061] FIGs. 24A-24C. Rheological and mechanical characterization of GDP sealant. (A) Time sweep of precursor solutions of GelMAG, GP (with 2% v / v pDDA), GD (with 0.1% w / v DMA), and GDP (with 0.1% w / v DMA and 2% v / v pDDA) that were crosslinked with visible light for 4 min, depicting storage modulus (G’) and loss modulus (G”) over 10 min. (B) Frequency sweep displaying the elastic behavior of engineered hydrogels. (C) Strain sweep measurements depicting the linear viscoelastic region of all hydrogel formulations.

[0062] FIGs. 25A-25E. In vitro and ex vivo adhesion characterization of GDP sealant. (A) Burst pressure graph of an ex vivo lung injury model involving attaching an unpunctured pig lung to a ventilator and creating a 10 mm wide pleural defect, applying prepolymer solution of GelMAG or GDP, photocrosslinking with visible light to form hydrogel sealants, and ventilating the lung with gradually increasing pressures until hydrogel burst. (B) Ex vivo burst pressures of hydrogels or Evicel used as a commercial control. Analysis by one-way ANOVA with Tukey’s post-hoc multiple comparisons test. Representative SEM images from the cross-section of GDP sealant adhered onto porcine (C) lung and (D) skin tissue. (E) Schematic of potential chemical interactions occurring between the GDP hydrogel and tissue surface. Data are represented as mean ± SD. Analysis by two-way ANOVA with Tukey’s post-hoc multiple comparisons test. *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001. n=3 biological replicates per group.

[0063] FIGs. 26A-26F. In vitro antibacterial properties and biocompatibility of GDP sealant. In vitro antimicrobial tests: (A) Bacterial concentration measured by optical density (OD) at 625 nm of P. aeruginosa during 5 days of culture with either GDP hydrogels prepared with various concentrations of DMA and pDDA, broad-spectrum antibiotic ciprofloxacin, or commercial wound dressing AquaDerm™. (B) Survival rate of P. aeruginosa after 5 days of treatment. (C) OD measurements of MRS A during 5 days of culture with either GDP hydrogels prepared with various concentrations of DMA and pDDA, broad-spectrum antibiotic ciprofloxacin, or commercial wound dressing AquaDerm™. (D) Survival rate of MRSA after 5 days of treatments. In vitro biocompatibility assessment: (E) Representative images live / dead stained images from NH43T3 cells exposed to GelMAG or GDP through Transwell® inserts for 5 days. Control cells were cultured without hydrogel exposure. (F) Representative actin / DAPI stained images of NIH3T3 cells cultured with GelMAG or GDP for 5 days. Data are represented as mean ± SD. Analysis by two-way ANOVA with Tukey’s post-hoc multiple comparisons test. *P < 0.05, **P < 0.01, ****p < 0.0001. n=3 biological replicates per group. Attorney Docket No. 50835-0006W01

[0064] FIG. 27.JH NMR of the prepolymer and hydrogels of GelMAG, GP, GD, or GDP. Magnified portion of the spectrum indicates the methacryloyl proton peaks on prepolymers and hydrogels.

[0065] FIG. 28. FTIR spectra of G, GP, GD, and GDP hydrogels depicting potential chemical interactions within the matrix, including hydrogen bonding and cation-7t bonding.

[0066] FIG. 29. Ultimate tensile strength of GDP hydrogels prepared with varying amounts of pDDA and DMA. Data are represented as mean ± SD. Analysis by two-way ANOVA with Tukey’s post-hoc multiple comparisons test. *P < 0.05, **P < 0.01, ***p < 0.001. n=3 per biological group.

[0067] FIG. 30. Energy loss after 12 rounds of cyclic compression on GDP hydrogels prepared with varying amounts of pDDA and DMA. Data are represented as mean ± SD. Analysis by two-way ANOVA with Tukey’s post-hoc multiple comparisons test. *P < 0.05. n=3 per biological group.

[0068] FIG. 31. In vitro wound closure adhesion energy of GDP hydrogels prepared with varying amounts of pDDA and DMA. Data are represented as mean ± SD. Analysis by two- way ANOVA with Tukey’s post-hoc multiple comparisons test. *P < 0.05. n=3 per biological group.

[0069] FIGs. 32A-32C. In vitro antibacterial evaluation of bacterial growth after treatment using spread plate method. (A) Representative images of agar plates used to calculate CFU after P. aeruginosa or MRS A were treated with either GDP hydrogels formed with varying concentrations of DMA and pDDA, broad-spectrum antibiotic ciprofloxacin, or commercial wound dressing AquaDerm™. (B) Concentration of P. aeruginosa or MRS A after treatment calculated as CFU / mL based on a spread plate method. (C) Log reduction of bacterial viability after treatment relative to untreated control. Data are represented as mean ± SD. Analysis by one-way ANOVA with Tukey’s post-hoc multiple comparisons test. *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001. n=3 per biological group. Scale bar = 1 cm.

[0070] FIG. 33. Minimum inhibitory concentration (MIC) of broad-spectrum antibiotic ciprofloxacin and GDP against either P. aeruginosa or MRSA after 24 h incubation. Data are represented as mean ± SD. Analysis by one-way ANOVA with Tukey’s post-hoc multiple comparisons test. ***P < 0.001. n=3 per biological group.

[0071] FIG. 34. Representative images of live / dead staining of either P. aeruginosa or MRSA after 5 days of either no treatment (control) or treatment with GelMAG, GP, GD, GDP, ciprofloxacin, or AquaDerm™. Live cells were stained green and dead cells were stained red. Attorney Docket No. 50835-0006W01

[0072] FIG. 35. Zeta potential measurements of DI water that was incubated with either GelMAG or GDP hydrogels to monitor the rate of pDDA release from the hydrogel matrix. Data are represented as mean ± SD. Analysis by one-way ANOVA with Tukey’s post-hoc multiple comparisons test. *P < 0.05, **P < 0.01, ****p < 0.0001. n=3 per biological group.

[0073] FIG. 36. Number of NIH3T3 cells per unit area after incubation with GelMAG or GDP after 1 and 5 days. Control represents cells without hydrogel exposure. Data are represented as mean ± SD. Analysis by two-way ANOVA with Tukey’s post-hoc multiple comparisons test. n=3 per biological group.

[0074] FIGs. 37A-37B. Physical health of pigs with lacerate lungs that were treated with GDP hydrogel. The pigs that had lung lacerations treated by GDP sealant were monitored to assess physical health after their procedure by measuring (A) body weight after surgery (day 0) and during recovery (days 7 and 14 post-operation) and (B) freedom from pneumothorax assessed through thoracic ultrasound after surgery (day 0) and during recovery (day 14 postoperation). Data are represented as mean ± SD. n=3 per biological group.

[0075] DETAILED DESCRIPTION

[0076] The compositions described herein include hemostatic, ionically conductive, biocompatible, biodegradable, adhesive, and antibacterial gelatin-based hydrogels comprising DMA and pDDA. In some examples, the hemostatic and sealant compositions described herein can be used to induce hemostasis in a wound and seal the wound. Methods of using and / or preparing these hemostatic and sealant compositions are also provided herein. Some embodiments of the compositions and methods described herein may provide one or more of the following advantages.

[0077] Certain embodiments of the present disclosure include photocrosslinkable gelatinbased adhesive hydrogels comprising DMA and pDDA. As discussed above, there is currently an unmet need for hemostatic biomaterials suited for multi-dimensional wound sealing and repair on elastic organs. The compositions and methods of the present disclosure address this need.

[0078] For example, the present disclosure provides an optimized formulation of the engineered hemostatic sealant comprising photocrosslinkable GelMAG, DMA, and pDDA. The multi-component hemostatic sealant composition described herein can serve as an antibacterial and stretchable adhesive hemostat on traumatic injuries sustained on internal and external elastic organs. In some embodiments, the hemostatic sealant compositions of the disclosure are biocompatible and biodegradable. Biocompatibility and biodegradability of the Attorney Docket No. 50835-0006W01 disclosed compositions were tested and proven through in vitro cell studies using NIH3T3 cells and in vivo subcutaneous implantation in rats. In some embodiments, the compositions of the disclosure promote cell communication and migration when applied on injured tissue. In some embodiments, this cell communication and migration may in turn improve, support, and / or accelerate tissue regeneration and wound healing processes. In some embodiments, the hemostatic sealant compositions of the disclosure are also highly adhesive. The adhesivity of the disclosed compositions was characterized through in vitro wound closure adhesion tests on skin tissue and ex vivo burst pressure tests on ventilated pig lungs.

[0079] Furthermore, in some embodiments, the hemostatic sealant compositions of the disclosure exhibit biomimetic mechanophysical properties including extensibility, Young’s modulus, and conductivity. In some embodiments, the hemostatic sealant compositions of the disclosure also display prolonged antibacterial resistance against Gram-negative and Grampositive strains of bacteria. Therefore, hemostatic sealant compositions described herein are multifunctional hydrogels that may address various the clinical needs of wound treatment and management. Some embodiments described herein may provide hemostatic and sealant hydrogels that have improved mechanical properties over commercially available surgical adhesives, such as CoSeal™, Progel™, and Evicel®. In some embodiments, the hemostatic and sealant compositions and hydrogels of the disclosure have superior mechanical properties including toughness, tensile strength, ultimate strength, stiffness, stretchability, and adhesiveness, that when combined, can provide a tissue sealant capable of withstanding pressures exerted by dynamic organs such as the lung and heart, as described herein in Examples 10, 14, and 15.

[0080] Compositions

[0081] The present disclosure features hemostatic and sealant compositions that can include one or more of glycidyl methacrylated gelatin (GelMAG), methacrylated dopamine (DMA), poly(diallyldimethylammonium chloride) (pDDA), and a visible light-activated photoinitiator. The present disclosure features hemostatic and sealant hydrogel precursor compositions that comprise the hemostatic and sealant compositions. Additionally, the present disclosure features hemostatic and sealant hydrogels that are formed by photocrosslinking the hemostatic and sealant hydrogel precursor compositions as is described elsewhere herein. The hemostatic and sealant compositions, hydrogel precursor compositions, and hemostatic and sealant hydrogels of the disclosure are ionically conductive, biocompatible, biodegradable, adhesive, and antibacterial. The terms “GDP,” “GDP Attorney Docket No. 50835-0006W01 composition,” “GDP adhesive,” and “GDP hydrogel,” as used herein, refer to the hemostatic and sealant composition, the hemostatic and sealant hydrogel precursor composition, or the hemostatic and sealant hydrogel of the disclosure comprising GelMAG, DMA, and pDDA.

[0082] In some embodiments, to synthesize the GDP hydrogels, gelatin is modified to produce glycidyl methacrylated gelatin (GelMAG) and then grafted with methacrylic anhydride-modified dopamine (DMA). In some embodiments, DMA facilitates wet adhesion. In some embodiments, a composite is further formed upon mixing GelMAG and DMA with pDDA. In some embodiments, pDDA provides hemostatic and antibacterial activity.

[0083] Gelatin is a derivative from collagen, which is the main structural component of the cornea. Gelatin has strong adhesive properties to cells and tissue due to the presence of RGD motifs in gelatin, a denatured form of collagen that is chemically modified to form a light- activated precursor. In some embodiments, a chemically modified gelatin can be included in the hemostatic and sealant compositions of the present disclosure. In some embodiments, the chemically modified gelatin is modified with glycidyl methacrylate (GMA) to form GelMAG, a photocrosslinkable derivative of gelatin. In some embodiments, the chemical modification of gelatin can be performed by a one-step synthesis reaction of gelatin with GMA, as described in Example 1.

[0084] As described in Example 2 and Equation 1, the degree of methacrylation (DM) of GelMAG can be defined as the ratio of methacrylate groups to the free amine groups in gelatin prior to the reaction. In some embodiments, the hemostatic and sealant compositions include and / or are composed of GelMAG having a degree of methacryloyl substitution of about 50%. In some embodiments, the hemostatic and sealant compositions include and / or are composed of GelMAG with a degree of methacryloyl substitution ranging from at least about 40-60% (e.g., about 40% to about 45%, about 40% to about 50%, about 40% to about 55%, about 40% to about 60%, about 45% to about 50%, about 45% to about 55%, about 45% to about 60%, about 50% to about 55%, or about 50% to about 60%.

[0085] In some embodiments, the concentration of GelMAG in the GDP compositions is about 20% weight per volume (w / v). In some embodiments, the concentration of GelMAG in the GDP compositions ranges from about 15% to about 25% (w / v) (e.g., about 15% to about 20%, about 15% to about 20%, about 15% to about 25%, or about 20% to about 25% (w / v)).

[0086] DMA is a compound that is synthesized through a reaction between methacrylic anhydride (MA) and dopamine hydrochloride, as described in Example 1 and as shown in FIG. 1 A-ii. DMA is similar to marine mussel adhesive due to the presence of catechol groups, which are key to the adhesive properties of both substances. Marine mussels produce Attorney Docket No. 50835-0006W01 byssal threads, which are strong, fibrous structures containing catechol groups that anchor them to surfaces, as shown in FIG. 1 A-ii. In some embodiments, the DMA is conjugated to a backbone of the GelMAG. In some embodiments, the concentration of DMA in the GDP compositions is about 0.1% (w / v). In some embodiments, the concentration of DMA in the GDP compositions ranges from about 0.05% to about 0.15% (w / v) (e.g., about 0.05% to about 0.1%, about 0.05% to about 0.15%, or about 0.1% to about 0.15% (w / v)).

[0087] In some embodiments, the GDP compositions include pDDA. pDDA is a polyelectrolyte, cationic polymer. In some embodiments, pDDA provides hemostatic and antibacterial properties to the GDP compositions. In some embodiments, the concentration of pDDA in the GDP compositions is about 2% (v / v). In some embodiments, the concentration of pDDA in the GDP compositions ranges from about 0.5% to about 2% (v / v) (e.g., about 0.5% to about 2%, about 0.5% to about 2.5%, about 0.5% to about 3%, about 0.5% to about 3.5%, about 0.5% to about 4%, about 0.5% to about 4.5%, about 0.5% to about 5%, about 0.5% to about 10%, about 0.5% to about 15%, about 0.5% to about 20%, about 0.5% to about 24%, about 1% to about 2%, about 1% to about 2.5%, about 1% to about 3%, about 1% to about 1%, about 1% to about 4%, about 1% to about 4.5%, about 1% to about 5%, about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 2% to about 2.5%, about 2% to about 3%, about 2% to about 1%, about 2% to about 4%, about 2% to about 4.5%, about 2% to about 5%, about 2% to about 10%, about 2% to about 15%, about 2% to about 20%, or about 2% to about 25%).

[0088] Generally, a hydrogel may be formed by using at least one, or one or more types of hydrogel precursors, and setting or solidifying the one or more types of hydrogel precursors in an aqueous solution to form a three-dimensional network, wherein formation of the three- dimensional network may cause the one or more types of hydrogel precursors to gel. As used herein, the term “hemostatic and sealant hydrogel precursor composition” refers to any of the hemostatic and sealant compositions described herein that may be used to form a hydrogel.

[0089] In some embodiments, the GDP compositions can include a photoinitiator that can be used to further activate polymerization and solidification of the hemostatic and sealant hydrogel precursor compositions when it is in a non-solid (e.g., viscous liquid, gel, liquid, or solution) form. In some embodiments, exposing the hemostatic and sealant hydrogel precursor composition to light activates the photoinitiator, triggering the formation of free- radicals, resulting in vinyl-bond crosslinking between methacrylate groups, and thus polymerization of the hemostatic and sealant hydrogel precursor composition. The Attorney Docket No. 50835-0006W01 polymerization in turn, results in a physical change of the hemostatic and sealant hydrogel precursor composition (i.e., changing from a solution to a hydrogel).

[0090] In some embodiments, the GDP compositions are photo-crosslinked to form GDP hydrogels. In some embodiments, the GDP compositions include a photoinitiator solution. In some embodiments, the photoinitiator solution comprises Eosin Y, triethanolamine (TEA), N-vinyl-8-caprolactam (VC), or any combination thereof. In some embodiments, the GDP compositions include a photoinitiator solution. In some embodiments, the photoinitiator solution comprises Eosin Y, TEA, and VC.

[0091] In some embodiments, the photoinitiator solution includes a light-activated photoinitiator. In some embodiments, the light-activated photoinitiator includes an ultraviolet light-activated photoinitiator. In some embodiment, the light-activated photoinitiator includes a near-infrared (NIR) light-activated photoinitiator. In some embodiment, the light-activated photoinitiator includes a visible light-activated photoinitiator. In some embodiments, the light-activated photoinitiator can includes a blue light-activated photoinitiator. In some embodiments, the visible light-activated photoinitiator includes triethanolamine, N- vinylcaprolactam, riboflavin, 2-hydroxy-4’-(2- hydroxyethoxy)-2-methylpropiophenone, Eosin Y disodium salt, 4,6-trimethylbenzoylphosphinate, triethanol amine, dl-2,3- diketo- 1,7,7-trimethylnorcamphane (CQ), 1 -phenyl- 1,2-propadi one (PPD), 2,4,6- trimethylbenzoyldiphenylphosphine oxide (TPO), bis(2,6-dichlorobenzoyl)-(4- propylphenyl)phosphine oxide, 4,4’-bis(dimethylamino)benzophenone, 4,4’- bis(diethylamino)benzophenone, 2- chlorothioxanthen-9-one, 4- (dimethylamino)benzophenone, phenanthrenequinone, ferrocene, diphenyl(2,4,6 trimethylbenzoyl)phosphine oxide / 2-hydroxy-2-methylpropiophenone (50 / 50 blend), dibenzosuberenone, (benzene) tricarbonylchromium, resazurin, resorufin, benzoyltrimethylgermane, derivatives thereof, or any combinations thereof. In some embodiments, the visible light-activated photoinitiator includes a mixture of triethanolamine, N-vinylcaprolactam, riboflavin, 2-hydroxy-4’-(2- hydroxy ethoxy)-2-methylpropiophenone, and Eosin Y disodium salt. In some embodiments, the visible light-activated photoinitiator comprises a mixture of two or more elements selected from triethanolamine, N- vinylcaprolactam, riboflavin, 2-hydroxy-4’-(2- hydroxyethoxy)-2-methylpropiophenone, and Eosin Y di sodium salt.

[0092] Different types of light sources can be used to photo-crosslink the hydrogel precursor composition (e.g., the composition in a solution form). Non-limiting examples of light sources that can be used to polymerize the composition include visible light sources (e.g., white or blue light), ultraviolet light sources, near-infrared light sources, and fluorescent light Attorney Docket No. 50835-0006W01 sources. In some embodiments, the composition includes a visible light-activated photoinitiator that can be activated upon exposure of light having a wavelength between about 450 nanometers (nm) to 550 nm. In some embodiments, the hemostatic and sealant hydrogel precursor composition is photo-crosslinked by activating the visible light-activated photoinitiator upon exposure of light having a wavelength between about 400 nanometers (nm) to about 750 nm (e.g., about 400 nm to about 450 nm, about 400 nm to about 500 nm, about 400 nm to about 550 nm, about 400 nm to about 600 nm, about 400 nm to about 650 nm, about 400 nm to about 700 nm, about 400 nm to about 750 nm, about 450 nm to about 500 nm, about 450 nm to about 550 nm, about 450 nm to about 600 nm, about 450 nm to about 650 nm, about 450 nm to about 700 nm, about 450 nm to about 750 nm, about 500 nm to about 550 nm, about 500 nm to about 600 nm, about 500 nm to about 650 nm, about 500 nm to about 700 nm, or about 500 nm to about 750 nm). In some embodiments, the visible light-activated photoinitiator can be activated upon exposure of light having a wavelength of about 400 nm. In some embodiments, the visible light-activated photoinitiator can be activated upon exposure of light having a wavelength of about 450 nm. In some embodiments, the visible light-activated photoinitiator can be activated upon exposure of light having a wavelength of about 500 nm. In some embodiments, the visible light-activated photoinitiator can be activated upon exposure of light having a wavelength of about 550 nm.

[0093] Pharmaceutical Compositions

[0094] Certain embodiments of the present disclosure include the hemostatic and sealant compositions and a pharmaceutically acceptable carrier. As used herein, the expression “pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation and is compatible with administration to a subject, for example a human. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits. Examples of pharmaceutically acceptable carriers include, but are not limited to, a solvent or dispersing medium containing, for example, water, pH buffered solutions (e.g., Attorney Docket No. 50835-0006W01 phosphate buffered saline (PBS), HEPES, TES, MOPS, etc.), isotonic saline, Ringer’s solution, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), alginic acid, ethyl alcohol, and suitable mixtures thereof. In some embodiments, the pharmaceutically acceptable carrier can be a pH-buffered solution (e.g. PBS).

[0095] In some embodiments, the pharmaceutically acceptable carrier is a topical carrier. In some embodiments, the GDP composition is formulated for topical use. In some embodiments, the GDP composition is topically administered to a tissue (e.g., a tissue having a wound) of a patient. In some embodiments, the GDP composition can be applied to a surface of a wound in a tissue (e.g., a surface of a wound in an organ) of a patient to induce hemostasis and act as a sealant.

[0096] In some embodiments, the GDP pharmaceutical compositions may be provided in a kit. In such aspects, the kit may comprise a syringe prefilled with the GDP pharmaceutical composition, a hypodermic syringe, and instructions for use. In some embodiments, the GDP pharmaceutical compositions may be provided as a single or multiple dose pharmaceutical composition in a pre-filled syringe. In some embodiments, the GDP pharmaceutical compositions may be provided as a single or multiple dose pharmaceutical composition in a vial. In some embodiments, the GDP pharmaceutical composition is suitable for diagnostic or therapeutic use in vitro, in vivo, and / or ex vivo.

[0097] Physical Properties of Hemostatic and Sealant Compositions

[0098] The physical properties of the compositions of the disclosure, including but not limited to stiffness, elasticity, toughness, adhesion, burst pressure, and conductivity, can be finely tuned by modulating the concentration of one or more of the components of the hemostatic and sealant composition (e.g., GelMAG, DMA, pDDA, and / or the photoinitiator).

[0099] In some embodiments, these physical properties can be altered by changing the density (e.g., the number) of functional groups (e.g., methacrylated group) per polymer chain (e.g., per molecule). For example, in some embodiments, different types of functional groups can be used to modify the polymers, such as glycidyl methacrylate, which can be used for photo-crosslinking properties, or polyelectrolytes, which can be used for charge-based interactions and coacervation. In some embodiments, these physical properties can also be altered by varying the molecular weight (e.g., the polymer chain length) of the polymers used to synthesize the hemostatic and sealant compositions disclosed herein.

[0100] Alternatively, or in combination to the polymer concentration modulation, the physical properties of the hemostatic and sealant compositions can also be finely tuned by Attorney Docket No. 50835-0006W01 controlling the light exposure time (e.g., the polymerization time). In some embodiments, the hemostatic and sealant composition is exposed to a light source for about 4 minutes. In some embodiments, the hemostatic and sealant composition is exposed to a light source for about a period ranging from about 15 seconds to 15 minutes. In some embodiments, the hemostatic and sealant composition is exposed to a light source for about a period ranging from about 1 to 10 minutes. In some embodiments, the hemostatic and sealant composition is exposed to a light source for about 30 seconds to about 4 minutes, about 1 to about 4 minutes, about 2 to about 4 minutes, about 3 to about 4 minutes, about 4 to about 5 minutes, about 4 to about 6 minutes, about 4 to about 7 minutes, about 4 to about 8 minutes, about 4 to about 9 minutes, or about 4 to about 10 minutes.

[0101] Important mechanical properties of the composition include stiffness, elasticity, toughness, adhesion, burst pressure, and conductivity. In some embodiments, the mechanical properties (e.g., stiffness, elasticity, conductivity) of the GDP hydrogels can be varied by altering the concentration of DMA and pDDA within the GDP hydrogel. In some embodiments, the adhesivity (e.g., adhesion strength) and the burst pressure of the GDP hydrogels can be varied by changing the concentration of DMA.

[0102] In some embodiments, the GDP hydrogel has a Young’s modulus of about 30 kilopascals (kPa) to about 200 kPa (e.g., about 30 kPa to about 35 kPa, about 30 kPa to about 40 kPa, about 30 kPa to about 45 kPa, about 30 kPa to about 50 kPa, about 30 kPa to about

[0103] 55 kPa, about 30 kPa to about 60 kPa, about 30 kPa to about 65 kPa, about 30 kPa to about

[0104] 70 kPa, about 30 kPa to about 75 kPa, about 30 kPa to about 80 kPa, about 30 kPa to about

[0105] 85 kPa, about 30 kPa to about 90 kPa, about 30 kPa to about 95 kPa, about 30 kPa to about

[0106] 100 kPa, about 30 kPa to about 110 kPa, about 30 kPa to about 120 kPa, about 30 kPa to about 130 kPa, about 30 kPa to about 140 kPa, about 30 kPa to about 150 kPa, about 30 kPa to about 160 kPa, about 30 kPa to about 170 kPa, about 30 kPa to about 180 kPa, about 30 kPa to about 190 kPa, or about 30 kPa to about 200 kPa). In some embodiments, the GDP hydrogel has a Young’s modulus of about 30 kPa. In some embodiments, the GDP hydrogel has a Young’s modulus of about 40 kPa. In some embodiments, the GDP hydrogel has a Young’s modulus of about 20 kPa.

[0107] In some embodiments, the GDP hydrogel has an ultimate strength of about 60 kilopascals (kPa) to about 150 kPa (e.g., about 60 kPa to about 65 kPa, about 60 kPa to about 70 kPa, about 60 kPa to about 75 kPa, about 60 kPa to about 80 kPa, about 60 kPa to about 85 kPa, about 60 kPa to about 90 kPa, about 60 kPa to about 95 kPa, about 60 kPa to about 100 kPa, about 60 kPa to about 110 kPa, about 60 kPa to about 120 kPa, about 60 kPa to Attorney Docket No. 50835-0006W01 about 130 kPa, about 60 kPa to about 140 kPa, about 60 kPa to about 150 kPa, about 70 kPa to about 75 kPa, about 70 kPa to about 80 kPa, about 70 kPa to about 85 kPa, about 70 kPa to about 90 kPa, about 70 kPa to about 95 kPa, about 70 kPa to about 100 kPa, about 70 kPa to about 110 kPa, about 70 kPa to about 120 kPa, about 70 kPa to about 130 kPa, about 70 kPa to about 140 kPa, about 70 kPa to about 150 kPa, about 80 kPa to about 85 kPa, about 80 kPa to about 90 kPa, about 80 kPa to about 95 kPa, about 80 kPa to about 100 kPa, about 80 kPa to about 110 kPa, about 80 kPa to about 120 kPa, about 80 kPa to about 130 kPa, about 80 kPa to about 140 kPa, about 80 kPa to about 150 kPa, about 90 kPa to about 95 kPa, about 90 kPa to about 100 kPa, about 90 kPa to about 110 kPa, about 90 kPa to about 120 kPa, about 90 kPa to about 130 kPa, about 90 kPa to about 140 kPa, about 90 kPa to about 150 kPa, about 100 kPa to about 110 kPa, about 100 kPa to about 120 kPa, about 100 kPa to about 130 kPa, about 100 kPa to about 140 kPa, or about 100 kPa to about 150 kPa). In some embodiments, the GDP hydrogel has an ultimate strength of about 100 kPa. In some embodiments, the GDP hydrogel has an ultimate strength of about 90 kPa. In some embodiments, the GDP hydrogel has an ultimate strength of about 110 kPa.

[0108] In some embodiments, the GDP hydrogel has a stretchability ranging from about 80% to about 220% (e.g., about 80% to about 210%, about 80% to about 210%, about 90% to about 210%, about 100% to about 210%, about 110% to about 210%, about 120% to about 210%, about 130% to about 210%, about 140% to about 210%, about 150% to about 210%, about 160% to about 210%, about 170% to about 210%, about 180% to about 210%, about 190% to about 210%, about 200% to about 210%, or about 210% to about 220%). In some embodiments, the GDP hydrogel has a stretchability of about 210%.

[0109] In some embodiments, the GDP hydrogel has a toughness ranging from about 20 kilojoules per cubic meter (kJ / m3) to about 105 kJ / m3(e.g., about 20 kJ / m3to about 90 kJ / m3, about 30 kJ / m3to about 90 kJ / m3, about 40 kJ / m3to about 90 kJ / m3, about 50 kJ / m3to about 90 kJ / m3, about 60 kJ / m3to about 90 kJ / m3, about 70 kJ / m3to about 90 kJ / m3, about 80 kJ / m3to about 90 kJ / m3, about 90 kJ / m3to about 95 kJ / m3, about 90 kJ / m3to about 100 kJ / m3, about 90 kJ / m3to about 105 kJ / m3,). In some embodiments, the GDP hydrogel has a toughness ranging from about 90 kJ / m3. In some embodiments, the GDP hydrogel has a toughness ranging from about 80 kJ / m3. In some embodiments, the GDP hydrogel has a toughness ranging from about 100 kJ / m3.

[0110] In some embodiments, the GDP hydrogel has a compression modulus of about 30 kilopascals (kPa) to about 80 kPa (e.g., about 30 kPa to about 40 kPa, about 30 kPa to about 45 kPa, about 30 kPa to about 50 kPa, about 30 kPa to about 60 kPa, about 30 kPa to about Attorney Docket No. 50835-0006W01

[0111] 70 kPa, about 30 kPa to about 80 kPa, about 50 kPa to about 60 kPa, about 50 kPa to about 70 kPa, or about 50 kPa to about 80 kPa). In some embodiments, the GDP hydrogel has a compression modulus of about 50 kPa. In some embodiments, the GDP hydrogel has a compression modulus of about 40 kPa. In some embodiments, the GDP hydrogel has a compression modulus of about 60 kPa.

[0112] In some embodiments, cyclic compression testing of GDP hydrogels can be used to determine energy loss, as described in Example 3. In cyclic compression testing, energy loss refers to the energy dissipated during each loading and unloading cycle. This energy dissipation is typically represented by the area enclosed within the hysteresis loop on a stressstrain curve. The energy loss can provide insights into the material’s behavior under repeated loading, such as fatigue damage and material degradation. In some embodiments, the GDP hydrogel has an energy loss ranging from about 10% to about 30% (e.g., about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, or about 10% to about 30%, about 20% to about 25%, or about 20% to about 30%). In some embodiments, the GDP hydrogel has an energy loss of about 25%. In some embodiments, the GDP hydrogel has an energy loss of about 20%. In some embodiments, the GDP hydrogel has an energy loss of about 30%.

[0113] In some embodiments, the GDP hydrogel has an adhesion strength of about 20 kilopascals (kPa) to about 50 kPa (e.g., about 20 kPa to about 25 kPa, about 20 kPa to about 30 kPa, about 20 kPa to about 35 kPa, about 20 kPa to about 40 kPa, about 20 kPa to about 45 kPa, about 20 kPa to about 50 kPa, about 25 kPa to about 30 kPa, about 25 kPa to about 35 kPa, about 25 kPa to about 40 kPa, about 25 kPa to about 45 kPa, about 25 kPa to about 50 kPa, about 30 kPa to about 35 kPa, about 30 kPa to about 40 kPa, about 30 kPa to about 45 kPa, about 30 kPa to about 50 kPa, about 35 kPa to about 40 kPa, about 35 kPa to about 45 kPa, about 35 kPa to about 50 kPa, about 40 kPa to about 45 kPa, about 40 kPa to about 50 kPa, or about 45 kPa to about 50 kPa). In some embodiments, the GDP hydrogel has an adhesion strength of about 45 kPa. In some embodiments, the GDP hydrogel has an adhesion strength of about 40 kPa. In some embodiments, the GDP hydrogel has an adhesion strength of about 50 kPa.

[0114] Adhesion energy measures the energy required to separate two bonded surfaces. It quantifies the strength of the adhesive bond by indicating how much energy is needed to overcome the forces holding the surfaces together. This property is crucial in applications where strong and durable bonds are essential, such as in the GDP sealant compositions of the disclosure. In some embodiments, the GDP hydrogel has an adhesion energy of about 10 J / m3to about 50 J / m3(e.g., about 10 J / m3to about 20 J / m3, about 10 J / m3to about 30 J / m3, about Attorney Docket No. 50835-0006W01

[0115] 10 J / m3to about 40 J / m3, or about 10 J / m3to about 50 J / m3). In some embodiments, the GDP hydrogel has an adhesion energy of about 10 J / m3.

[0116] In some embodiments, the GDP hydrogel has strong adhesive properties, especially in wet environments. Typically, to measure the adhesive strength of a sealant, an in vitro burst pressure test can be conducted. As discussed in Example 3, burst pressure can be tested in vitro by sealing punctured and pressurized collagen sheets with GDP hydrogels and ex vivo by a ventilator-assisted pig lung. In some embodiments, the GDP hydrogel has a burst pressure of about 20 kPa to about 60 kPa (e.g., about 20 kPa to about 30 kPa, about 20 kPa to about 35 kPa, about 20 kPa to about 40 kPa, about 20 kPa to about 45 kPa, about 20 kPa to about 55 kPa, about 20 kPa to about 60 kPa, about 30 kPa to about 35 kPa, about 30 kPa to about 40 kPa, about 30 kPa to about 45 kPa, about 30 kPa to about 50 kPa, about 30 kPa to about 55 kPa, about 30 kPa to about 60 kPa, about 35 kPa to about 40 kPa, about 35 kPa to about 45 kPa, about 35 kPa to about 50 kPa, about 35 kPa to about 55 kPa, about 35 kPa to about 60 kPa, about 40 kPa to about 45 kPa, about 40 kPa to about 50 kPa, about 40 kPa to about 55 kPa, about 40 kPa to about 60 kPa, about 45 kPa to about 50 kPa, about 45 kPa to about 55 kPa, or about 45 kPa to about 60 kPa). In some embodiments, the composition has a burst pressure of about 50 kPa. In some embodiments, the composition has a burst strength of about 45 kPa. In some embodiments, the composition has a burst strength of about 55 kPa.

[0117] The degradation rate of the composition can be controlled based on the concentration of one or more polymers added (e.g., HAGM and / or GelMA). In some embodiments, the composition has a degradation rate of about 60% after 28 days of implantation. In some embodiments, the composition has a degradation rate of about 50% to about 70%, about 55% to about 70%, about 60% to about 70%, or about 65% to about 70%, after 28 days of implantation.

[0118] In some embodiments, the GDP hydrogel of the disclosure has a conductivity ranging from about 0.1 Siemens per meter (S / m) to about 1 S / m (e.g., about 0.1 S / m to about 0.2 S / m, about 0.1 S / m to about 0.3 S / m, about 0.1 S / m to about 0.4 S / m, about 0.1 S / m to about 0.5 S / m, about 0.1 S / m to about 0.6 S / m, about 0.1 S / m to about 0.7 S / m, about 0.2 S / m to about 0.3 S / m, about 0.2 S / m to about 0.4 S / m, about 0.2 S / m to about 0.5 S / m, about 0.2 S / m to about 0.6 S / m, about 0.2 S / m to about 0.7 S / m, 0.3 S / m to about 0.4 S / m, about 0.3 S / m to about 0.5 S / m, about 0.3 S / m to about 0.6 S / m, about 0.3 S / m to about 0.7 S / m, about 0.4 S / m to about 0.5 S / m, about 0.4 S / m to about 0.6 S / m, about 0.4 S / m to about 0.7 S / m, about 0.5 S / m to about 0.6 S / m, about 0.5 S / m to about 0.7 S / m, about 0.6 S / m to about 0.7 S / m, about 0.7 S / m to about 0.8 S / m, about 0.7 S / m to about 0.9 S / m, or about 0.7 S / m to about 0.8 S / m). Attorney Docket No. 50835-0006W01

[0119] In some embodiments, the GDP hydrogel of the disclosure has a conductivity of about 0.7 S / m. In some embodiments, the GDP hydrogel of the disclosure has a conductivity of about 0.8 S / m. In some embodiments, the GDP hydrogel of the disclosure has a conductivity of about 0.9 S / m.

[0120] Methods of Treatment Using GDP Hydrogels

[0121] Certain embodiments of the present disclosure include methods of sealing a wound in a tissue of a subject. The methods include the steps of contacting the wound of the subject with a therapeutically effective amount of the hemostatic and sealant hydrogel precursor composition of the disclosure, and photo-crosslinking the hemostatic hydrogel precursor composition by exposing the hydrogel precursor to a visible light, thereby forming the hemostatic and sealant hydrogel. The term “therapeutically effective amount” refers to the amount of the GDP composition or GDP hydrogel that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. For example, in some embodiments, a therapeutically effective amount of the GDP composition can be the amount of the GDP composition or GDP hydrogel that will induce hemostasis in a wound and / or seal the wound.

[0122] In some embodiments, the GDP hydrogel precursor compositions can be injected into the wound in the tissue using a syringe. In some embodiments, the precursor GDP hydrogel precursor compositions can be applied onto a surface of the wound by using a syringe, a pipette (e.g., a Pasteur pipette), a brush, a dropper bottle or dropper tube configured to dispense a viscous fluid (e.g., having an aperture at a distal end that is large enough to dispense a viscous fluid), or any other suitable device or tool. In some embodiments, the precursor GDP hydrogel precursor compositions can be applied onto a surface of the wound using a spray nozzle such that the GDP hydrogel precursor compositions is sprayed onto the surface of the wound. In some embodiments, the GDP hydrogel precursor compositions can be applied as a drop (e.g., when in a viscous, non-solid state) onto the wound without the need for an applicator. Exposure to visible light can permit crosslinking to provide an adhesive solid hydrogel with hemostatic and sealant properties. By adjusting the light exposure time, the polymerization of the GDP compositions of the disclosure can be finely controlled, allowing for a precise application, as compared to commercially available tissue sealants.

[0123] In some embodiments, the GDP hydrogel is used to protect the wound, promote faster healing, reduce a risk of infection, and act as a protective barrier. In some embodiments, the Attorney Docket No. 50835-0006W01

[0124] GDP hydrogel remains localized on, seals, and forms a surface barrier over a wound, an incision, an injury, and / or a laceration once applied. In some embodiments, upon photocrosslinking, the precursor composition becomes a solid and transparent hydrogel, forming a antibacterial, biocompatible, hemostatic, and adhesive sealant on the wound surface.

[0125] In some embodiments, the methods of sealing or treating a wound or an injury in a tissue of a subject using the GDP hydrogels of the disclosure include sealing or treating the wound or the injury in various tissue and organ types. For example, in some embodiments, the tissue can be an epithelial tissue, a connective tissue, a muscle tissue, a nervous tissue, or any combination thereof. In some embodiments, the tissue is a nerve. In some embodiments, the organ can be a lung. In some embodiments, the organ can be a heart. In some embodiments, the organ can be a liver. In some embodiments, the organ can be a brain, a spinal cord, a cranial nerve, a spinal nerve, an eye, an esophagus, a thyroid, a thymus, a stomach, a small intestine, a large intestine, a pancreas, a spleen, a kidney, a ureter, a bladder, a urethra, an ovary, a fallopian tube, a uterus, a cervix, a prostate, a testes, or any combination thereof.

[0126] The GDP compositions of the disclosure can be used to treat and seal tissue incisions, cuts, wounds, and / or injuries having a length or diameter of less than about 1 mm to about 20 mm. In some embodiments, the GDP compositions of the disclosure can be applied both under “normal” (e.g., in-the-office or operating room) settings, or under emergency “in-in- field” settings. Various providers, physicians, and, in select cases, physician assistants and paramedics (e.g., in the combat theater) can apply the GDP compositions described herein to seal wounds or injuries and promote their healing.

[0127] Methods of Preparing GDP Hydrogels

[0128] Provided herein are methods of preparing any of the GDP compositions and GDP hydrogels disclosed herein. The methods include dissolving the GelMAG in a solution including the visible light-activated photoinitiator, mixing the DMA with the solution comprising the visible light-activated photoinitiator and the dissolved GelMA, incubating the solution comprising the visible light-activated photoinitiator, the dissolved GelMA, and the DMA for at least twenty four hours, mixing the pDDA with the solution comprising the visible light-activated photoinitiator, the dissolved GelMA, and the DMA, and photocrosslinking the solution by exposing the solution to a visible light after mixing the pDDA with the solution, thereby forming the hemostatic hydrogel. Attorney Docket No. 50835-0006W01

[0129] In some embodiments, the methods can include synthesizing GelMAG prior to dissolving GelMAG in a solution including the visible light-activated photoinitiator. For example, in some embodiments, the GelMAG can be synthesized by chemically modifying gelatin with GMA through a one-step reaction, as described in Example 1. In some embodiments, once synthesized, GelMAG is dissolved in a photoinitiator solution comprising Eosin Y, TEA, and N-vinyl-s-caprolactam at 37 °C.

[0130] In some embodiments, the methods can include synthesizing DMA prior to mixing DMA in the solution including the visible light-activated photoinitiator and the dissolved GelMAG. For example, in some embodiments, the GelMAG can be synthesized by chemically modifying gelatin with GMA through a one-step reaction, as described in Example 1. Once GelMAG is fully dissolved, the method includes the step of adding DMA, quickly mixing, and and incubating overnight to conjugate DMA onto the GelMAG backbone.

[0131] Next, in some embodiments, the method includes adding pDDA and quickly mixing to ensure homogenous distribution of the polyelectrolyte. In some embodiments, the GDP hydrogel precursor can be poured into a mold (e.g., a polydimethylsiloxane (PDMS) mold) of any suitable size and shape and crosslinked with visible light. In some embodiments, the GDP hydrogel precursor is photo-crosslinked for four minutes. In some embodiments, the GDP hydrogel precursor changes from a solution form to a hydrogel form after photocrosslinking, as described previously herein.

[0132] EXAMPLES

[0133] Certain embodiments of the present disclosure are further described in the following examples, which do not limit the scope of any embodiments described in the claims.

[0134] Example 1 - Synthesis of GelMAG / DMA / pDDA (GDP) Bioadhesive Hydrogels Materials

[0135] Gelatin from porcine skin (Gel strength 300, Type A), methacrylic anhydride (MA), glycidyl methacrylate (GMA), poly(diallyldimethylammonium chloride) (pDDA), Eosin Y, and triethanolamine (TEA), type II collagenase, ethyl acetate, and hexane were purchased from Sigma Aldrich. Dopamine hydrochloride, N-vinyl-s-caprolactam (VC), Dulbecco’s phosphate buffered saline (DPBS), PrestoBlue™ reagent, and Abeam Anti-CD68 antibody were purchased from Thermo Fisher Scientific. Dulbecco’s Modified Eagle Medium (DMEM) was purchased from ATCC, fetal bovine serum was purchased from Corning, and Attorney Docket No. 50835-0006W01 penicillin / streptomycin was purchased from Life Technologies®. Commercial live / dead kits (calcein AM and ethidium homodimer), Al exaFlour® 594 (phalloidin), and 4’,6-diamidino-2- phenylindole dihydrochloride (DAP I) were purchased from Invitrogen®. Mayer’s hematoxylin was purchased from Electron Microscopy Sciences®. Nuclear magnetic resonance (NMR) solvents deuterated dimethyl sulfoxide (DMSO-d6) and deuterium oxide (D2O) were purchased from Fisher Scientific.

[0136] Synthesis of glycidyl methacrylate-modified gelatin (GelMAG)

[0137] Gelatin was chemically modified with GMA to produce GelMAG through a one-step reaction. First, 10% (w / v) porcine gelatin was dissolved in DPBS at 60 °C while under constant, vigorous stirring to make a gelatin solution. Once fully dissolved, 0.16% (v / v) GMA was added dropwise at 60 °C under stirred conditions and left for four hours. The mixture was then diluted (2X) with DPBS to quench the methacrylation. The solution was dialyzed with a 12 to 14 kilodalton (kDa) molecular weight cutoff (MWCO) dialysis tubing for 7 days against deionized water at 50 °C to remove impurities like unreacted GMA and salts. The clear dialyzed solution was then frozen at -80 °C overnight and then lyophilized for

[0138] 7 days until a white foam-like solid was produced. GelMAG was stored at 4 °C.

[0139] Synthesis of methacrylic anhydride-modified dopamine (DMA)

[0140] Dopamine was chemically modified with MA to produce DMA using a modified synthesis that was previously reported. Briefly, dopamine hydrochloride at 5% (w / v) was produced in a mixture of borax and sodium bicarbonate solution (5:2) under nitrogenated conditions at room temperature. Subsequently 25% (v / v) of MA tetrahydrofuran solution (0.2 ml / ml) was added dropwise to the mixture. During the reaction, the pH was maintained above

[0141] 8 by adjustment with sodium hydroxide. The reaction continued overnight and then was washed in triplicate with ethyl acetate. Afterwards, the pH of the aqueous phase was reduced to below 2 by adjustment with hydrochloric acid and the organic layer was separated, and concentrated by rotary evaporator, and mixed with cool hexane to precipitate DMA. The precipitated DMA was then further purified by using cooled hexane, dried under vacuum conditions, and stored at 4 °C.

[0142] Preparation of GelMAG / DMA / pDDA (GDP) hydrogel

[0143] Hydrogels were prepared by dissolving 20% (w / v) GelMAG in a photoinitiator solution of 0.08% (w / v) Eosin Y, 0.9% (v / v) triethanolamine (TEA), and 0.9% (w / v) N-vinyl- Attorney Docket No. 50835-0006W01 s-caprolactam at 37 °C. Once fully dissolved, 0.1% (w / v) DMA was added, quickly mixed, and incubated overnight to conjugate DMA onto the GelMAG backbone. Next, pDDA was added at concentrations of 0.5, 1, or 2% (v / v) and quickly mixed to ensure homogenous distribution of the polyelectrolyte. The prepolymer solutions with GelMAG, DMA, and pDDA (GDP) or just GelMAG and pDDA (GP) were crosslinked in a poly dimethylsiloxane (PDMS) mold for 4 minutes with visible light (450-550 nm) using a LS1000 Focal Seal Xenon Light Source (100 mW / cm2, Genzyme).

[0144] Example 2 - Physicochemical Characterization Methods of GDP Hydrogels

[0145] Proton nuclear magnetic resonance (}H NMR) spectroscopy

[0146] 'HNMR analysis was conducted on prepolymer solutions and hydrogels to calculate both the degree of methacrylation (DM) and the degree of crosslinking (DC). Samples were frozen at -80 °C overnight, lyophilized for two days, and then fully dissolved with a concentration of 10 mg / mL in the NMR solvent at 50 °C. The spectra were obtained in deuterated dimethyl sulfoxide (DMSO-d6) using a 400 MHz Bruker AV400 spectrometer (64 scans). All spectra were processed with phase and baseline corrections and assigned a reference point at the residual singlet peak of DMSO-d6 at 2.54 ppm before analysis. The DM was calculated by finding the ratio of amine protons on the lysine residues on the GelMAG backbone to free amine protons on the gelatin backbone. The two vinylic protons residing on the methacryloyl group of GelMAG give rise to two separate singlet peaks at 5.74 and 6.13 ppm. The peak at 2.78 ppm corresponding to the primary amine protons in gelatin and the secondary amine protons in GelMAG are integrated to determine DM according to Eq. 1. 100 Eq. 1

[0147] 1H NMR analysis was conducted on dopamine hydrochloride and DMA with the same methods for sample preparation and spectra processing. The methacryloyl protons on DMA give rise to two peaks at 5.33 and 5.64 ppm.XH NMR analysis was also conducted on GelMAG prepolymer solution and hydrogels with and without DMA and pDDA to calculate the different DCs using Eq. 2. 100 Eq. 2 Attorney Docket No. 50835-0006W01

[0148] Their spectra were taken with the same spectrometer instrument and processed similarly to previous samples.

[0149] Scanning electron microscopy (SEM)

[0150] SEM images were taken of the hydrogel surface after bacterial culture. Hydrogels were washed thrice with DPBS, fixed in 2.5% (v / v) glutaraldehyde and 4% (v / v) paraformaldehyde, washed again, and then serially dehydrated in ethanol (30% to 100% (v / v)). The samples then underwent critical drying (Tousimis Autosam dri®-810 Critical Point Dryer), gold sputtering (Denton BenchTop™ Turbo-IV Evaporator), and SEM imaging (Zeiss Supra® 40 variable pressure (VP) SEM).

[0151] Zeta potential test

[0152] Zeta potential of prepolymer solutions (50 pg / mL in deionized (DI) water) was taken (Malvern Zetasizer® Nano-Z) with the universal DTS1070 folded capillary cells and DTS (Nano) software (version 4.20) at 25 °C. (N=4)

[0153] Conductivity test

[0154] Hydrogel conductivity was measured after photocrosslinking and dipping into DI water, which facilitated ionic conductance. The hydrogels were placed between two gold nanochips and conductivity was measured with CorrWare® potentiostat software for electrochemical analysis (-0.4 open circuit (OC) to 0.4 OC, 50 mV / S). The slope of electrical current and voltage data determined the conductivity (N=4).

[0155] Swelling ratio

[0156] Swelling ratios of all hydrogels were obtained by incubating hydrogels in DPBS at 37°C for 48 hours. The dry mass (Wo) and mass taken at predetermined time points (Wi) were used in Eq. 3 to assess the swelling profile. (N=4)

[0157] In vitro enzymatic degradation

[0158] Degradation ratios of all samples were obtained by incubating the hydrogels in collagenase type II at a concentration of 2 U / mL for four weeks at 37 °C. Collagenase Attorney Docket No. 50835-0006W01 solution was refreshed every two to three days to maintain constant degradation activity. The degradation profile was assessed using Eq. 4 and then normalized to account for initial swelling. (N=4) 100 Eq. 4

[0159] Example 3 - Mechanical Characterization Methods of GDP Hydrogels Tensile test

[0160] For tensile tests, hydrogels made of 80 pL precursor solution in a rectangular polydimethylsiloxane (PDMS) mold (8 mm length, 5 mm width, 1 mm depth) were crosslinked using the aforementioned procedure. Dimensions were confirmed using a digital caliper and then hydrogels were secured in tensile tape, placed in the Instron® 5944 mechanical tester, and pulled to failure at a strain rate of 1 mm / min while data were collected on Bluehill® Universal software. Tensile strength and stretchability were recorded at the stress and strain, respectively, at failure. Young’s modulus was defined as the slope of the initial linear portion of the stress-strain curve, specifically at 6-10% of maximum strain. Toughness was measured as the area under the stress-strain curve.

[0161] For compression tests, hydrogels made of 80 pL precursor solution in a cylindrical PDMS mold (5 mm diameter, 2 mm depth), where dimensions were confirmed with a digital caliper, were placed on the Instron® compression plates and compressed to failure at a rate of 1 mm / min while data were recorded Bluehill® Universal software. Compressive modulus was taken as the slope of the stress-strain curve at 97-99% of maximum strain. Cyclic compression testing was separately performed by compressing the samples to 50% at a rate of 1 mm / min for 12 cycles. Energy loss was measured from the last cycle using Eq. 5 where the loading curve represents the compressed sample and unloading curve is the decompressed sample. (N=4)

[0162] In vitro wound closure

[0163] Wound closure tests based on American Society for Testing and Materials (ASTM) F2458 were conducted with some modifications. Briefly, porcine skin tissue was rid of hair and fat, cut into rectangular pieces (3 cm x 1 cm), and secured with superglue to glass slides. At the junction of two tissue pieces, 100 pL of prepolymer solution was applied in a square (1 cm x 1cm) and visible light-cured for 4 minutes to prepare a hydrogel. The glass slides Attorney Docket No. 50835-0006W01 were fixed to the Instron® 5940 mechanical tester where they were pulled apart at a rate of 1 mm / min until hydrogel failure. Adhesion strength data were collected with Bluehill® Universal software at maximum stress and the adhesion energy was taken as the area under the curve of force versus displacement. (N=4)

[0164] Burst pressure testing

[0165] Burst pressure tests based on ASTM F2054 were conducted with some modifications. A custom-built burst pressure device consisting of a steel base, top holder, syringe pump, pressure sensor, and computer with data collecting software (PASCO Capstone™) was assembled. A dry collagen sheet was dampened in water and dried thoroughly with a Kimwipe™ wipe. The collagen sheet was secured in the holder and a puncture (diameter: 1 mm) was made in the center of the collagen sheet. Then 60 pL of prepolymer solution was applied and photocured to form a hydrogel. The simulated sealed wound was then pressurized with 10 mL / min of air flowrate through the syringe pump. (N=4)

[0166] Ex vivo burst pressure on ventilated pig lung

[0167] Burst pressure tests based on ASTM F2054 were conducted on a ventilator-assisted pig lung. The pig lung was connected to a Veterinary Anesthesia Ventilator (Hallowell EMC) regulating compressed air at a respiratory rate 12 bpm and a maximum working pressure of 60 cmH20. The ventilator was connected to a pressure sensor and computer with data collecting software (PASCO Capstone™). Injuries (lacerations and punctures of various dimensions) were made on the lung and sealed with hydrogel. Respiratory volume was steadily increased until an air leak was detected by submersion of the lung into a water bath. (N=4)

[0168] Example 4 - Bacteria Survival and Zone of Inhibition

[0169] Two strains of bacteria (P. aeruginosa and MRS A) were cultured. A bacterial broth was prepared by inoculating one colony of each strain into their respective culture media and incubating overnight at 35 °C. Bacterial concentration was assessed with optical density (OD) readings at 625 nm using the Biotek™ Eon Microplate Spectrophotometer. For the survival assay, both bacterial broths were diluted to OD 0.06, sterilized hydrogels were added to the suspension, and the cultures were incubated at 35 °C. Bacterial survival was measured at specific timepoints with OD measurements. Colony forming units (CFU) were calculated by plating the hydrogel-treated bacterial suspension on to agar plates and incubating overnight at Attorney Docket No. 50835-0006W01

[0170] 35 °C. The CFU were counted on the following day. For the zone of inhibition test, a bacterial suspension with OD 0.06 was evenly spread onto an agar plate. Cylindrical hydrogels (diameter: 5 mm, height: 2 mm) were prepared, submerged in PBS for one hour, lightly dried, and then placed onto the agar plate. The plates were incubated overnight at 35 °C. The following day, the zone of inhibition, formed between the edge of the hydrogel and the start of the bacteria coverage, was measured using a digital caliper. (N=3)

[0171] Example 5 - In Vitro Hemostatic Test

[0172] Citrated human fresh whole blood (ZenBio, Inc.) from a diabetic patient was activated with 0.1 M calcium chloride in a 9: 1 ratio (blood CaCh). Before activating the blood, equal amounts of hydrogel prepared at the bottom of a well plate were prewarmed for 5 min. The activated blood mixture was vigorously stirred for 5 seconds and then added to the well plates. At specific time points, the blood clotting was quenched with saline solution and nonclotted liquid was removed to determine clotting time. Simultaneously, 2 pL of the nonclotted liquid was placed into the Nanodrop™ One / OnecMicrovolume UV-Vis spectrophotometer (ThermoFisher Scientific) to measure hemoglobin concentration at 540 nm, representing clot density. A baseline correction at 750 nm was included for the entire spectrum. Blood clotting index (BCI) was determined at for all samples at the timepoint when the fast clotting sample had fully clotted using Eq. 6 where A is absorbance at 540 nm. Clotting weight was measured at this same timepoint. (N=4)

[0173] Example 6 - In Vitro Biocompatibility Tests

[0174] Cellular biocompatibility studies were conducted on mouse embryonic lung fibroblast cells (3T3-Swiss albino cell line, ATCC®). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, ATCC®) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin antibiotics. Fibroblast cells were cultured on a Falcon® polystyrene tissue culture flask with a vented cap (Coming, Inc.) and incubated at 37 °C with 5% CO2 infusion. Cells were cultured with GelMAG (G) and GelMAG / DMA / 2% pDDA (G / D / p2) as well as without hydrogel treatment as a control. Cells were seeded at a density of 1 x 104cells / mL in 24-well plates, hydrogels were loaded into a transwell insert, and both cells and hydrogels were covered entirely with DMEM.

[0175] Viability of 3T3 cells was measured using a live / dead kit (Invitrogen®) according to manufacturer instructions. Briefly, cells were stained with 0.05% Calcein-AM and 0.2% ethidium homodimer-1 in DPBS for 20 min at 37°C. Fluorescent imaging was performed on Attorney Docket No. 50835-0006W01 days 1, 3, and 5 post-seeding using AxioObserver Z1 inverted microscope (Carl Zeiss AG). Live cells appeared green and apoptotic cells appeared red. Cell viability was determined by dividing the number of live cells by the total cell count. (N=3)

[0176] Spreading and morphology of the 3T3 cells were observed by conducting Actin / DAPI staining. Cells were first fixed with 4% (v / v) paraformaldehyde (10 min), washed with 0.5% (v / v) Triton-X (10 min), and then blocked with 1% (v / v) BSA (30 min). Cells were then stained with 0.1% (v / v) Phalloidin and 0.05% (v / v) DAPI in DPBS (10 min), washed three times with DPBS, and then imaged with the AxioObserver Z1 inverted microscope (Carl Zeiss AG). Actin filaments were stained green and nuclei were stained blue. Cell spreading was quantified by positively stained F-actin per unit area. (N=3)

[0177] As evidenced by the apparent increasing amounts of nuclei per unit area, the cell number after exposure to GDP increased from 408 ± 125 cells / mm2at day 1 to 1998 ± 272 cells / mm2at day 5 (P < 0.0001) (FIG. 36). Metabolic activity was quantified using a PrestoBlue™ assay (Life Technologies™). Cells were incubated with 10% (v / v) PrestoBlue™ solution in DMEM 45 minutes at 37 °C. Then, fluorescence was measured at 600 nm using Gen5 3.04 Microplate Reader and Imaging Software and Synergy LX Multimode Reader. (N=3)

[0178] Example 7 - In Vivo Biocompatibility, Biodegradation, and Hemostasis Tests in Rats

[0179] Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) (ARC-2021-113) at University of California at Los Angeles (UCLA). Male Wistar rats (-200 g) were purchased from Charles River Laboratories (Boston, MA). Rats were given anesthesia by inhalation of isoflurane (4%) which was maintained at 1.5% during surgery. To assess biocompatibility and biodegradability with subcutaneous implantations, eight 1 cm incisions were made on the dorsal skin of the rats and subcutaneous pockets were created with blunt, curved scissors. Hydrogels, which were prepared in a sterile manner in a cylindrical mold (5 mm diameter, 2 mm depth), lyophilized, and UV-sterilized, were implanted into the pockets before incisions were closed with 4-0 polypropylene sutures (AD Surgical). At days 7 and 28 post-operation, rats were euthanized by CO2 asphyxiation and hydrogels were explanted with or without tissue for histological analysis or biodegradation measurements, respectively.

[0180] To assess hemostatic efficiency in a liver hemorrhage model, rats received the same general anesthesia and underwent a median laparotomy which exposed the liver and surrounding area using a wound retractor. A 2 mm deep puncture was performed on the liver Attorney Docket No. 50835-0006W01 and immediately treated with sterile prepolymer solution that was photocured into a hydrogel. An injury group with no hydrogel treatment was used as a control. Filter paper was used to collect blood for 10 minutes post-injury and was later dried to measure total blood loss.

[0181] Afterwards, the abdominal wound was closed by suturing (4-0 polypropylene) the peritoneum and abdominal skin separately. At day 14 post-operation, rats were euthanized and hydrogel with surrounding tissue were explanted for histological analysis.

[0182] Hemostatic performance was also performed on a tail amputation model, wherein the rats received general anesthesia before 6 cm of the tail were transected. Hydrogel was immediately applied while the injury control group received no hydrogel treatment. Blood loss was collected on filter paper for 10 minutes. Rats were euthanized before recovering from anesthesia.

[0183] Histological analyses were conducted on explanted hydrogels (and tissue) to assess biocompatibility through the natural inflammatory response. Explanted hydrogels were first fixed in 4% (v / v) paraformaldehyde for four hours and then incubated in 15% followed by 30% (w / v) sucrose solution at 4 °C overnight. Hydrogels were embedded in Optimal Cutting Temperature (O.C.T) compound, frozen in liquid nitrogen, and then sectioned into 10 pm thick sections using a Leica CM1950 cryostat. The sections were contained on positively charged slides for hematoxylin and eosin (H&E) staining and immunohistological (IMH) staining. IMH staining was performed using anti-CD68 and anti-CD45 as primary antibodies (Abeam, Inc.) with Alexa Fluor 594-conjugated Goat anti-Rabbit IgG (H+L) (Invitrogen™) as a secondary antibody. All samples were stained with DAPI and fluorescence imaging was performed using the Axi oOb server Z1 inverted microscope (Carl Zeiss AG).

[0184] Statistical analysis

[0185] Results were presented as mean ± standard deviation (SD) (*P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001). One-way or two-way analysis of variance (ANOVA) t-tests were performed followed by Tukey’s test for statistical analysis (GraphPad Prism® Version 8.4.3).

[0186] Example 8 - Formation and Physical Characterization of GDP Hydrogel

[0187] The GDP hydrogel was formed by first synthesizing its constituents: GelMAG, DMA, and pDDA. GelMAG was synthesized through a one-step reaction between gelatin and glycidyl methacrylate (GMA) (FIG. lA-i). The synthesis of GelMAG was confirmed by proton nuclear magnetic resonance (1H NMR) through the emergence of peaks at 5.74 and 6.13 ppm corresponding to the two vinylic methacryloyl protons (FIG. 10). Furthermore, the Attorney Docket No. 50835-0006W01 amine protons on GelMAG lysine residues, present at 2.78 ppm, indicated a 50% degree of methacryloyl substitution (Eq. 1). Similarly, DMA was synthesized through a reaction between methacrylic anhydride (MA) and dopamine hydrochloride (FIG. lA-ii). Two methacryloyl proton peaks at 5.33 and 5.64 ppm confirmed the production of DMA (FIG. 11). Lastly, poly electrolyte pDDA was utilized for its cation- saturated backbone that likely provided the composite with hemostatic and antibacterial properties (FIG. lA-iii).

[0188] The combination of GelMAG, DMA, and pDDA in a photoinitiator solution of Eosin Y, triethanolamine (TEA), and N-vi ny I caprolactam (VC) formed hydrogels after 4 min of photocrosslinking with visible light (FIG. IB). The surface charge was taken of pDDA as well as GelMAG-pDDA (GP) and GDP hydrogels in anionic precursor solution (FIG. 1C). Even in hydrogels containing negatively charged DMA, the surface charge drastically increased after adding higher concentrations of pDDA. Numerous forces strengthened the resulting hydrogel including covalent bonding between GelMAG methacryloyl residues, electrostatic interactions between pDDA cations and charged functional groups on GelMAG, and cation-7t interactions between the quaternary ammonium of pDDA and aromatic ring of DMA (FIG. ID)

[0189] JH NMR of GDP prepolymer and hydrogel showed that the incorporation of DMA resulted in aromatic proton peaks at 7.44 ppm while the addition of pDDA resulted in quaternary ammonium-adjacent methyl proton peaks at 2.97 ppm (FIG. lE-i). Photocrosslinking diminished the methacryloyl proton peaks of GelMAG at 5.70 and 6.08 ppm, indicating hydrogel formation and covalent bond formation between adjacent moi eties and, thereby, hydrogel formation. However, the presence of either pDDA or DMA in GelMAG-pDDA (GP) or GelMAG-DMA (GD) hydrogels, respectively, seemed to reduce the extent of methacryloyl peak consumption, indicating lower degrees of crosslinking (FIGs. lE-ii and FIG. 27). The crosslinking density of GelMAG hydrogels containing either DMA or pDDA decreased, likely due to their steric blocking of methacryloyl covalent selfinteractions (FIGs. 12-14). Accordingly, the GelMAG hydrogel was characterized to have 85% degree of crosslinking, which was higher than the 64% or 59% degree of crosslinking in the GP or GD gels, respectively (FIG. IF). Nevertheless, when all three components were combined to form the GDP precursor, there was a higher (87%) degree of crosslinking in the resulting hydrogel.

[0190] The degree of crosslinking decreased by 21.2% and 25.5% when pDDA or DMA, respectively, were added to GelMAG hydrogels (FIG. IF). In another study, decreased degree of crosslinking was observed when free and self-polymerized dopamine was added to Attorney Docket No. 50835-0006W01 a gelatin and quatemized chitosan matrix, weakening the mechanical and adhesive properties of the hydrogel. On the contrary, the GDP composite displayed an abundance of electrostatic and cation-7t interactions that increased the degree of crosslinking in the matrix to 87.2% (FIG. IF). Not only did the DMA-pDDA interactions free up methacryloyl residues for covalent bonding, but they also reduced the scavenging effect DMA had on the photoinitiators to permit less obstructed photocrosslinking. Therefore, a robustly entangled matrix was formed within the GDP adhesive.

[0191] Numerous physical and chemical interactions between the constituents, including covalent bonding between GelMAG methacryloyl residues as well as cation-7t interactions between pDDA and DMA, likely occurred during the crosslinking of the GDP sealant (FIG. ID). The possibility of physical crosslinking in the hydrogels via electrostatic interactions was assessed through zeta potential measurements of various prepolymer solutions (FIG. 1C)

[0192] The photoinitiator solution (EosinY / TEA / VC), GelMAG, and GD prepolymers all exhibited negative zeta potentials while pure pDDA as well as prepolymer solutions containing pDDA had increasingly positive zeta potentials corresponding to higher pDDA concentration. Due to the high surface charge of pDDA-containing solutions, there was likely electrostatic interactions in their resulting hydrogels. In addition to increasing the extent of physical crosslinking, the presence of both pDDA and DMA also impacted the ionic conductivity of the GDP composite. Compared to GelMAG (0.11 ± 0.017 S / m), GD hydrogels exhibited 0.43 ± 0.065 S / m conductivity, likely due to ability of anionic DMA to form hydrogen bonding and electrostatic interactions with water (P < 0.001) (FIG. 3G).

[0193] After the addition of 1% and 2% (v / v) pDDA to the GDP hydrogels, their conductivities increased to 0.78 ± 0.13 S / m (P < 0.001) and 0.68 ± 0.051 S / m (P < 0.01), respectively. In addition to their higher zeta potential compared to gels without pDDA, the GDP hydrogels likely exhibited higher ionic conductivity due to the doping effect that DMA has on pDDA (77). Since the conductivity of GDP containing 2% (v / v) pDDA was within the range of native electroactive tissues like muscle (0.04-0.5 S / m), cardiac (0.5 S / m), and nerve (0.08-1.3 S / m), it could ultimately facilitate cellular communication and tissue regeneration on their diverse biophysical environments (72).

[0194] In order to validate the potential chemical interactions that could occur within the hydrogel upon photocrosslinking, we conducted Fourier transform infrared (FTIR) spectroscopy on GelMAG, GP, GD, and GDP gels (FIG. 28). There were consistent peaks across all samples, indicating that modifying GelMAG with DMA or pDDA did not alter the Attorney Docket No. 50835-0006W01 bulk chemical backbone. In particular, all hydrogels contained the broad peak around 3300 cm'1, representative of O-H or N-H hydrogen bond stretching vibrations. Also, all samples contained the characteristic amide peaks (1650 and 1540 cm'1) that are associated with gelatin. GDP seemed to have lower transmittance of a peak at 600 cm'1, which is absent in all other formulations and may be due to non-covalent bonding between the aromatic group of DMA and the ammonium group of pDDA. The bands corresponding to the resulting cation-7t bonds could exist in the fingerprint region (73).

[0195] Example 9 - Mechanical Characterization of GDP Hydrogels

[0196] Hydrogels developed for wound healing must adapt to tissue terrains of dynamic rigidity and mobility. However, there are limited multifunctional hemostatic bioadhesives with biomimetic mechanical properties that support the physiological activity of elastic organs. Even commercialized tissue sealants such as TISSEEL, CoSeal™, and Dermabond® have very limited mechanics (less than 10 kPa strength and 15% stretchability), increasing the possibility of inadequate wound closure. Herein, to emphasize the conformability of GDP adhesive to injuries on soft, sturdy, static, or motive organs, the mechanical properties were characterized using a universal testing machine. Pure GelMAG possessed a stiff matrix, rendering a Young’s modulus of 176 ± 10 kPa which decreased to 79 ± 3 kPa in GelMAG- DMA (GD) (P < 0.0001) (FIGs. 2A).

[0197] To assess the conformability of GDP sealants to injuries on dynamic organs, their mechanical properties were characterized using a rheometer and an Instron mechanical tester. Time sweet rheological measurements were first conducted to monitor the evolution of the storage modulus (G’) and loss modulus (G”) during hydrogel formation (FIG. 24A). After 4 min or crosslinking with visible light, the moduli of all hydrogel formulations stabilized at around 50-70 kPa, indicating rapid and efficient crosslinking. Since no visible difference was noted between GelMAG, GP (with 2% v / v pDDA), GD (with 0.1% w / v DMA), and GDP (with 0.1% w / v DMA and 2% v / v pDDA) hydrogels, it could be inferred that neither the bulk hydrogel mechanics or the gelation kinetics were impacted by the additional components. On the other hand, frequency sweep measurements revealed more substantial differences in mechanical behavior between the hydrogels (FIG. 24B). While all samples exhibited higher G’ than G” across a broad frequency range, indicating predominantly elastic behavior, the GelMAG, GP, and GDP hydrogels maintained the highest and most frequency -independent G’ values, emphasizing their structural integrity throughout dynamic stress and reduced susceptibility to network relaxation. Similarly, strain sweep measurements provided insight Attorney Docket No. 50835-0006W01 into the structural resilience of the hydrogels. While GelMAG, GP, and GDP gels maintained their elastic moduli within the linear viscoelastic region until 40-50% strain before structure breakdown, the GD hydrogel underwent mechanical failure around 15% strain (FIG. 24C). Extended linear viscoelastic regions could indicate higher crosslinking density and network cohesion, allowing the materials to accommodate substantial deformation before failure. Their ability to withstand higher strain supports their application in surgical settings where they must endure dynamic tissue deformations.

[0198] The decrease in crosslinking density after grafting DMA onto GelMAG resulted in a softer hydrogel (FIG. IF). The optimal GDP composite containing 0.1% (w / v) DMA and 2% (v / v) pDDA resulted in further softening of the matrix to 36 ± 2 kPa, within the range of muscle, cartilage, and most organs. Increasing the polyelectrolyte concentration in the hydrogels seemed to impart greater ductility while simultaneously enhancing ultimate strength, likely due to the greater amount of electrostatic and cation-7t interactions that reinforced the matrix. Therefore, even though an initial decrease in ultimate strength was observed after adding low concentrations (0.5% (v / v)) of pDDA to GP and GDP, increasing pDDA to 2% (v / v) bolstered ultimate strength in both hydrogels (FIG. IB). Therefore, the optimal GDP hydrogel displayed an ultimate strength of 100 ± 8 kPa.

[0199] Conjugating GelMAG and DMA decreased ultimate strength due to the impacted crosslinking density, but it also greatly enhanced hydrogel stretchability. GD, with an ultimate strain of 201 ± 8%, was able to dissipate the applied stress better than GelMAG (83 ± 7%) since it contained more non-covalent interactions and longer sidechains (P < 0.0001) (FIG. 2C). The GDP hydrogels displayed a slight initial decrease followed by subsequent increase in stretchability after adding higher amounts of pDDA, exemplified by 208 ± 15% extension in the ideal GDP composite (which contained 0.1% (w / v) DMA and 2% (v / v) pDDA). Energy dissipation through the plentiful but weak chemical interactions (e.g., hydrogen bonding, electrostatic interactions) not only enhanced stretchability but also produced a tough hydrogel. Even though the toughness of GelMAG (55 ± 9 kPa) decreased to 22 ± 3 kPa in GD, increasing pDDA concentration significantly enhanced toughness due to the exponentially increased amounts of electrostatic interactions (P < 0.05) (FIG. 2D). Therefore, the optimal GDP composite retained a toughness of 93 ± 12 kPa. Adding higher amounts of pDDA to GDP also decreased the compression modulus since the material became softer, resulting in a biologically relevant modulus of 47 ± 8 kPa (FIG. 2E). Furthermore, the optimal GDP hydrogel displayed 25 ± 4% energy loss after 12 cycles of compression, which was only slightly higher than GelMAG (14 ± 2%) (P < 0.05) (FIG. 2F). Attorney Docket No. 50835-0006W01

[0200] After adding 0.5% (v / v) pDDA to GDP sealant, the toughness dropped to 60 ± 5.2 kJ / m3(P < 0.001), and after increasing pDDA concentration in GDP to 2% (v / v), the toughness rose to 96 ± 8.3 kJ / m3(P < 0.001). Although adding DMA resulted in higher toughness, the ultimate strength of GD (102 ± 4.68) was lower than that of pure GelMAG (140 ± 13 kPa) (P < 0.01) (FIG. 29). Adding 0.5% (v / v) pDDA to GD hydrogel further reduced the ultimate strength but increasing pDDA concentration to 2% (v / v) restored its properties so that the GDP sealant retained 101 ± 6.5 kPa ultimate strength. Furthermore, since the lungs undergo continuous expansion and contraction, we assessed the extent of energy loss of the GDP hydrogels after cyclic deformation. Compared to the 10 ± 0.48% energy loss of pure GelMAG after 12 cycles of compression, the GDP sealant exhibited 22 ± 1.3% energy loss, both of which are relatively low (P < 0.05) (FIG. 30).

[0201] Mechanical mismatch is a limitation for many tissue sealants and hemostatic biomaterials. For example, commercial pleural air leak sealant Progel™, which claims high elasticity to support lung expansion, is notoriously known by surgeons to underperform. With only about 40 kPa ultimate strength and 25% stretchability, Progel™ is unsuited for wound sealing on most elastic organs. Furthermore, many tissue sealants utilizing catecholcontaining compounds to enhance adhesion suffer from weak mechanical properties. Therefore, the composition of the GDP adhesive was successfully tuned in order to balance catechol retention while rendering a mechanically soft, strong, stretchable, and tough hydrogel.

[0202] Example 10 - In Vitro and Ex Vivo Adhesion Characterization of GDP Hydrogel

[0203] Alongside mechanical tunability, robust adhesion of hydrogels to wet biological surfaces is essential for reliable wound sealing. Commercial sealants CoSeal™, Progel™, and Evicel® claim to be strong wet adhesives, but their adhesion strengths were very weak (under 15 kPa), which can dangerously complicate the treatment of hemorrhaging injuries. To display the strong adhesive properties of GDP, standard in vitro wound closure (ASTM F2458) and burst pressure (ASTM F2054) tests were performed. During wound closure, hydrogels glued two pieces of pig skin that were pulled apart by a universal tester (FIG. 3A). Adhesion energy was also found to be higher for GDP hydrogels (18 ± 3.6 J / m2) compared to GelMAG (7.6 ± 0.11 J / m2) or various GP samples (P < 0.05) (FIG. 31). Similar trends were observed during burst pressure test using punctured and pressurized collagen sheets. The adhesion strength of GD (40 ± 3 kPa) was significantly increased from that of GelMAG (25 ± 1 kPa) since DMA is a derivative of L-DOPA, a chemical responsible for the durable Attorney Docket No. 50835-0006W01 underwater adhesion of marine blue mussels (P < 0.0001) (FIG. 3B). After adding pDDA to GDP adhesives, the strength initially decreased but then further increased to 45 ± 1 kPa for the composition containing 2% (v / v) pDDA. All hydrogels displayed far higher adhesion strengths as compared to CoSeal™ (2 ± 0.3 kPa) and Progel™ (1 ± 0.7 kPa). Similarly, adhesion energy considerably increased from 9 ± 1 J / m3for GelMAG to 43 ± 5 J / m3for GD (P < 0.0001) (FIG. 3C). While GelMAG can form covalent and hydrogen bonding interactions with tissue moieties (e.g., hydroxyl, amino, thiol groups), there is more potential for adhesive interactions (e.g., Michael addition, Schiff base, electrostatic interactions) with the presence of DMA and pDDA (FIG. 25E).

[0204] After adding pDDA to GDP, adhesion energy between hydrogel and tissue decreased since matrix cohesion and potential for energy dissipation were increased. However, the adhesion energies of all hydrogels still surpassed that of CoSeal™ and Progel™. In DMA- containing hydrogels, both unoxidized catechol and oxidized quinone residues of DMA engaged with the tissue surface through Michael addition, hydrogen bonding, Schiff base, and covalent interactions which resulted in stronger adhesion properties (FIG. 3D). Furthermore, the substantial ^-conjugated electron density of DMA was thought to increase the strength of cation-7t interactions between DMA and PDDA in an aqueous environment, which is why the optimal GDP composite displayed such robust adhesion.

[0205] The adhesive properties of the GDP sealants were further characterized using ex vivo burst pressure tests on freshly isolated pig lungs. The unpunctured lungs were attached to a ventilator where a defect was made and sealed with the engineered hydrogels (FIG. 4Aii). Then, the lungs were cyclically pressurized with air and hydrogel burst pressure was recorded using a Pasco Capstone software (FIG. 25A). The optimized GDP sealant containing 0.1% (w / v) DMA and 2% (v / v) pDDA sealed a shallow pleural defect with 0.5 mm depth and 10 mm diameter with a burst pressure of 5.4 ± 0.06 kPa, which was significantly higher than the burst pressure of GelMAG at 3.7 ± 0.12 kPa (P < 0.001) and Evicel at 1.4 ± 0.36 kPa (P < 0.0001) (FIG. 25B). Burst pressure testing was also performed by sealing punctured and pressurized collagen sheets with hydrogels (FIG. 3E).

[0206] In a similar trend, the presence of both 0.1% (w / v) DMA and 2% (v / v) pDDA within GDP bolstered the burst pressure to 51 ± 5 kPa, which was much higher than that of GelMAG (29 ± 4 kPa) (P < 0.0001) (FIG. 3F). The optimized GDP sealant containing 0.1% (w / v) DMA and 2% (v / v) pDDA sealed a shallow pleural defect with 0.5 mm depth and 10 mm diameter with a burst pressure of 5.4 ± 0.06 kPa, which was significantly higher than the burst pressure of GelMAG at 3.7 ± 0.12 kPa (P < 0.001) and Evicel at 1.4 ± 0.36 kPa (P < Attorney Docket No. 50835-0006W01

[0207] 0.0001) (FIG. 25B). All engineered hydrogels displayed higher burst pressures than CoSeal™ (5 ± 0.6 kPa) and Progel™ (8 ± 1 kPa) (P < 0.0001) (FIG. 3F).

[0208] The strong tissue adhesion displayed by GDP adhesives far surpassed not only CoSeal™ and Progel™, but also other reported biomaterials using dopamine derivatives. For example, gelatin and dopamine that were coupled using l-ethyl-3-[2-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) had a burst pressure of only 19 kPa. Similarly, a conductive hydrogel based on gelatin-dopamine, hyaluronic acid, and polypyrrole had under 18 kPa adhesion to pig skin.

[0209] Here, dopamine was chelated to ferric ions which decreased capacity for tissue interactions. When hyaluronic acid was combined with dopamine and tannic acid, an adhesion strength of 11 kPa was reported. Even when synthetic polyacrylamide, often used for its superior mechanics and adhesion, was crosslinked with polydopamine to yield a highly stretchable hydrogel, only 15 kPa adhesion was noted. Therefore, to fully utilize the adhesion potential of catechol-containing compounds, the chemical microenvironment of the hydrogel must be carefully manipulated. In many cases, the adhesive moieties of dopamine were engaged within the matrix and unable to interact with tissue surfaces. Here, the cation-7t interactions in GDP strengthened the connection between pDDA and the aromatic ring of DMA but left the catechol groups free for tissue adhesion. Because of that, GDP showcased its potential to adhere to tissue surfaces better than most hemostatic biomaterials.

[0210] The addition of polyelectrolyte pDDA also imparted notable ionic conductivity to GDP, making it physically adaptable to electroactive tissues. The conductivity of GP adhesives with higher concentrations of pDDA was slightly but steadily increased from that of GelMAG (0.11 ± 0.01 S / m) (FIG. 3G). In the GD hydrogel, anionic DMA enhanced ionic conductivity to 0.43 ± 0.06 S / m due to its greater potential for hydrogen bonding and electrostatic interactions with water molecules (FIG. 3H). As for GDP adhesives with higher concentrations of pDDA, conductivity was significantly enhanced to 0.78 ± 0.13 S / m (for GDP with 1% (v / v) pDDA) and 0.68 ± 0.08 S / m (for GDP with 2% (v / v) pDDA). Both conductivity and surface charge were bolstered in GDP adhesives since DMA acted as a doping agent for pDDA (FIG. 15). The insignificant decrease in conductivity of the optimal GDP adhesive with 2% (v / v) pDDA was likely due to a saturation of DMA-induced doping and subsequent quenching of pDDA cations with DMA anions. GDP conductivity was within the range of native electroactive tissue (e.g., skin, muscles, cartilage, nerve).

[0211] Unlike other developed electroactive biomaterials, retaining strong conductivity in GDP adhesives notably did not affect other essential characteristics such as mechanics, Attorney Docket No. 50835-0006W01 adhesion, or biocompatibility. Previously, electropolymerized polydopamine and polypyrrole displayed 0.45 S / m conductivity, but the application was ultimately limited by weak mechanics (under 10 Pa strength and 10% strain) and poor adhesion (38 Pa). Another conductive hydrogel using methacrylated silk and polyacrylic acid polymerized with dopamine, ferric ions, and peroxide, possessed suitable mechanics but provided only 10 kPa adhesion. Hydrogels also harnessed conductivity through the incorporation of carbon-based materials. A dopamine-grafted gelatin hydrogel combined with graphene oxide (GO) exhibited high conductivity but had weak mechanical and adhesive strength (both under 20 kPa). In another study, gelatin-dopamine was combined with chitosan and polydopamine- grafted carbon nanotubes (CNTs) to produce a conductive hydrogel but it had limited adhesion (under 7 kPa in wet conditions). Furthermore, inorganic materials like GO and CNTs pose a strong risk of cytotoxicity, which can limit their clinical application. On the contrary, the GDP adhesive possessed biomimetic conductivity as well as excellent mechanical and adhesive properties that can promote wound sealing and repair on elastic and dynamic organs.

[0212] The adhesive properties of GDP were further exemplified in ex vivo lung injury models using ventilated pig lungs. The unpunctured lungs were attached to a ventilator where a defect was made and sealed with the hydrogel (FIGs. 4A - i-iv). Then, the lungs were pressurized with air and hydrogel burst pressure was recorded using a PASCO Capstone software (FIG. 4B). The optimal GDP composition (with 0.1% (w / v) DMA and 2% (v / v) pDDA) sealed a shallow pleural defect with 0.5 mm depth and 10 mm diameter with a burst pressure of 5.4 ± 0.1 kPa (FIG. 4C). This was significantly higher than the sealing efficacy of GelMAG (3.6 ± 0.1 kPa) and CoSeal™ (2.5 ± 0.4 kPa) (P < 0.001 between GDP and GelMAG and P < 0.0001 between GDP and CoSeal™). Larger and deeper injuries mimicking stab or bullet wounds were also tested to exemplify the air-tight sealing potential of GDP adhesive for clinical translation. Lacerations with 2.5 mm depth and 10, 15, or 20 mm length were created and sealed (FIG. 4D). Punctures of 1.5 mm depth and 10, 15, or 20 mm diameter were also tested (FIG. 4E). As pressure in the lungs was gradually and cyclically increased, GDP effectively sealed lacerations of 10 mm and 15 mm length without affecting the maximum pressure a pig lung can withstand (FIG. 4F). For the 20 mm laceration, a slight decrease in burst pressure was observed as the hydrogel detached after multiple cycles of ventilation at 3.5 kPa. When applied on deep punctures, GDP sealed a 10 mm diameter injury without affecting normal lung expansion. Only a slight drop in burst pressure was observed when GDP sealed a 15 mm diameter puncture. Therefore, GDP Attorney Docket No. 50835-0006W01 adhesive showcased robust adhesion and desirable air-leak sealing of large injuries on highly elastic organs.

[0213] Previous studies have also tested the sealing efficacy of biopolymer-based sealants on 10 mm wide pleural defects on ex vivo pig lungs. A methacrylated tropoelastin (MeTro) sealant, resembling the human elastin protein, displayed only 2.92 ± 0.49 kPa burst pressure for this type of injury. Meanwhile, GDP adhesive had far higher burst pressures against this shallow pleural defect as well as against injuries of larger dimensions, showcasing its sealing efficacy of clinically relevant traumatic wounds.

[0214] In order to observe the robust adhesiveness of GDP on various biological surfaces, we conducted scanning electron microscopy (7S) imaging on the intersections of GDP-sealed tissues. For example, we crosslinked GDP on either freshly isolated soft porcine lung tissue (FIG. 25C) or stiff porcine skin tissue (FIG. 25D), and in either case, the engineered hydrogel displayed bioadhesion through mechanical interlocking with the tissue surfaces. The porous GDP matrix seamlessly integrated with the tissue, supporting its application as a surgical sealant for various topologies, regardless of their stiffness.

[0215] Example 11 - Fast Acting and Long-Term Antibacterial Ability of GDP Hydrogels

[0216] Traumatic injuries incur significant risk of sepsis, which is known to increase medical cost three-fold and is responsible for 10% of mortality after trauma, thus demanding better therapeutics. Many hydrogels utilize charged polymers (e.g., chitosan, polyaniline) and / or inorganic compounds (e.g., CNTs, GO, kaolin, zeolites) to provide bactericidal wound healing platforms. Although their charge-dense nature facilitates antibacterial activity, it may also result in cytotoxicity.

[0217] To emphasize the rapid bactericidal nature of GDP, antibacterial tests were conducted against widely pathogenic Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). Bacteria that was not treated with hydrogels was used as a control, which had considerable viability and colony forming units (CFU) (FIGs. 5A-5B).

[0218] GDP hydrogels prepared with varying concentrations of DMA (0-0.1% w / v) and pDDA (0-2% v / v) were incubated with either strain of bacteria for a period of 5 days, after which bacterial survival was measured either through spectrophotometric analysis of optical density (OD) at 625 nm or using the spread plate method to count colony forming units (CFU). The broad-spectrum antibiotic ciprofloxacin was used against both bacteria as a positive control, and a commercial wound dressing AquaDerm™ was also compared to the Attorney Docket No. 50835-0006W01 engineered GDP hydrogels. While the untreated / 5, aeruginosa continued to proliferate throughout the incubation period, bacteria cultured with any formulation of GDP hydrogels, antibiotics, or AquaDerm™ experienced lower OD (FIG. 26A). Nevertheless, GelMAG, GP, GD, and GDP hydrogels containing lower concentrations of pDDA (0.5% v / v) could not inhibit bacterial growth, resulting in high OD (0.5-1.1) by day 5. AquaDerm™ was also unable to inhibit bacterial proliferation, causing around 0.70 OD. On the other hand, GP and GDP hydrogels with higher concentrations of pDDA (1 or 2% v / v) exhibited low OD (0.1- 0.2) at day 1, which further decreased by day 5 of the assay. However, ciprofloxacin seemed to completely diminish OD from the start of the incubation, indicating minimal bacterial growth. After the 5-day culture, a spread plate method was utilized to count CFU (FIG. 32A), which was then used to calculate concentration (CFU / mL), survival rate, and log reduction of viable bacteria. The untreated / 5, aeruginosa control was 5.6 x 106CFU / mL whereas GDP treatment resulted in 8.7 x 105CFU / mL (P < 0.0001) (FIG. 32B), corresponding to a bacterial survival rate of 14.9 ± 6.4% (FIG. 26B) and a log reduction of 0.98 ± 0.25 (FIG. 32C). The antibacterial efficacy of GDP was only slightly lower than that of ciprofloxacin, which experienced 2.7 x 105CFU / mL, corresponding to 4.6 ± 1.9% survival (P = 0.99) and 1.5 ± 0.2 log reduction (P < 0.05). Bacteria treated with GDP exhibited a significantly lower survival rate compared to GelMAG (71.3 ± 8.2%, P < 0.001), GD (70.1 ± 7.8%, P < 0.001), or AquaDerm™ (59.8 ± 19%, P < 0.01). Furthermore, representative SEM images from the surface of GelMAG and GDP sealants after the antibacterial assay revealed far less P. aeruginosa present on the pDDA-containing hydrogel (FIG. 26C). GDP also caused a 10 mm zone of inhibition (ZOI) against / 5, aeruginosa, further demonstrating its bactericidal properties (FIG. 5D). Culturing the hydrogels with MRSA elicited a similar response. While GelMAG exhibited similar OD to the untreated control, GP and GDP with higher pDDA concentrations stopped proliferation after 1 day (FIG. 26C). They acted in a comparable manner to ciprofloxacin, which could immediately obstruct growth, and better than GelMAG, GD, GDP with lower pDDA concentration and AquaDerm™ treatments. After the 5-day culture, the untreated MRSA control was at a concentration of 7.5 x 106CFU / mL, whereas GDP treatment led to 7.3 x 105CFU / mL (P < 0.0001) (FIG. 32B), corresponding to 9.7 ± 1.1% survival (FIG. 26F) and 1.0 ± 0.07 log reduction (FIG. 32C). The survival rate of GDP -treated MRSA was comparable to that of ciprofloxacin-treated samples (12.4 ± 4.9%) (P = 0.99) and less that of GelMAG (53.9 ± 15.7%) (P < 0.05).

[0219] Similarly, SEM images revealed that GDP had less MRSA present on its surface compared to GelMAG (FIG. 5G). In addition, GDP caused a 7 mm ZOI against MRSA (FIG. 5H). Attorney Docket No. 50835-0006W01

[0220] Without the incorporation of pDDA, GelMAG and GD did not have a prolonged effect on the viability of P. aeruginosa during a 6-day incubation (FIG. 5A). However, GP and GDP containing pDDA changed this trend, as seen by their fast-acting and prolonged bactericidal effect that was also reflected in end-stage (day 6) CFU measurements (FIGs. 5A- 5B). To understand the mechanism of such strong antimicrobial activity, smaller amounts of pDDA were introduced to the GDP hydrogels and observed greater bacterial viability (FIGs. 16A-16B)

[0221] Representative scanning electron microscopy (SEM) images of GelMAG and GDP adhesive after the culture revealed significantly less bacteria present on the surface of the hydrogel containing pDDA (FIG. 5C). GDP also displayed proactive bactericidal activity by forming a 10 mm zone of inhibition (ZOI) against P. aeruginosa (FIG. 5D). Likewise, when incubated with MRSA, only hydrogels containing pDDA showed effective antibacterial activity and minimal CFU (FIGs. 5E-5F). Additionally, the surface of GDP adhesive was more inhospitable to MRSA growth compared to GelMAG, and its strong antibacterial nature facilitated a 7 mm ZOI (FIGs. 5G-5H).

[0222] Since GDP and ciprofloxacin exhibited the strongest antibacterial properties, we cultured P. aeruginosa or MRSA with a range of concentrations of either antimicrobial agent to calculate the minimum inhibitory concentration (MIC) required to prevent bacterial growth. After a 24 h incubation, we determined that 1.6-2.3 pg / mL ciprofloxacin was sufficient for inhibiting either strain of bacteria (FIG. 33). On the other hand, since the active ingredient in GDP is only a small portion of the bulk hydrogel, a MIC of 2083-2500 pg / mL was required. We further assessed GDP’s contact-activation mechanism of bacterial resistance through live / dead staining of bacterial cultures after 5 days of treatment with GelMAG, GD, GP, GDP, ciprofloxacin, AquaDerm™, or untreated control (FIG. 34).

[0223] For / < aeruginosa, the control, GelMAG, and GD samples showed predominantly green signals, indicating high bacterial survival. Bacteria treated with AquaDerm™ also showed high green signal. In contrast, the GP and GDP -treated bacteria displayed more pronounced red signals, comparable to those treated with ciprofloxacin, suggesting more prominent bactericidal activity. Likewise, for MRSA, the control, GelMAG, GD, and AquaDerm™ samples appeared to have a large number of live cells, whereas GP and GDP had a better antibacterial effect, similar to that of ciprofloxacin. Overall, live / dead staining revealed the potent antibacterial properties of GDP against both Gram-negative and Grampositive bacteria. The strong antibacterial nature of the GDP sealant could be due to the presence of cationic pDDA, which could likely aggregate anionic bacteria cell membranes Attorney Docket No. 50835-0006W01 and disrupt their growth as well as limit biofilm spreading (27, 22). As exemplified through zone of inhibition tests, pDDA likely leached out of the GDP matrix to interact with and inhibit bacterial growth.

[0224] To assess the rate at which pDDA was released from the hydrogel, we measured the zeta potential of DI water that was incubated with the hydrogel over a period of time. The progressively increasing zeta potential confirmed that pDDA was likely released from the hydrogel during the initial water absorption process (FIG. 35). Solution containing GDP exhibited around 1.3 mV zeta potential after 4 h of incubation and around 10.5 mV after 48 h, both of which were significantly higher than the solution containing pure GelMAG (around 0.12 mV after 4 h, P < 0.05, and around 3.2 mV after 48 h, P < 0.0001). Therefore, the GDP hydrogel could release pDDA for maximum bactericidal activity.

[0225] Most previously developed antibacterial biomaterials utilized dense ionic charge to combat microbial growth. In one case, an antibacterial bioadhesive comprising bacterial cellulose-grafted pDDA, polydopamine, and polyacrylamide was limited by weak adhesion (under 20 kPa). Another hemostatic bioadhesive based on methacrylated chitosan and levodopa-grafted N-hydroxymethyl acrylamide was able to resist bacterial growth and regulate hemostasis. However, synthetic polymers like acrylates and acrylamides have often presented concerns of cytotoxicity. Biocompatibility issues were also observed in a hydrogel comprising GelMA and adenine acrylate used to deliver copper ions as a means of hindering bacterial survival. Overall, antibacterial hydrogels often lack appropriate wet tissue adhesion or biocompatibility, rendering them unsuitable for sealing and protecting wounds against infection. However, GDP adhesive displayed a fast-acting bactericidal nature without compromising its notable adhesive properties.

[0226] Example 12 - Rapid Hemostatic Performance of GDP Hydrogels

[0227] Heavy hemorrhage must be controlled to prevent 50% of post-trauma mortality and for physiological recuperation of traumatic injuries. After demonstrating adequate mechanical and adhesion properties of GDP adhesive that would permit effective sealing of dynamic injuries, the in vitro hemostatic ability was assessed with a standard blood clotting assay using human fresh whole blood. Citrate-activated blood was treated with hemostatic hydrogels and quenched with saline solution at certain timepoints to visually quantify blood clotting efficacy (FIGs. 6A-6B). Untreated blood fully clotted within 58 ± 3 min, quantitatively confirmed by complete absorbance of hemoglobin, while treatment with GelMAG only slightly decreased the clotting time (FIG. 6C). When various amounts of Attorney Docket No. 50835-0006W01

[0228] DMA and pDDA were incorporated into the GDP hydrogels, blood coagulation was expedited by 10-25 min (FIGs. 17-18). The high surface charge of these biomaterials likely permitted adsorbing anionic erythrocytes, causing aggregation and faster clotting. Consequently, the optimal GDP adhesive cut down clotting time in half to 30 ± 5 min (P < 0.0001) (FIG. 6B). At this clotting time, GDP retained the heaviest clot compared to all other formulations (FIG. 6D). The GDP adhesive also greatly reduced the blood clotting index (BCI) of untreated whole blood to 8 ± 4% (FIG. 6E). Furthermore, the ionic charge of GDP enabled rapid swelling to 345 ± 35% after 4 h in an aqueous environment, which supported rapid blood absorbance, clot formation, and hemostasis (FIG. 19).

[0229] The hemostatic efficacy of GDP was also assessed using two in vivo models of injury performed in rats. In a tail amputation model, 6 cm of the tail was incised, causing profuse bleeding from the tail vein (FIG. 7A-i). The injury was then quickly treated with GelMAG or GDP precursor solutions that were photocrosslinked to form hemostatic hydrogels (FIG. 7A- ii). A commercial cellulose-based hemostatic agent Surgicel® was also assessed while untreated injuries were used as a control. Then, blood was collected for 10 min postamputation on pre-weighed filter paper (FIG. 7A-iii). While the untreated control group lost 3,733 ± 855 mg of blood, all treatment groups showed significantly less exsanguination (FIG. 7A-iv). GDP enabled rapid hemostasis (68 ± 18 mg blood loss) and outperformed both GelMAG (162 ± 19 mg, P < 0.05) and Surgicel® (184 ± 20 mg, P < 0.01). A similar trend was observed in the liver puncture model where the rat liver was incised 2 mm deep and treated with hemostatic hydrogels before measuring blood loss (FIGs. 7Bi-iii). Both the untreated control (83 ± 23 mg) and GelMAG treated (61 ± 16 mg) groups experienced profound bleeding while GDP adhesive resulted in only 12 ± 4 mg of blood loss, comparable to the effect of Surgicel® (7 ± 0.6 mg) (FIG. B-iv). In a similar mechanism to numerous chitosan-based hemostats, the charged nature of pDDA within the GDP adhesive expedited blood cell aggregation and clot formation.

[0230] To evaluate the long-term sealing efficacy and biocompatibility of GDP adhesive, histology and immunostaining was performed on hydrogel and liver tissue from rats after two weeks of recuperation. All animals survived and recovered from this traumatic injury with so signs of wound irritation. Hematoxylin and eosin (H&E) staining confirmed that both GelMAG and GDP showed excellent adhesion and retention onto the bloody injury (FIG. 7C). Additionally, there were no signs of fibrous encapsulation in the surrounding tissue, indicating no abnormal inflammatory response to the biomaterials. Immunostaining for lymphocytes (CD3) and macrophages (CD68) was conducted to further characterize the local Attorney Docket No. 50835-0006W01 immune response (FIGs. 7D-7E). There was slight infiltration of both markers in the liver tissue, but no negative reaction was observed in the rats. Therefore, the GDP adhesive facilitated rapid hemostasis and securely sealed the injury without causing cytotoxicity.

[0231] Hemostats with weak adhesive abilities can prove fatal. Previously, surgical hemostats Surgicel® and Oxycel® were used to control bleeding during thoracotomy procedures, but they were left in situ and later found detached from the injury site, causing severe medical complications. Due to their weak tissue adhesion, the hemostats migrated to the spinal cord and caused paraplegia in multiple patients. To overcome this problem, hemostatic patches have been developed with adhesive properties, but many require mechanical pressure to adhere to the injury. In doing so, they can cause damage to delicate soft tissue (e.g., blood vessels) and to non-compressible trauma (e.g., head, neck, torso). Other hemostatic hydrogels that formed and adhered in situ were used to treat rat tail and liver injury models. For example, a conductive and hemostatic hydrogel based on dopamine- grafted gelatin and graphene oxide (GO) decreased the amount of blood lost after liver puncture by 80% compared to the untreated control. An antibacterial and hemostatic hydrogel using chitin and gold nanoparticle-filled halloysite nanotubes (Au@HNT) decreased punctured liver bleeding by 55%. When used to control hemorrhage following rat tail amputation, the chitin hydrogel with Au@NHT reduced bleeding by 61%. Another biomaterial based on carboxymethyl chitosan, dextran, and polyglutamic acid prevented 69% of bleeding from the incised tail. In another case, 82% of blood loss was prevented with a hemostatic hydrogel comprising modified Pluronic F127, chitosan, silk, and tannic acid. The engineered GDP adhesive, however, prevented 86% of blood loss after liver puncture and 98% of bleeding after tail amputation in rats.

[0232] Example 13 - Biocompatibility and Biodegradability of GDP Hydrogel

[0233] A crucial limitation of most hemostatic bioadhesives is lack of biocompatibility and biodegradability. Therefore, the in vitro biocompatibility of GDP adhesive was extensively analyzed using transwell-mediated exposure to mouse embryonic fibroblast (3T3) cells. Live / dead staining of cells cultured with either GelMAG or GDP showed that they proliferated in a comparable manner to the untreated control after 1 and 5 days of incubation (FIGs. 21, 8A-i) Furthermore, quantitative analysis based on live / dead images found an average cell viability of 96 ± 1% across all samples (FIG. 8B). Actin / DAPI staining on 3T3 cells after hydrogel treatment revealed healthy cell spreading, morphology, and number Attorney Docket No. 50835-0006W01

[0234] (FIGs. 8A-ii, 21). Metabolic activity, quantified using a PrestoBlue™ assay, also steadily increased in all samples throughout the culture (FIG. 8C).

[0235] In vivo biocompatibility and biodegradability were also assessed using a rat subcutaneous implantation model. H&E staining of the hydrogel-tissue interface after 7 and 28 days of implantation depicted durable adhesion and retention of GelMAG and GDP adhesive to the tissue (FIGs. 8D, 22A-22B). Additionally, there was significant cell infiltration into the GDP matrix which had undergone 61 ± 3% biodegradation after 28 days of implantation, leaving a more porous scaffold (FIG. 8E). There was also no formation of fibrous capsules around the implant. This negligible immune response was further analyzed by conducting immunostaining for hematopoietic cells (CD45) and macrophages (CD68) (FIGs. 8F-8G). There was a notable presence of both markers at the tissue interface of GelMAG or GDP on post-operation day 7 (FIGs. 23A-23D). By day 28, however, the signs of inflammation had considerably reduced. Therefore, the multifunctional abilities of GDP adhesive did not sacrifice its biocompatibility and biodegradability.

[0236] Example 14 - In Vivo Hemostatic, Adhesion, and Biocompatibility of GDP Hydrogel on Pig Lung Injury

[0237] After assessing the biocompatibility and biodegradability of the GDP hydrogel using small animal models, the multifunctional behavior of the hemostatic sealant was assessed on a large lung injury model conducted in pigs. First, a lateral thoracotomy was performed to access the lung. Then a 15 mm long laceration and subsequent air leak was created, prepolymer solution was applied and photo-crosslinked to form a hydrogel, and then the leak was confirmed to be sealed with saline submersion (FIG. 9A). After the procedure, ultrasound was used to monitor the state of the lungs and hydrogel placement. The healthy lung ultrasound showed that there was no sign of post-operative pneumothorax, as indicated by the presence of a clear pleural line and A lines (FIG. 9B). After 14 days, the hydrogels and surrounding tissue were explanted and used for H&E analysis. Intersections of the H&E stained hydrogel and tissue showed robust adhesion and retention of the both GDP and TISSEEL® onto the lung for the duration of the experiment (FIGs. 9C-9D). However, while GDP did not cause any signs of fibrosis or collagen deposition - indicating no abnormal immune response from its presence - the tissue treated with TISSEEL® had severe fibrosis and potentially even necrosis. The amalgamation of cells in the region under the hydrogel may be a sign of lack of oxygen and nutrients to the underlying tissue which ultimately impacted the normally porous-looking lung tissue. After the creation of the laceration, the Attorney Docket No. 50835-0006W01 hemostatic performance of hemostatic sealants GDP and TISSEEL® or non-adhesive hemostat Surgicel™ was evaluated for 1 minute while the expelled blood was collected using filter paper (FIG. 9E-i). The hemostatic assay revealed that GDP caused significantly less blood loss when compared to the untreated injury or the injuries that were treated with TISSEEL® or Surgicel™ (FIG. 9E-ii). Along with the hemostatic performance, the longterm in vivo and ex vivo adhesion capabilities of the GDP hydrogel were evaluated.

[0238] After 14 days post-operation, the live pigs were ventilated with progressively increasing the pressures of air (FIG. IF-i) and monitored using ultrasound to assess for pneumothorax or failure of the hydrogel sealant. Even with maximum ventilator output and the maximum pressure that the lungs could withstand, the GDP hydrogel remained an effective sealant (FIG. 9F-ii) The pigs survived this recovery period without loss of body mass (FIG. 37A) or incidences of pneumothorax (FIG. 37B). After the animal was sacrificed, a thoracotomy was performed to access the operation site and another increase in ventilator-driven pressure test was conducted to assess the hydrogel’s sealing abilities (FIG. 9F-iii). Once again, the GDP hydrogel could withstand the maximum pressure possible, indicating its robust adhesion onto the injured lung. The hydrogel was then explanted with surrounding tissue for immunohistological analysis. Staining for lymphocytes (CD3) revealed that the tissue surrounding GDP did not experience many signs of inflammation (FIG. 9G). Similarly, there were few to no pan-macrophages (CD68) or activated macrophages (CD80), further demonstrating the biocompatibility of the hemostatic sealant (FIG. 9H). On the contrary, there was excessive lymphocyte and activated macrophage infiltration into the lung tissue treated with TISSEEL® even after 14 days post-operation (FIGs. 9G-9H). Overall, the GDP sealant was able to cause rapid hemostasis and seal the lacerated lung of the pigs without causing severe inflammation, unlike the commercial standard for hemostatic sealing.

[0239] Example 15 - GDP Hydrogels Exhibit Superior Performance

[0240] Hemostatic bioadhesives for trauma therapy have been heavily investigated and commercialized, however most have limited efficacy due to mechano-physical mismatching to the injury, weak adhesion to bloody tissue, or cytotoxicity. Therefore, an ideal platform for rapid hemostasis, dynamic wound sealing, and prolonged antibacterial resistance was engineered, thus minimizing the most common complications associated with trauma. GMA- grafted gelatin was mixed with MA-grafted dopamine and polyelectrolyte pDDA to produce the GDP adhesive. This composite was highly stretchable, strong, and conductive. It depicted robust in vitro adhesion on pig skin tissue (significantly higher than commercial lung sealants Attorney Docket No. 50835-0006W01

[0241] CoSeal™ and Progel™) and also acted as an air-tight sealant on clinically relevant, multidimensional ex vivo injuries on pig lungs. The addition of pDDA not only enabled numerous chemical interactions with DMA that bolstered the adhesion of the GDP hydrogel, but it also resulted in a prolonged antibacterial effect against P. aeruginosa and MRSA. There was also a fast-acting hemostatic effect noted in vitro on whole blood and in vivo on rat tail amputation and liver puncture models. Treatment of the bloody injuries with GDP resulted in minimal blood loss, comparable to or better than the commercial surgical hemostat Surgicel®. GDP was also confirmed to be biocompatible and biodegradable through in vitro assays using 3T3 cells as well as by in vivo subcutaneous implantation into rat dorsal tissue. Overall, GDP displayed great potential in controlling hemorrhage, sealing wounds on dynamic, elastic, and bloody surfaces, and providing a biologically safe scaffold for tissue regeneration, which makes it well-suited to treat a broad range of traumatic injuries.

[0242] OTHER EMBODIMENTS

[0243] It is to be understood that while certain embodiments have been described within the detailed description, the present disclosure is intended to illustrate and not limit the scope of any embodiment defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

Attorney Docket No. 50835-0006W01WHAT IS CLAIMED IS:

1. A hemostatic and sealant composition comprising: glycidyl methacrylated gelatin (GelMAG); methacrylated dopamine (DMA); poly(diallyldimethylammonium chloride) (pDDA); and a visible light-activated photoinitiator.

2. The hemostatic and sealant composition of claim 1, wherein the DMA is conjugated to a backbone of the GelMAG.

3. The hemostatic and sealant composition of claims 1 or 2, wherein the GelMAG is present at a concentration of 20% (w / v).

4. The hemostatic and sealant composition of any one of claims 1-3, wherein the GelMAG is present at a concentration ranging from 15% (w / v) to about 25% (w / v).

5. The hemostatic and sealant composition of any one of claims 1-4, wherein the DMA is present at a concentration ranging from about 0.05% (w / v) to about 0.15% (w / v).

6. The hemostatic and sealant composition of claim 5, wherein the DMA is present at a concentration of about 0.1% (w / v).

7. The hemostatic and sealant composition of any one of claims 1-6, wherein the pDDA is present at a concentration ranging from about 0.5% (v / v) to about 25% (v / v).

8. The hemostatic and sealant composition of claim 7, wherein the pDDA is present at a concentration of about 2% (v / v).

9. The hemostatic and sealant composition of any one of claims 1-8, wherein the GelMAG has a degree of methacryloyl substitution of about 50%.

10. The hemostatic and sealant composition of any one of claims 1-9, wherein the GelMAG has a degree of methacryloyl substitution between about 40% and about 60%.Attorney Docket No. 50835-0006W0111. The hemostatic and sealant composition of any one of claims 1-10, wherein the photoinitiator comprises Eosin Y, triethanolamine (TEA), N-vinyl-s-caprolactam (VC), or any combination thereof.

12. The hemostatic and sealant composition of any one of claims 1-11, wherein the composition is in a form of a solution or a hydrogel.

13. The hemostatic and sealant composition of any one of claims 1-12, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.

14. The hemostatic and sealant composition of any one of claims 1-13, wherein the hemostatic and sealant composition is formulated for topical use.

15. The hemostatic and sealant composition of any one of claims 1-14, wherein the visible light-activated photoinitiator is activated upon exposure of light having a wavelength between about 450 nanometers (nm) to about 550 nm.

16. The hemostatic and sealant composition of any one of claims 1-15, wherein the hemostatic and sealant composition is ionically conductive, biocompatible, biodegradable, adhesive, and antibacterial.

17. A hemostatic and sealant hydrogel precursor composition comprising the hemostatic and sealant composition of any one of claims 1-16.

18. A hemostatic and sealant hydrogel formed by photo-crosslinking the hemostatic and sealant hydrogel precursor composition of claim 17.

19. The hemostatic and sealant hydrogel of claim 18, wherein the hemostatic hydrogel has: (i) a Young’s modulus ranging from about 30 kilopascals (kPa) to about 200 kPa;(ii) an ultimate strength ranging from about 60 kPa to about 150 kPa; (iii) a stretchability ranging from about 80% to about 220%; (iv) a toughness of about 20 kilojoules per cubic meter (kJ / m3) to about 105 kJ / m3; (v) a compression modulus of ranging from about 30 kPa to about 80 kPa; (vi) an energy loss of about 10% to about 30%; (vii) an adhesion strength ranging from about 20kPa to about 50 kPa; (viii) an adhesion energy ranging from about 10 joules per cubic meter (J / m3) to about 50 J / m3;(ix) a burst pressure ranging from about 20 kPa to about 60 kPa; (x) a conductivityAttorney Docket No. 50835-0006W01 ranging from about 0.1 Siemens per meter (S / m) to about 1 S / m; or (xi) any combination of (i)-(iv).

20. A method of sealing a wound in a tissue of a subject, the method comprising: contacting the wound of the subject with a therapeutically effective amount of the hemostatic and sealant hydrogel precursor composition of claim 17; and photo-crosslinking the hemostatic hydrogel precursor composition by exposing the hemostatic hydrogel precursor composition to a visible light, thereby forming a hemostatic hydrogel.

21. A method of preparing a hemostatic and sealant hydrogel using the hemostatic and sealant composition of any one of claims 1-16, the method comprising: dissolving the GelMAG in a solution comprising the visible light-activated photoinitiator; mixing the DMA with the solution comprising the visible light-activated photoinitiator and the dissolved GelMAG; incubating the solution comprising the visible light-activated photoinitiator, the dissolved GelMAG, and the DMA for at least twenty four hours; mixing the pDDA with the solution comprising the visible light-activated photoinitiator, the dissolved GelMAG, and the DMA; and photo-crosslinking the solution by exposing the solution to a visible light after mixing the pDDA with the solution, thereby forming the hemostatic and sealant hydrogel.

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